Laboratory Handbook Full

LABORATORY HANDBOOK
FOR DAIRY INDUSTRY
National Dairy Development Board (NDDB)

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LABORATORY HANDBOOK
F O R D A I R Y I N D U S T R Y
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Preface
National Dairy Development Board has compiled all method adopted and approved by Nepal
Bureau of Standard and Metrology as well as selected additional suitable analytical methods into
a Laboratory Handbook. This Laboratory Handbook is aimed at fulfilling the need for
standardized analytical methods used for analyses of milk and milk products.
With this new handbook the methodology used at dairy plants should therefore in the future be
the same as the methodology used at public laboratories controlling the quality of milk and milk
products marketed in the country.
The outline and format of the Laboratory Handbook follow international standards and its
contents and analytical methods have all been discussed, evaluated and amended in cooperation
with representatives from Nepal Bureau of Standard and Metrology, Department of Food
Technology & Quality Control and Dairy Development Corporation.
The methodologies included follow the standard methodologies of:
• Nepal Bureau of Standard and Metrology (NBSM)

• Bureau of Indian Standards (BIS)
• Food and Agriculture Organization of the United Nations (FAO)
• Association of Official Analytical Chemists (AOAC)
• Dairy Development Corporation (DDC)
The priority given to the mentioned standards are as follows:
• 1st priority is given to Nepal Bureau of Standard

• 2nd priority is given to Bureau of Indian Standard
• 3rd priority is given to International Dairy Federation
• AOAC, FAO and DDC methods are included in the handbook where no methods were
available from NBSM, BIS or IDF
NDDB wishes to than everyone who has been involved in the preparation of this Laboratory
Handbook. A special thank is given to Ms. Anjuman Shrestha, Microbiologist NDDB/DSP and
Ms. Inger Waldhauer, Milk Quality Advisor NDDB/DSP for all their great work in the
compilation of the Laboratory Handbook.
NDDB
Kathmandu, March 2001
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Table of contents
SN Contents Page
1. Chapter 1 – SAMPLING TECHNIQUE……………………………………………... 1
2. Chapter 2 – DISINFECTION AND STERILIZATION…………………………….. 13
3. Chapter 3 – RECOMMENDED SAMPLING PLAN………………………………... 17
4. Chapter 4 – EXAMPLES OF REGISTRATION AND REPORTING SYSTEM…. 21
5. Chapter 5 – RAW MILK……………………………………………………………… 44
5.1 Determination of Sensory Quality of milk……………………………………………... 45
5.2 Determination of Temperature of Milk…………………………………………………. 47
5.3 Determination of pH of Milk……………………………………………………………. 48
5.4 COB Test in Milk……………………………………………………………………….. 49
5.5 Alcohol Test of Milk……………………………………………………………………. 50
5.6 Determination of Titrable Acidity in Milk……………………………………………… 51
5.7 Determination of Fat Content in Milk…………………………………………………... 52
5.8 Determination of Specific Gravity of Milk……………………………………………... 55
5.9 Determination of Solids Not Fat Percentage in Milk…………………………………… 56
5.10 Determination of Water Adulteration by Freezing Point……………………………….. 58
5.11 Detection of Sugar Adulteration in Milk……………………………………………….. 59
5.12 Detection of Starch Adulteration in Milk……………………………………………….. 60
5.13 Detection of Carbohydrate in Milk……………………………………………………… 61
5.14 Determination of Neutralizer, Sodium Bicarbonate…………………………………….. 62
5.15 Detection of Salt Adulteration in Milk………………………………………………….. 63
5.16 Detection of Formaldehyde in Milk…………………………………………………….. 64
5.17 Detection of Formaldehyde in Milk…………………………………………………….. 65
5.18 Detection of Urea adulteration in Milk…………………………………………………. 66
5.19 Detection of Hydrogen Peroxide Preservatives in Milk………………………………… 67
5.20 Detection of Neutralizer Caustic Soda in Milk…………………………………………. 68
6. Chapter 6 – PASTEURIZED MILK…………………………………………………. 69

6.1 Determination of Sensory Quality of Pasteurized Milk………………………………… 70
6.2 Determination of Fat Content in Pasteurized Milk……………………………………… 72
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6.3 Determination of Solids Not Fat in Pasteurized Milk…………………………………... 74
6.4 Determination of Acidity in Pasteurized Milk………………………………………….. 76
6.5 Determination of Phosphatase in Milk…………………………………………………. 77
6.6 Determination of Phosphatase in Milk…………………………………………………. 79
7. Chapter 7 – CREAM………………………………………………………………….. 80
7.1 Determination of Sensory Quality of Cream……………………………………………. 81
7.2 Determination of Fat Content in Cream………………………………………………… 83
7.3 Determination of Fat Content in Cream………………………………………………… 84
7.4 Determination of Acidity in Cream…………………………………………………….. 86
8. Chapter 8 – BUTTER…………………………………………………………………. 87
8.1 Determination of Sensory Quality of Butter……………………………………………. 88
8.2 Determination of Fat in Butter by Gerber Method…………………………………….. 90
8.3 Determination of Fat Content in Butter………………………………………………… 91
8.4 Determination of Fat by Soxhlet Extraction Method…………………………………… 92
8.5 Determination of Moisture Content in Butter…………………………………………… 94
8.6 Determination of Moisture Content in Butter…………………………………………… 95
8.7 Determination of Water, SNF & Fat in the Same Test Sample…………………………. 96
8.8 Determination of Salt Content in Butter………………………………………………… 99
8.9 Determination of Salt Content in Butter………………………………………………… 101
8.10 Determination of Solids Not Fat Content in Butter…………………………………….. 102
8.11 Detection of Coloring Matter in Butter………………………………………………… 104
8.12 Determination of Titrable Acidity in Butter……………………………………………. 105
8.13 Determination of Rancidity (Peroxide Value) in Butter………………………………… 106
9. Chapter 9 – GHEE……………………………………………………………………... 108

9.1 Determination of Sensory Quality of Ghee……………………………………………... 109
9.2 Determination of Moisture in Ghee (Routine Method)…………………………………. 110
9.3 Determination of Moisture in Ghee (Reference Method)………………………………. 111
9.4 Determination of Acidity (Free Fatty Acid) in Ghee…………………………………… 112
9.5 Determination of Refractive Index of Ghee……………………………………………. 113
9.6 Determination of RM Value (Reichert-Meissel) in Ghee………………………………. 115
9.7 Determination of Insoluble Impurities in Ghee………………………………………… 117
9.8 Determination of Melting Point of Ghee……………………………………………….. 118
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9.9 Determination of Rancidity (Peroxide Value) in Ghee…………………………………. 120
9.10 Determination of Vegetable Fat in Ghee……………………………………………….. 122
10. Chapter 10 – ICE CREAM……………………………………………………………. 126

10.1 Determination of Sensory Quality of Ice Cream………………………………………... 127
10.2 Determination of Fat Content in Ice Cream…………………………………………….. 129
10.3 Determination of Fat Content in Ice Cream…………………………………………….. 130
10.4 Determination of Total Solids Content…………………………………………………. 131
10.7 Determination of Titrable Acidity in Ice Cream………………………………………… 135
10.8 Determination of Overrun in Ice Cream………………………………………………… 136
10.9 Determination of Percentage Overrun in Ice Cream……………………………………. 138
10.10 Determination of Phosphatase in Ice Cream……………………………………………. 139
10.11 Determination of Sucrose Content in Ice Cream……………………………………….. 140
11. Chapter 11 – PANEER……………………………………………………………….. 142

11.1 Determination of Sensory Quality of Paneer…………………………………………… 143
11.2 Determination of Titrable Acidity in Paneer…………………………………………… 144
11.3 Determination of Fat Content in Paneer………………………………………………… 145
11.4 Determination of Moisture in Paneer…………………………………………………... 146
12. Chapter 12 – CHHANA………………………………………………………………. 147

12.1 Determination of Sensory Quality of Chhana………………………………………….. 148
12.2 Determination of Moisture of Chhana………………………………………………….. 149
12.3 Determination of Fat Content in Chhana……………………………………………….. 150
13. Chapter 13 – YOGHURT…………………………………………………………….. 151

13.1 Determination of Sensory Quality of Yoghurt…………………………………………. 152
13.2 Determination of Fat Content in Yoghurt……………………………………………….. 154
13.3 Determination of Titrable Acidity in Yoghurt…………………………………………... 155
13.4 Determination of percentage of Acidity in Yoghurt……………………………………. 156
13.5 Determination of Total Solids Content in Yoghurt……………………………………... 157
13.6 Determination of Total Solids Content in Yoghurt…………………………………….. 158
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14. Chapter 14 – CHEESE………………………………………………………………… 160
14.1 Determination of Sensory Quality of Cheese…………………………………………… 161
14.2 Determination of Salt Content in Cheese……………………………………………….. 165
14.3 Determination of Salt Content in Cheese……………………………………………….. 167
14.4 Determination of Chloride Content in Cheese………………………………………….. 168
14.6 Determination of Fat Content in Cheese……………………………………………….. 172
14.7 Determination of pH of Cheese………………………………………………………… 174
14.8 Determination of Nitrogen Content in Cheese…………………………………………. 175
14.9 Determination of Protein in Cheese……………………………………………………. 179
15. Chapter 15 – KHOA…………………………………………………………………… 181

15.1 Determination of Sensory Quality of Khoa…………………………………………….. 182
15.2 Determination of Titrable Acidity of Khoa…………………………………………….. 183
15.3 Determination of Moisture of Khoa……………………………………………………. 185
15.4 Determination of Fat Content in Khoa…………………………………………………. 186
16. Chapter 16 – SKIM MILK AND WHOLE MILK POWDER……………………… 187

16.1 Determination of Sensory Quality of Milk Powder…………………………………….. 188
16.2 Determination of Fat Content of Dried Milk…………………………………………… 190
16.3 Determination of Water Content in Dried Milk………………………………………… 194
16.4 Determination of Moisture and Total Solids…………………………………………… 195
16.5 Determination of Titrable Acidity……………………………………………………… 196
16.6 Determination of Titrable Acidity………………………………………………………. 197
16.7 Determination of Solubility Index of Milk Powder…………………………………….. 199
16.8 Determination of Dispersibility and Wettability of Instant Dried Milk………………… 201
17. Chapter 17 – SWEETENED CONDENSED MILK………………………………… 204

17.1 Determination of Sensory Quality……………………………………………………… 205
17.2 Determination of Fat Content…………………………………………………………… 206
17.3 Determination of Total Milk Solids……………………………………………………. 211
17.5 Determination of Total Solids Content………………………………………………….. 215
17.6 Determination of Sucrose Content……………………………………………………… 217
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17.7 Determination of Sucrose……………………………………………………………….. 221
17.8 Determination of Titrable Acidity………………………………………………………. 227
18 Chapter 18 – BUTTERMILK…………………………………………………………. 228

18.1 Determination of Fat Content in Buttermilk……………………………………………. 229
19. Chapter 19 – MICROBIOLOGICAL ANALYSIS………………………………….. 233

19.1 Diluent, Culture Medium & Reagent for Microbiological Tests……………………….. 234
19.2 Methylene Blue Reduction Test for Milk………………………………………………. 237
19.3 Dilution Techniques…………………………………………………………………….. 239
19.4 Total Plate Count……………………………………………………………………….. 241
19.5 Enumeration of Coliforms……………………………………………………………… 243
19.6 Enumeration of Yeasts and Molds……………………………………………………… 246
19.7 Enumeration of Yeasts and Molds……………………………………………………… 247
19.8 Method for Yeast and Mold Count of Foodstuff……………………………………….. 249
19.9 Detection of Penicillin in Milk by Disk Assay Technique……………………………… 252
19.10 Detection of Mastitis in Milk…………………………………………………………… 257
20. Chapter 20 – HYGIENIC CONDITIONS IN DAIRY INDUSTRY………………... 259
21. Chapter 21 – RECOMMENDATIO FOR ESTABLISHMENT OF 267

LABORATORY………………………………………………………………………..
22. Chapter 22 – GOOD LABORATORY PROCEDURES……………………………. 282
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Chapter 1
SAMPLING TECHNIQUE
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Sampling Technique
Aim: To accurately sample milk and dairy products.

Principle: Correct sampling is an operation that requires most careful attention. The accurate
sampling of milk and dairy products is important because only a sample truly representative of
the whole and collected in the correct way can form basis for reliable results of the tests to be
carried out afterwards. There are separate methods for sampling these products depending on the
type of test to be carried out later. This International Standard gives guidance on methods of
sampling milk and milk products for microbiological, chemical, physical and sensory analysis.
The following sections describe how the sampling is carried out.
General requirements:
Sampling personnel
An authorized person, properly trained in the appropriate technique shall perform sampling. The
person shall be free from any infectious disease. A person experienced in the technique of
sampling shall always undertake sampling for microbiological examination for microbiological
purposes.
Sealing and labeling of samples
Samples shall be sealed (in the case of an agreement between the parties concerned) and a label
attached, reproducing integrally the identification of product, the nature of the product and at
least the identification number, name and signature of the person responsible for taking the
samples. If necessary, additional information may be included, such as the purpose of sampling,
the mass or volume of sample and the unit from which the sample was taken and the condition of
product and storage conditions at the moment of sampling.
Apparatus:
Sampling equipment
Sampling equipment shall be made of stainless steel or other suitable material of adequate
strength, which does not bring about a change in the sample which could affect the results of
subsequent examinations. All surfaces shall be smooth and free from crevices. All corners shall
be rounded. The equipment shall be dry prior to use.
Sampling for microbiological examination
Sampling equipment shall be clean and sterilized prior to use. Disposable plastic equipment shall
be sterile. If possible, sterilization shall be performed by one of the two following methods:
Method A: Exposure to hot air at 170-175°C for not less than 2 hours.
Method B: Exposure to steam at 121±1°C for not less than 20 min in an autoclave.
After sterilization by method A or method B, sampling equipment shall be stored under sterile
conditions prior to use.
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If, in a particular situation, sterilization by method A or method B is impossible, the following
alternative methods, which shall be regarded as secondary methods only, can be used, provided
that the sampling equipment is used immediately after sterilization:
Method C: Exposure to suitable flame so that all working surfaces of the sampling equipment
come in contact with the flame.
Method D: Immersion in at least 70% v/v ethanol solution
Method E: Ignition with 96% v/v ethanol (be aware that 96% ethanol is hygroscopic and may
change its concentration during use for a longer time)
Method F: Exposure to a sufficient dose of γ-radiation.
After sterilization by method C or method E, sampling equipment shall be cooled under sterile
conditions or, in case of method D, be rinsed with the ethanol solution before sampling.
Sampling for chemical and physical analysis and for sensory examination.
Sampling equipment shall be clean and dry and shall not influence the properties, such as odor,
flavor or consistency and composition of the product. In some cases sterile equipment is required
to avoid microbial contamination of t he product.
Sample containers
Sample containers and closures shall be of materials and construction which adequately protect
the sample and which do not bring about a change in the sample which could affect the results of
subsequent analyses or examinations. Materials which are appropriate include glass, some metals
(for example stainless steel) and some plastics (for example polypropylene). The containers
should preferably be opaque. If necessary, transparent filled containers shall be stored in a dark
place. Containers and closures shall be dry, clean and either sterile or suitable for sterilization by
one of the methods A, B, C or D.
The shape and capacity of the containers shall be appropriate to the particular requirements for
the product to be sampled. Single-service plastic containers as well as aluminum foil of adequate
strength (sterile and non-sterile) and suitable plastic bags with appropriate methods of closure
may also be used.
Containers other than plastic bags shall be securely closed either by means of a suitable stopper
or by means of a screw-cap of the metal material, having if necessary, a liquid tight plastic liner
which is insoluble, non absorbent and grease proof and which will not influence the composition,
properties or the odor and flavor of the sample. If stoppers are used, they shall be made for or
covered with non-absorbent, odorless and flavorless material. Containers for samples for
microbiological examinations shall not be closed with cork stoppers or caps with cork seals, even
if provided with liners. Containers for solid, semi solid or viscous products shall be wide
mouthed. In case of small retail containers these are considered as sample containers; the sample
shall consist of the contents of one or more intact, unopened containers.
Sampling Technique:
Sampling shall be done in such a way as to get representative samples of the product. If samples
for microbiological, chemical and physical analyses and sensory examinations are taken
separately, samples for microbiological examinations shall be taken first using aseptic techniques
and sterilized equipment and containers. Care shall be taken to ensure that when taking samples
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for sensory examinations the flavor of the samples is not adversely affected by sterilization of
sampling equipment or sampling corks, for example flaming with ethanol. The precise method of
sampling and the mass oar volume of product to be taken vary with the nature of the product and
the purpose for which samples are required.
Preservation of samples
Preservatives shall normally not be added to samples intended for microbiological or sensory
examination. Preservatives may be added to some products, provided that:
• An instruction to do so is issued by the testing laboratory,
• The preservative is of a nature that does not interfere with subsequent analyses and testing
of texture and flavor shall not be performed, and
• The nature and quantity of preservative are stated in the sampling report and preferably
indicated on the label.
Storage and transport of samples
Storage and dispatch of the samples shall be such that the state of the sample at the time of
sampling is not adversely affected to any considerable extent. During transport, where necessary,
precaution should be taken to prevent exposure to off odors, direct sunlight and other adverse
conditions.
If cooling is necessary the minimum requirements to be met are the temperature ranges which are
either legally requested or prescribed by the manufacturer. The storage temperature after sample
should be attained as quickly as possible. The time and temperature shall be considered in
combination and not independently. Samples shall be dispatched immediately after sampling to
the testing laboratory. The time for dispatch of the sample to the testing laboratory shall be as
short as possible, preferably within 24 hours. If requested, samples shall be dispatched as
instructed by the testing laboratory.
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Milk and Liquid Milk Products
Applicability:
The instructions given in this clause are applicable to raw and heat-treated milk (except raw milk
from individual animals and raw milk taken within quality payment schemes), whole, partly
skimmed and skimmed milk, flavored milk, cream, fermented milk, buttermilk, liquid whey and
similar products.
Apparatus for manual mixing
Agitation for mixing liquids in bulk shall have a surface sufficient to produce disturbance of the
products. In view of the different shapes and sizes of containers, no specific design of agitators
can be recommended for all purposes but they shall be designed in such a way as to avoid
damage of the inner surface of the container during mixing.
Apparatus for manual agitation in small vessels
For mixing liquids in small vessels (for example in buckets and cans) a stirrer (plunger) of the
design and dimensions as shown in figure 1 is suitable. The length shall be adjusted to the depth
of the vessel.
300 to 1000mm
12.5mm
Figure 1: Recommended agitator (plunger) for cans and buckets
at least 2000mm
30mm
Figure 2: Suitable agitator (plunger) for road, rail and farm tanks
Apparatus for manual mixing in large vessels
A stirrer (plunger) of the design and dimensions as shown in figure 2 is suitable for use for larger
vessels (for example, road and farm tanks).
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Apparatus for mechanical agitation
Built-in agitators
The product to be mixed in the dark or vessel determines the technical data and construction of
built-in agitators. Various types of agitators are used but descriptions of them are beyond the
scope of this international standard.
Removal agitators
Removal agitators are mostly provided with a propeller and are introduced into transport tanks
and road and rail tanks through the manhole. Best stirring results are achieved at a depth
corresponding to 0.7 of the filling height. It is recommended that the stirrer be inclined 5-20° as
this allows, besides a vertical mixing of the liquor liquid also a horizontal movement.
Apparatus for sampling
A dipper of the shape and size shown in figure 3 is suitable to be used for sampling. The tapered
form of the cup permits nesting of the dippers.
Capacity not
less than 50ml
Figure 3: Suitable dipper for liquids

Sample containers
The capacity of the sample containers shall be such that they are almost completely filled by the
sample and allow proper mixing of the content before testing but avoid churning during transport.
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Sampling:
Thoroughly mix all liquids by inverting, stirring by pouring to and from one product container to
another of the same volume until sufficient homogeneity is obtained. The volume of the sample
taken shall not be less than 100ml.
Take samples for microbiological examination always first using aseptic techniques and
whenever possible from the same product containers as those taken for chemical and physical
analyses and for sensory examination. Sterile sampling equipment and sample containers shall be
applied using aseptic technique.
Sampling for chemical and physical analyses and for sensory examination
In certain cases sampling equipment and sample containers shall be sterile for chemical and
physical analyses and sensory examination. A method of mixing shall be considered efficient if
the difference in fat content between two samples taken under these conditions is less than 0.1%.
Small vessels, milk buckets and cans
Thoroughly mix the milk for example by transfer, stirring or plunging (plunger).
Milk tanks and vats
Mechanically agitate the milk for at least 5 min, until sufficient homogeneity is obtained. If the
tank is equipped with a periodical, time programmed agitation system, sampling may be carried
out after only a short duration (1-2 min). In those instances where the propeller of the agitator is
close to the surface of the milk, do not use the agitator since this is likely to lead to the formation
of foam.
Weighing bowl
It is essential for the milk to be adequately mixed in the weighing bowl if a representative sample
is to be obtained. The degree of mixing achieved when milk is tipped into the weighing bowl
varies and does not allow proper weighing. The amount of additional mixing shall be determined
by experiment. When the volume of milk to be sampled exceeds the capacity of the weighing
bowl, a sample representative of the whole consignment shall be obtained.
Large vessels, storage, rail and road tanks
In each case, thoroughly mix the milk by an appropriate method before sampling for example
mechanical agitation, stirring with clean compressed without foaming or by plunging (plunger).
Mixing using a plunger or a removable agitator to be used in road, rail tanks or vessels of similar
size shall be performed.
When samples are taken within 30min after filling the container, mix the milk for at least 5 min
by plunging or stirring with an agitator. When the milk has been stored in the tank for a longer
period of time mixing shall be extended to at least 15 min.
When the tank is completely filled, as is normally the case with transport, road and rail tanks,
proper mixing of milk showing pronounced creaming phenomenon can only be achieved by
mechanical agitation.
[Source: International Dairy Federation (IDF) Standard, 50C: 1995]
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Butter and Related Products
Applicability
The instructions given in this clause are applicable to butter, butter with additives, half-cream
milk and half-fat butter and similar products
Sampling equipment (see for milk)
• Butter triers of sufficient length to pass diagonally to the bottom of the product container
and of a dimension suited for the purpose envisaged.
• Spatula, broad-bladed
• Knife, of suitable size
Sampling
The size of the sample shall not be less than 50g
whenever possible, from the sample product containers as those taken for chemical and physical
analyses and for sensory examination.
Use the spatula to remove the surface layer of the product from the sampling area to a depth of
not less than 5mm. use sterile trier each time for taking the core of the product. For
microbiological examination of the surface, sample shall be performed according to special
instructions depending on the purpose envisaged.
Sampling for chemical, physical analysis and for sensory examination
Take for a number of sensory examinations and especially for physical analysis, a sample of 2kg.
In certain cases sampling equipment and sample containers shall be sterile for chemical, physical
analysis and for sensory examination.
Retail containers (with content of 1kg or less)
The content of the intact and unopened container constitutes the sample. Take one or more
containers to obtain a sample of not less than 50g.
Products in bulk or packets (with content of more than 1kg)
Pass the butter trier of suitable size from the edge diagonally through the product, ensuring that
the trier does not penetrate the bottom surface. Rotate the trier through a half turn and withdraw it
with the core.
Discard the upper 25mm of the core. Remove the rest of the core by means of a spatula from the
trier and transfer it either directly or after wrapping in aluminum foil to the container. The
temperature of the butter, the sampling room and the butter trier should be about the same.
Sampling of butter stored under deep freezing conditions requires special care and experience.
Large containers (for sample sizes of more than 2kg)
For sampling from a large container or sample sizes of more than 2kg cut with a knife a block of
the product which will fit into the sample box, wrap the block in aluminum foil and place it in the
box. Avoid deformation of the product during cutting and wrapping.
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Butterfat (Butteroil) and Related Products
Applicability
The instructions given in this clause are applicable to anhydrous milkfat, butterfat, butteroil and
similar products.
Sampling equipment
• Butter triers: Of sufficient length to pass diagonally to the bottom of the product
container and of a dimension suited for the purpose envisaged.
• Spatula: Broad-bladed
• Agitator (plunger)
• Dipper: Of capacity 25-100ml
Sample container
The capacity of the sample containers shall be such that they are almost completely filled by the
sample and allow proper mixing of the contents before testing. The size of the sample shall not be
less than 50g.
whenever possible, from the same product containers as those taken for chemical and physical
analyses and for sensory examination. Sterilize the sampling equipment and sample containers as
described for liquid milk. Use spatula to remove the surface layer of the product from the
sampling area to a depth of not less than 5mm and proceed using aseptic technique.
Sampling for chemical and physical analyses and for sensory examination
In certain cases sampling equipment and sample containers shall be sterile for chemical, physical
analysis and for sensory examination.
Retail containers (with a content of 1kg or less)
The content of intact and unopened containers to obtain a sample of 200g.
Product in bulk
Liquid products: Thoroughly mix the product by plunging or by mechanical agitation until
sufficient homogeneity is obtained.
Solid products: Take as in solid butter sample.
[Source: International Dairy Federation (IDF) Standard 50C: 1995]
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Cheese
Applicability
The instructions given in this clause are applicable to cheese, in particular hard, extra hard
cheese, semi-hard, semi-soft, soft cheese, processed cheese preparations, flavored processed
cheese and cheese products.
Sampling equipment and chemicals
Cheese triers: Of shape and size appropriate to the cheese to be sampled
Knife: With pointed blade and smooth surface
Spatula
Cutting wire: Of sufficient/appropriate size and strength
Sealing compounds: For example a mixture of paraffin, wax and beeswax, prepared by heating
which shall be in compliance with the food law of the specific country
Ethanol: Undenatured, 70%, v/v
Sample container: (See as in liquid milk)
Sampling
The size of the sample shall be less than 100g. Immediately after sampling, place the samples
(cores, slices, sectors, entire small cheese, etc.) in a sample container of suitable size and shape.
The sample may be cut into pieces for insertion into the container, but it shall not be compressed
or ground. Storage of cheese samples tightly wrapped in aluminum foil inside or even outside a
sample container is especially suitable to prevent molding of the cheese surface.
Unless otherwise specified, whatever the method of sampling used, the sample shall include any
surface layer of the cheese (such as mold and rind). If it is necessary to examine the surface layer
(for example to examine surface flora), special sampling instructions shall be observed according
to the purpose envisaged. Take the heterogeneity of the product into account when collecting
samples.
Sampling of cheese other than fresh cheese and cheese sold in brine, oil, etc.
Take samples for microbiological examination first using aseptic techniques and whenever
possible, from the same cheese as those taken for chemical, physical analyses and for sensory
examination. Sampling is performed, depending upon the shape, mass and type, by taking an
entire cheese, packed or pre-packed portions or a sector, slices or cores as shown in figure 8.
Sampling by taking an entire cheese or cheese pre-packs
This method is normally used for small cheeses, small portions of cheese or pre-packed cheeses.
Take a sufficient number of packets or portions to obtain a sample of not less than 100g. place the
sample in the original packaging in the sample container (plastic bags, etc.).
Sampling by taking sectors, slices or cores
Remove any outer wrapping from the cheese; inner wrapping, such as for example wax or plastic
film is not removed.
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Sampling by cutting sectors or slices
Cut the sample by means of a knife of sufficient size or a cutting wire. The sectors or slices shall
be of sufficient thickness.
Sampling by taking cores
Take samples for microbiological examination first using aseptic techniques and whenever
possible, from the same product (core) as those taken for chemical and physical analyses and for
sensory evaluation.
The amount of sample taken for surface samples may be smaller than 100g. The sampling
equipment and containers shall be sterile. Thoroughly wipe the cheese surface as far as necessary
at the sampling site with undenatured ethanol. For taking cores take a short core of a larger
diameter first. To do this insert a trier into the cheese to a depth of 25mm. Rotate the trier through
one complete turn and withdraw it with the core. Keep this core and use it later to close the core
hole. If the core is required as a surface sample, immediately place it in a sample container. Then
insert a smaller trier of sufficient length through the inner surface of the cheese exposed by the
core hole. Rotate the trier through one complete turn and withdraw it with the core. With the aid
of a knife transfer the core to the sample container. Repeat this procedure until a sample of not
less than 100g is obtained. Then close the core hole by means of the first outer core: if the latter is
requested as a surface sample, seal the core hole with a suitable sealing compound.
Sampling for chemical and physical analysis and for sensory examination
Take a core by inserting a trier of sufficient length into the cheese. Rotate the trier through one
complete turn and withdraw it with the core. Close the core hole with the outer plug
(approximately 10-20mm) and if the outer plug is required as a surface sample, seal the core hole
with a suitable sealing compound. Wrap the cores in aluminum foil before placing them in
sample container if analysis is not performed immediately after sampling.
Sampling of fresh cheese
For sampling of fresh cheese the containers shall be intact and unopened. The containers shall not be
opened until immediately before analysis. Take a sufficient number of sample containers to obtain a
sample of not less than 100g. Containers from which portions are taken shall be taken as a whole.
Sampling of cheese sold in brine, oil, etc.
Sample cheese by taking fragments of at least 100g each (without brine, oil, etc.). During storage
in brine in particular, the composition of the cheese will change, depending on time and
temperature. The testing laboratory shall specify whether the sample shall include brine, oil, etc.,
or not. Normally brine, oil, etc., are include; whenever possible, the original ratio of cheese and
liquid shall be maintained and the latter shall completely cover the cheese. If brine is included,
take a sufficient amount of brine so that the cheese in completely covered. If brine is not
included, the cheese or cheese fragments shall be dried with filter paper and placed in the sample
container.
Note: Indicate in the sampling report whether the sample has been taken with or without brine,
oil, etc.
11
Paneer
Draw enough sub-samples to give a total sample of at least 50g. Employ one of the following
techniques depending upon the shape, mass and type of paneer:
1. Sampling by cutting
2. Sampling by means of a trier
3. Taking a complete paneer as a sample
1. Sampling by cutting: Using a knife with a pointed blade, make two cuts radiating from
the center of the paneer (if the paneer has a circular base), or parallel to the sides (if the
base is straight-sided). The size of the piece thus obtained should be such that, after
removal of any inedible surface layers, the remaining edible portion would weigh not less
than 50g.
2. Sampling by means of a trier: According to the shape, mass and type of paneer, one of
the following sampling techniques should be employed:
• The trier may be inserted obliquely towards the center of the paneer, once or several
times, into one of the flat surfaces, at a point not less than 10cm from the edge.
• The trier may be inserted perpendicular into one face and passed through the center of
the paneer to reach the opposite face.
• The trier may be inserted horizontally into the vertical face of the paneer, midway
between the two faces, towards the center of the paneer.
• For large paneers, the outer 2cm or more of the plug containing the rind may be used
for closing the hole made in the paneer. In this case, the remainder of the plug or
plugs then constitutes the sample. The plug-holes should be closed with greater care
and, if possible, they should be sealed over with an approved sealing compound.
3. Sampling by taking an entire paneer: This method should normally be reserved for
small paneer and for wrapped portions of paneer packaged in small containers.
[Source: International Organization for Standardization (ISO/R) 707-1968 (E)]
12
13
Chapter 2
DISINFECTION AND STERILIZATION
14
15
2.1 Maintenance of Laboratory Equipment
Equipment for chemical and sensory testing

It is not necessary to sterilize the equipment but it should at all times be kept clean and free from
all traces of milk residues (especially fat residues can be difficult to remove) and chemicals that
might interfere with the result of a test.
Rinse well and drain before use and at the end of the working day clean all equipment in a
detergent, rinse in distilled water and dry.
Equipment for bacteriological testing
1. Rinse in cold water

2. Wash and brush in hot water containing detergent in 1% solution (i.e., washing soda,
Teepol)
3. Rinse in hot distilled water and examine for cleanliness
4. Allow to dry upside-down in dust-free surroundings
5. Gerber butyrometers and stoppers should not be exposed to strong alkalis like soda, as a
precipitate may be formed in the narrow stem of the tube. Use instead Teepol
6. After use, pipettes should be immediately placed (vertically) in a cylinder containing
sanitizing solution (200ppm chlorine solution plus 1% Teepol). Cotton plugs should be
removed before sanitizing
7. Routine sanitizing should always be done before any kind of sterilization
[Source: Quality Control Handbook, Dairy Development Corporation]
- 14 -
2.2 Sterilization of Laboratory Equipment
The equipment must be dry before the sterilization begins. Pipettes should have a small piece of
colon put into the mouth tip and the pipettes should then be put into a canister before sterilization
Petri plates should be assembled (lid and bottom together) and other glassware and utensils
should be packed in brown paper and eventually sealed with autoclave tape before sterilization.
Sterilization shall be performed by one of the following methods:
1. Exposure to hot air at 170-175°C for not less than 1 hour

2. Exposure to steam at 121±1°C for not less than 20 min in an autoclave. The equipment
shall be dry when used.
After sterilization, equipment may be stored prior to use if kept under sterile conditions.
If an autoclave or a hot air oven is not available or the material cannot stand the above mentioned
methods, the following alternative methods are recommended, provided that the equipment is
used immediately after sterilization.
1. Exposure to saturated steam at 100°C for 1 hour

2. Immersion in boiling water for at least 1 min? (or 10 min?)
3. Immersion in 70% (v/v) ethanol solution and ignition to burn the alcohol
4. Exposure to suitable flame so that all working surfaces contact with the flame
5. Immersion of equipment in a solution with 400ppm active chlorine for at least 10min
15
2.3 Disinfection of Laboratory Tables, etc.
When doing bacteriological tests it is necessary to clean the tables and then wipe them with a
piece of cloth moistened in a 200ppm chlorine solution (1 teaspoonful bleaching powder in a
bucket of water, about 5 liters of warm water) or with ethanol. This solution can also be used to
disinfect hands before the work starts.
If germ falls show heavy contamination with mold and bacteria in the air, it will certainly help to
spray the room with a 1000ppm chlorine solution (1 teaspoonful in 1 liter water) by using a small
manual hand spray (the type for cleaning windows). After spraying leave the room for 10 min –
the smell is very unpleasant.
16
17
Chapter 3
RECOMMENDED SAMPLING PLAN
18
19
Recommended Sampling Plan
Aim: To accurately and timely sample and analyze milk and milk products at intermediate stage
of processing and final product.
Principle: The accurate and timely sampling and immediate analysis of the samples obtained at
various stages of processing through to the final product is very important for maintaining the
quality of the product. Protocols are established as to when, where, and how samples are to be
drawn and analyzed. Strictly adhering to these protocols will not only help avoid problems (and
therefore expense) but will also lead to product of consistent quality.
S.N. Product Analysis Sampling Site Frequency of Testing
1 Raw milk Sensory test 1 sample from bulk 1 sample/Daily
Temperature & Quantity container/chilling
Alcohol test center
Acidity test
Fat test 1 sample from bulk
container/individual
SNF test
farmer
Adulteration test
MBRT test 1 sample from bulk 1 sample/Daily
TPC at 30°C container/chilling
center
1 sample from bulk
container/individual
farmer
2 Pasteurized Sensory test 1 sample from bulk 2 samples/Daily
milk Acidity test tank
Fat test
SNF test 1 sample from filled
Phosphatase test pouch
Coliform count
TPC at 30°C 1 sample from bulk 4 samples/Week
MBRT test tank/twice in a week
1 sample from filled
pouch/twice in a
week
3 Milk Total pouches produced After milk filling Daily
packaging How many (%) leaking from filling section
immediately
How many leaking after
12 hours
- 18 -
4 Butter Sensory test 1 sample from churn 2 samples per batch
Moisture, % (intermediate stage of
Fat, % production)
Salt, %
Curd, % 1 sample from packed
Peroxide value (rancidity) product
Acidity, %
Detection of coloring
matters
Weight of 5 packages
Coliform count
Yeast/mold count
5 Cream Sensory test 1 sample from bulk 2 samples per batch
Fat, % tank
Acidity, %
Coliform 1 sample from filled
TPC bottle
Yeast/mold count
6 Ice cream Sensory test fat, % 1 sample from bulk 2 samples per batch
Total solids, % container
Acidity, %
Sucrose, % 1 sample from
Overrun, % individual cups
Phosphatase test
Coliform count
TPC
Yeast/mold count
7 Yoghurt Sensory test 1 sample from 1 sample per batch
Fat, % individual container
Total solids, %
Acidity, %
Coliform count
Yeast/mold count
- 19 -
8 Ghee Sensory test 1 sample from 3 samples per batch
Moisture, % bulk tank
FFA, % 1 sample from
Peroxide value (rancidity) filled bottle
RM value 1 sample from
Melting point polythene
packed product
Vegetable fat adulteration test
Refractive index
Insoluble impurities
9 Paneer Sensory 1 sample from 1 sample per batch
Fat, % individual
Moisture, % packed product
Acidity, %
TPC
Coliform count
Yeast/mold count
10 Cheese Sensory test 1 sample from 1 sample per batch
Fat, % individual cake
Moisture, %
Salt content, %
pH
protein, %
coliform count
Yeast/mold count
11 Skim and Sensory test 2 samples from 2 sample per batch
whole milk Fat test individual bags purchased and
powder Moisture and total solids reconstituted
Titrable acidity
Solubility index
Solubility percent
Coliform count
Total plate count
- 20 -
21
Chapter 4
EXAMPLES OF REGISTRATION &

REPORTING SYSTEM
22
23
Examples of Registration and Reporting System
Aim: To register the observed result of individual products in organized tables.

Principle: The observed results should be registered in proper, managed result recording sheets.
The quality control staff should fill up the result first into daily sheets according to the product
result-result registering sheet and after one week or month he/she should have to compile all the
results of a week or month of the products into their respective weekly or monthly registering
sheets.
Weekly/Monthly Reporting
In the following sheets the total value observed in a week or month is divided by number of
observation done in the same period to calculate the average value.
For example, 30 is the total value observed in a week or month and 6 is the number of
observations done in the same period. So the average is calculated as follows:
30
Average = =5
6
Maximum value is the highest value observed beyond the required value. Similarly the minimum
value is the lowest value observed below the required value.
For example, if in fat test, 3.5% and 2.8% are observed during a month and we require 3.0% by
standard of Food Act, 3.5% becomes the maximum value and 2.8% becomes the minimum value.
Note: The compiler of this book has not mentioned standard deviations and the level of
significances. The tables are also not numbered and captioned as required by the convention,
which may lead to difficulty on the part of the user of this book ----- Basanta R.
1. Raw Milk
Analysis Suppliers
1 2 3 4 5 6
Temperature when received
Sensory test
COB test
Alcohol test
Acidity, %
Fat, %
SNF, %
Adulteration test
MBRT test
TPC
Antibiotic test
- 22 -
2. Pasteurized Milk
Analysis No. of samples Average Maximum value Minimum value
Acidity, %
Fat, %
SNF, %
Coliform count
TPC, 30°C
MBRT test
Volume (pouches
Phosphatase
Sensory test (comments):
Pasteurization temperature
3. Butter
Moisture, %
Fat, %
Salt, %
Rancidity (peroxide)
Acidity, %
Curd, %
Coloring matter
Weight
Coliform count
Yeast/mold count
Sensory test (comments:
4. Ice Cream
Fat, %
Total Solids, %
Acidity, %
Sucrose, %
Overrun, %
Phosphatase test
Coliform count
TPC at 30°C
Yeast/mold count
23
5. Yoghurt
Fat, %
Total solids, %
Acidity, %
Coliform count
Yeast/mold count
6. Ghee
Moisture, %
FFA, %
Rancidity (peroxide)
RM value
Melting point
Refractive index
7. Paneer
Fat, %
Moisture, %
Acidity, %
TPC at 30°C
Coliform count
Yeast/mold count
8. Cheese
Moisture, %
pH
salt, %
fat, %
protein, %
coliform
yeast/mold count
24
9. Cream
Fat, %
Acidity, %
Coliform count
TPC
Yeast/mold count
10. Skim & Whole Milk Powder

Fat test
Moisture
Total solids
Titrable acidity
Solubility index
Solubility percent
Coliform count
Total plate count
25
Raw Milk
Physical, Chemical & Microbiological Analysis
Daily/Weekly Reporting Date: .
Date Quantity Sensory Temp. COB Alcohol Fat, % Lacto SNF, Acidity, Adultera- TPC, MBRT Remarks
Kg Test °C Reading % % tion Test 30°C Test
Signature:_________________________
26
Pasteurized Milk
Physical and Chemical Analyses at Intermediate Stage of Processing
Daily Reporting Date: .
Date Sample No. Tank No. Temp. °C Sensory Fat, % Lacto SNF, % Acidity, % Pasteurization Remarks
Test Reading Temp
Signature:_________________________
27
Pasteurized Milk
Physical and Chemical Analyses of Final Product
Daily Reporting Date: .
Date Sample No. Volume in Sensory Fat, % Lacto SNF, % Acidity, % Phosphatase Remarks
Pouch Test Reading Test
Signature:_________________________
28
Pasteurized Milk
Microbiological Analysis of Final Packed Product
Date Sample No. Volume in Pouch Coliform Test TPC at 30°C Remarks
Signature:_________________________
29
Pasteurized Milk
Milk Packaging and Analysis of Final Product
Date Total Pouches Produced Leaking Immediately, % Leaking after 12 hours Length of Film of Pouch Remarks
Signature:_________________________
30
Butter
Weekly Reporting Date: .
Date Sample Batch No. Weight, g Sensory Fat, % Moisture, Salt, % Peroxide Acidity, % Remarks
No. Test % Value
Signature:_________________________
31
Butter
Microbiological Analysis of Final Product
Date Sample No. Batch No. Coliform Count Yeast/Mold Count Remarks
Signature:_________________________
32
Cream
Date Sample No. Batch No. Weight, g Sensory Test Fat, % Acidity, % Remarks
Signature:_________________________
33
Cream
Date Sample No. Batch No. Coliform Count Sensory Test TPC Yeast/Mold Remarks
Count
Signature:_________________________
34
Ice Cream
Date Sample No. Batch No. Sensory Fat, % Total Solids, Acidity, Sucrose, Overrun, Remarks
Test % % % %
Signature:_________________________
35
Ice Cream
Date Sample No. Batch No. Coliform Test Sensory Test TPC Yeast/Mold Count Remarks
Signature:_________________________
36
Yoghurt
Date Sample No. Batch No. Sensory Test Fat, % Total Solids, % Acidity, % Remarks
Signature:_________________________
37
Yoghurt
Date Sample No. Batch No. Coliform Test Yeast/Mold Count Remarks
Signature:_________________________
38
Ghee
Date Sample Batch Sensory Moisture, FFA, % Peroxide RM Melting Insoluble Refractive Remarks
No. No. Test % Value Value point Impurities Index
Signature:_________________________
39
Paneer
Date Sample No. Batch No. Sensory Test Body & Texture Fat, % Moisture, % Remarks
Signature:_________________________
40
Paneer
Date Sample No. Batch No. TPC Coliform Count Yeast/Mold Count Remarks
Signature:_________________________
41
Cheese
Date Sample No. Batch No. Sensory Test Fat, % Moisture, % Salt, % pH Protein, % Remarks
Signature:_________________________
42
Cheese
Date Sample No. Batch No. Coliform Count Yeast/Mold Count Remarks
Signature:_________________________
43
Chapter 5
RAW MILK
44
45
5.1 Determination of Sensory Quality of Raw Milk
Aim: To determine the sensory quality of raw milk

Principle: Judging the quality of milk by its taste and smell requires considerable skill, which
could only be acquired by practice. Sensory tests are used in all dairies and an experienced person
can pick out bad samples with a high degree of accuracy. The used of sensory technique is very
useful for preliminary assessment of milk quality and handling practice. Milk is smelled and
observed visually to see if there are any sensory defects.
Apparatus: Plunger
Chemicals: Not required
Preparation of samples:
In the case of large containers, a sample of at least 500ml should be taken. During the sensory
evaluation, the samples should have a temperature of 16±2°C.
Procedure: The sensory evaluation of liquid milk should be carried out in relation to appearance
and flavor (odor and taste).
The evaluation of appearance is carried out with separate scoring; involving the filling of the
milk, color, visible purity, presence of foreign matters, spots of mold and phase separation.
Examine in the opened container, if necessary by pouring out the milk for the bulk container.
The evaluation of flavor is made by smelling and tasting the liquids milk.
Deferent categories of liquid milk should be evaluated in accordance with the table of
international standard.
Note: For tanker, perform the above practice for both the chambers.
- 45 -
Expression of Result:
Record the observations for all the samples in the data record sheet.
International Table of Liquid Milk Quality Terms
1. Appearance 2. Flavor
Untypical color Watery
Clotted Bitter
Curdy Cooked
Brown color Smoked
Foreign matter Burnt
Protein or fat flocs Chemical flavor
Protein or fat on the wall Feed flavor
Cream layer Foreign matter
Cream lumps Light induced flavor
Sedimentation Fruity
Ropy/String Malty
Separation of phases Metallic
Oily
Oxidized
Salty
Acid
Sour
Tallowy
Yeasty
Rancid
Unclean
Stale/Old
46
5.2 Determination of Temperature of Milk
Aim: To determine the temperature of milk

Principle: The temperature of milk is determined with a standard thermometer. Bulk raw milk,
when received from a chilling station in the factory shall not have a temperature more than 7°C.
Apparatus: Standard thermometer
Procedure:
1. For bulk milk (from farmer’s containers, cans, tankers, storage tanks, etc.), use a standard
thermometer and take the temperature reading of the sample as soon as it is drawn from
the bulk.
2. Also for packaged milk from market, use a standard thermometer. To avoid chances of
temperature increase, take the temperature reading as soon as possible.
[Source: Handbook of Food analysis, BIS SP: 18 (Part XI) -1981]
47
5.3 Determination of pH of Milk
Aim: To determine pH of milk

Principle: pH may be defined as the negative logarithm of the hydrogen ion concentration. The
measurement of pH is finding increasing use in the dairy industry since it provides in many cases
a more meaningful measurement than titrable acidity. pH values ranging from 0 to 6 are acid
while those ranging from 8 to 14 are alkaline, the value 7 being neutral.
Apparatus: pH meter (the pH meter is a device capable of measuring small difference in voltage
developed between two electrodes immersed in the sample. The electrical changes are converted
into direct pH readings. The pH meter is standardized by the use of standard buffer solutions
which should be carefully handled to prevent contamination. For fresh cheese, special cheese
electrodes are available), beaker, grater.
Chemicals: Not required (what about buffer for the standardization of pH meter?--- Basanta R)
Procedure:
1. Pour the sample into a clean, dry beaker.
2. Carefully press electrodes into the milk-containing beaker and determine the pH of milk
directly from the pH meter (what about the number of readings, agitation of the milk,
washing of pH meter, standardization of pH meter, etc.?---Basanta R).
The pH of the milk sample comes on the digital screen of the pH meter
[Source: Laboratory Guide in Dairy Chemistry Practicals, FAO]
48
5.4 Determination of Suitability of Milk for Pasteurization by Clot-on-
Boiling (COB) Test
Aim: To determine the suitability of milk for pasteurization by COB test

Principle: This is a quick test to determine developed acidity and the suitability of milk for
processing. Milk sample that clots or precipitates when heated to boiling temperature is not
suitable for further processing. The average acidity at with milk clots on boiling is 0.23% (as
lactic acid), but milk varies widely in this respect.
Apparatus: Test tubes preferably with a mark at 5ml, and burner
Procedure:
1. Transfer 5ml of the sample to the test tube.
2. Boil over flame for few seconds
3. Remove the tube and rotate it in an almost horizontal position and examine the film of
milk or side of the test tube for any precipitated particles.
The formation of clot is indicative of a positive test. COB test is positive also when milk on heat
treatment changes as follows:
• Coagulates
• Thickens
• Lumps
• Flakes
• Grains
• Precipitates
Milk that gives a positive COB is not suitable for distribution as liquid milk or for processing.
[Source: Bureau of Indian Standards, IS: 1479 (Part-I) – 1960]
49
5.5 Determination of Heat Resistance of Milk by Alcohol Test
Aim: To determine the heat resistance of milk by alcohol test

Principle: This test is like COB test regarding the suitability of milk for heat treatment but is
more sensitive than the COB test. As milk gradually sours and acid is formed, proteins become
susceptible to alcohol precipitation. This happens shortly before it loses it stability to
pasteurization.
Apparatus: Test tubes, pipettes (two 2-ml pipettes for milk and alcohol)
Chemicals: 72% (v/v) ethyl alcohol
Procedure:
1. Pipette 2ml of milk sample in a test tube.
2. Add 2ml of ethyl alcohol.
3. Mix the contents by shaking gently.
4. Observe carefully at the inner surface of the test tube.
The milk is not suitable for heat treatment or the milk is alcohol positive, if there is any trace of
following:
• Coagulation
• Thickening
• Lumping
• Flaking
• Graining
• Precipitation
• Dense curd formation
Note: This method could have some constraints for buffalo milk
[Source: Bureau of Indian Standards, IS 1479 (Part-I) – 1960]
50
5.6 Determination of Titrable Acidity in Milk
Aim: To determine the acidity of milk

Principle: The pH value for fresh milk is normally about 6.6 at 25°C. this value indicates that
milk has a “natural acidity”. Acidity is determined by titration with alkali (sodium hydroxide) in
the presence of an indicator (phenolphthalein) and hence the term “titrable acidity”. The natural
acidity of milk is much more uniform. The higher the SNF in milk, the higher the natural acidity
and vice-versa. The titrable acidity of cow milk varies on an average from 0.13 to 0.14% and
buffalo milk form 0.14 to 0.15%. Developed or real acidity is due to lactic acid, formed as a
result of bacterial action on lactose of milk. Hence titrable acidity of stored milk is equal to the
sum of natural acidity and developed acidity. The titrable acidity is usually expressed as a
percentage of lactic acid. Colostrum has a high natural acidity in part because of its very high
protein content (Sukumar De).
Apparatus: Glass burette (50ml capacity with 0.1ml graduation), porcelain dish – white
bottomed – 100ml capacity, 1ml pipette, 10ml pipette, stirring rods.
Chemicals: 0.1N sodium hydroxide (NaOH) solution (Dissolve equal parts of sodium hydroxide,
sticks or pellets, in equal parts of water in a flask. Tightly stopper the flask with a rubber bung
and allow any insoluble sodium carbonate to settle down for 3-4 days. Use the clear supernatant
liquid for preparing the standard 0.1N solution. About 8ml of stock solution is required per liter
of distilled water. The solution should be accurately standardized against acid potassium
phthalate or oxalic acid), 1% phenolphthalein indicator.
Procedure:
1. Mix the sample avoiding incorporation of air bubbles and transfer 10ml with the pipette to
the porcelain dish.
2. Add 4 drops of 1% phenolphthalein indicator to the milk.
3. Titrate with 0.1N sodium hydroxide solution until slight pink color appears.
4. Note the volume of 0.1N sodium hydroxide solution used in the titration.
volume of 0.1N NaOH × 0.009
Percent acidity in milk = ×100%
volume of milk sample
0.009 is a conversion factor from milliliter of 0.1N NaOH to gram of lactic acid.
[Source: Nepal Bureau of Standard NS: 24-2039]
51
5.7 Determination of Fat in Milk
Aim: To determine the percentage of fat in milk by Gerber method

Principle: When sulfuric acid is added to milk, the proteins will be digested thus destroying the
membranes of the fat globules. The free fat is then released. The fat remains in liquid due to heat
produced and during centrifugation the fat being lighter will be separated on top of the solution.
Addition of amyl alcohol has the function of separation fat from other components.
Apparatus: Gerber centrifuge (1100±100 rev/min), Gerber butyrometer for milk (0-10% scale
with 0.1% mark), hot water bath maintained at 65±2°C, 10ml measure for acid, 1ml measure for
amyl alcohol, 10.75ml pipette for milk at 27°C, butyrometer stoppers, butyrometer stand, key for
butyrometer stoppers.
Chemicals: Gerber sulfuric acid of density 1.820-1.825 at 20°C corresponding to concentration
of 90-91% by weight – clear and colorless, amyl alcohol of density 0.811g/ml at 20°C – clear and
colorless.
Sample preparation: If the sample is fresh, warm it to approximately 27°C and mix thoroughly
but do not shake it so vigorously as to cause frothing or churning of the fat. Pour the sample into
another clean dry vessel and back to the original. Repeat this process of pouring back and forth
until a homogeneous mixture is obtained. Allow the sample to stand for three or four minutes
after mixing to allow air bubble to rise; invert the sample bottle three or four times immediately
prior to taking milk for the test.
Procedure:
1. Transfer 10ml of sulfuric acid into the butyrometer with the help of a 10ml pipette or the
automatic tilt measure for sulfuric acid by taking care not to wet the neck of the
butyrometer with the sulfuric acid. The sulfuric acid should be clean and absolutely free
from fat residues.
2. Measure 10.75ml of a well-mixed sample of milk and transfer it carefully to the
butyrometer without allowing it to mix with the acid. This is done by allowing the jet of
milk from the pipette to hit the inside wall f the butyrometer by holding the pipette in a
slanting manner and resting the tip end on the mouth of the butyrometer (see figure 4).
3. Measure 1ml amyl alcohol into the butyrometer with the help of a 1ml pipette or the
automatic measure for amyl alcohol. Do not wet the butyrometer with alcohol.
4. Close the butyrometer firmly with the stopper without disturbing the contents.
5. Shake the butyrometer carefully, without inverting it, until the contents are thoroughly
mixed, the curd is dissolved and no white particles are seen in the liquid. Then gently
invert the butyrometer a few times to mix the contents thoroughly.
6. Transfer the butyrometer quickly, with the bulb uppermost, into a water bath having a
temperature of 65°C and leave it there for not less than 5 min. Take care to have the water
level in the bath above the top of the fat column in the butyrometer. Meanwhile, adjust the
stopper so that the fat column shall be on the scale after centrifuging.
7. Place the butyrometer in the centrifuge and balance it. Centrifuge for 5 min
(1100±100rpm).
52
8. After centrifuging, temper the butyrometer in the water bath at 65°C for 5 min.
9. Adjust the fat column with the scale on butyrometer and read off the top of the fat column
(bottom of the meniscus). Read to the nearest tenth of a percentage.
Record the fat percentage.
pipette
Gerber butyrometer
Figure 4: Position of pipette in butyrometer
53
φ 15 max
Small bulb
Matt surface
% 10
6
X X
5
65 min
cross section at XX 4
Graduated tube 2
φ 25 max
Large bulb
Neck 14.5 +
−1
φ int. 11.3 +
− 0.3
All dimensions in mm
Figure 5: 0 to 10% butyrometer with corrugated neck
54
5.8 Determination of Specific Gravity of Milk
Aim: To determine the specific gravity of milk

Principle: The specific gravity of milk is the ration of its weight to the weight of an equal
volume of water at 16°C. it is considered that specific gravity of water at 16°C is 1. The average
specific gravity of normal milk is 1.032 at 16°C.
The specific gravity of milk may be determined by a hydrometer. The hydrometer works on the
principle that “if a body floats in a liquid it is buoyed up by a force equal to the weight of the
liquid it displaces”. A special form of hydrometer used for taking the specific gravity of milk is
called “lactometer”.
Apparatus: Lactometer, thermometer, water bath, and lactometer jar.
Procedure:
Mix the milk sample thoroughly. This can be done by gently shaking/inverting (but not
incorporating air) the sample bottle.
Adjust the temperature of milk sample to get the temperature between 10 and 20°C. Note the
exact temperature of the milk by means of a thermometer.
Transfer the milk into the lactometer jar and immerse the lactometer in the milk sample slowly.
The take the reading at the top of the meniscus after the lactometer comes to rest.
Lactometer Reading
Specific Gravity of Milk = +1
1000
[Source: Bureau of Indian Standards, IS: 1478 (Part-I) – 1960]
55
5.9 Determination of Solids-Not-Fat Percentage in Raw Milk
Aim: To determine the percentage of solid-not-fat in milk by using a lactometer.

Principle: The main constituents of milk are water, fat, proteins, lactose and ash. The
constituents other than water are known as “total solids”. The total solids minus the fat is called
“solids-not-fat (SNF)”. The SNF is calculated using a formula that contains fat percentage and
corrected lactometer reading.
Apparatus: Lactometer (ISI certified/marked only), lactometer jar, thermometer (0-100°C),
apparatus for Gerber fat test.
Chemicals: Not required.
Procedure:
1. Determine the fat percentage in milk as described in the section “Determination of Fat in
Milk”.
2. Adjust the temperature of the milk sample to 27°C.
3. Mix the contents of the sample bottle by inverting the bottle gently 5 times. Be careful in
order to avoid the formation of air bubbles and foam.
4. Invert the sample bottle gently two to three times. Pour enough milk into the lactometer
jar but avoid the formation of air bubbles. Insure that some milk overflows when the
lactometer is inserted.
5. Insert the lactometer gently to wet the stem not more than a short length about 3mm
beyond position of equilibrium. The lactometer should float freely and shall not touch the
sides of the jar.
6. Allow the lactometer to remain steady in the milk. Take the reading within 30 seconds.
See the result at level and take the reading at the point of the lactometer where the surface
of the milk cuts across the stem of the lactometer.
7. Note the temperature of the milk.
8. Obtain the corrected lactometer reading, CLR, by applying the correction factor shown in
the temperature correction chart (next page).
Expression of result:
Calculate solids-not-fat (SNF) using the formula:
CLR
SNF% = + 0.25 × F + 0.5
4
Where, F = milk fat percentage
56
Correction to lactometer readings taken at temperatures other than 27°C
Temperature of observation, °C Correction
25.0 -0.7
25.5 -0.5
26.0 -0.3
26.5 -0.2
27.0 0
27.5 +0.2
28.0 +0.3
28.5 +0.5
29.0 +0.7
1.029
Figure 6: Method of taking lactometer reading
[Source: Quality Handbook Dairy Development Corporation]
57
5.10 Detection of Water Adulteration by Freezing Point
Aim: To determine the milk for water adulteration.

Principle: The freezing point of milk is normally between -0.55 and -0.53°C. Addition of water
will give higher freezing points while addition of milk solids, salts, sugar, etc., will lower the
freezing point significantly as will the souring of milk. The freezing point is also affected by
vacuum treatment, sterilization or freezing of milk. The freezing point determination is regarded
as one of the most accurate tests for detecting adulteration with water and for determining the
amount of water added because the rise of freezing point is proportional to the dilution.
Apparatus: Cryoscope thermometer (-1.2 to +0.4°C; 1/100 with certificate), isolated Dewar
flask, freezing tube 100-120ml with mark for 75ml, precooling bath, volumetric flasks-100ml,
laboratory balance (at least decimals), agitator.
Procedure:
1. A freezing mixture is prepared by dissolving 80g NaCl in one liter water in a bucket with
3kg finely crushed ice. Temperature of solution is between -3 and -5°C. The mixture is
transferred to the Dewar flask only.
2. Transfer 75ml milk to the test tube. Cool it first in ice water in the precooling bath and
then in the freezing mixture in the Dewar flask.
3. Place a thermometer in the milk; agitate the milk vigorously all the time while following
the change of temperature.
4. At first is noted a steady decrease in the temperature, then a sudden rise (first sub-cooling,
the crystallization) and the temperature will remain constant for some time, the latter
being the freezing temperature of the milk sample, which is carefully read and noted.
5. Calibration A: Exactly 7.00g of sucrose is dissolved in distilled water to the 100ml level
of volumetric flask. This solution has a freezing point of -0.422°C.
6. Calibration B: Exactly 10.00g of sucrose is similarly dissolved. This will have a freezing
point of -0.621°C.
(a − b × 0.199 + 0.422)
Freezing point = ??????
c−b
a = noted freezing point of milk
b = noted freezing point of calibration solution containing 7.00g sucrose
c = noted freezing point of calibration solution containing 10.00g sucrose
Estimation of percent water added in the milk is given by:
−0.53 ×100
% water added = − 100
Freezing point of the sample
58
5.11 Detection of Sugar (Sucrose) Adulteration in Raw Milk
Aim: To determine adulteration of milk with sugar (sucrose) at receiving station.

Principle: Cane sugar may be added to milk to raise the density to prevent detection of
extraneous matter. Adulteration with sugar in milk can be detected by two methods. One is
enzyme detection method and the other is resorcinol method. Enzyme detection method is a quick
method but is very expensive. Resorcinol method is easy, quick, cheap, and commonly used. In
this method, resorcinol change color of sugar-adulterated milk from white to red.
Apparatus: Test tube, hot plate, beaker
Chemicals: Concentrated hydrochloric acid, resorcinol crystals.
Procedure:
1. Take 10ml of milk in a test tube
2. Add 1ml conc. Hydrochloric acid
3. Add 100mg resorcinol
4. Shake it vigorously and boil it for 10 min in boiling water bath
5. Note the change in color of milk sample
Presence of red color indicates cane sugar adulteration
[Source: Handbook of Food analysis, BIS, SP: 18 (Part XI) – 1981]
59
5.12 Detection of Starch Adulteration in Raw Milk
Aim: To determine starch adulteration in raw milk.

Principle: Starch in a form of cereal flours or rice washed water may be added to raise the
density of milk to prevent detection of added water. The presence of starch, in the form of cereal
flours or rice washed water is detected by the simple iodine test of starch.
Apparatus: Test tube, heater or burner, pipette
Chemicals: 1% iodine solution (in potassium iodide solution).
Procedure:
1. Take about 3ml of milk sample in a clear test tube

2. Boil the milk over flame and cool it
3. Add 1-3 drops of 1% iodine solution and mix well
4. Note the change in color
Presence of blue color indicates that the milk is adulterated with starch
[Source: Handbook of Food Analysis BIS, SP: 18 (Part XI) – 1981]
60
5.13 Detection of Carbohydrate Adulteration in Milk
Aim: To determine carbohydrate in milk.

Principle: The presence of carbohydrate is determined by using a solution of cupric acetate as
suggested by Barfoed.
Apparatus: Test tube, water bath, filter paper
Chemicals: Cupric acetate solution (Dissolve 45g of neutral crystallized cupric acetate in 900ml
water and filter it. To the filtrate is added 1.2ml of 50% acetic acid and the solution diluted to 1
liter). A portion of the reagent, heated on the water bath, should show no reduction (yellow or red
color).
Procedure:
1. To 5ml of cupric acetate solution in a test tube, add 5ml of the milk sample and place in a
boiling water bath for 3.5 min
2. Examine for precipitated cuprous oxide, viewing tube against a blank background in a
good light
3. If none is found, let the tube stand at room temperature for 5 to 10 min, pour out the
liquid and carefully through a filter paper
Note any traces of cuprous oxide remains adhering to the test tube or to the paper after rinsing
with water. The presence of residue of cuprous oxide (red) indicates carbohydrate adulteration.
(Since milk also contains carbohydrate (lactose) and can be broken down to monosaccharides
upon prolonged boiling under acidic condition, the of boiling in the above test appears to be very
critical ---Basanta R)
[Source: Food analysis by A. G. Woodman, 1941]
61
5.14 Detection of Use of Neutralizer (Sodium bicarbonate)
Aim: To determine sodium bicarbonate neutralizer in raw milk.

Principle: Neutralizer in the form of limewater or sodium bicarbonate may be added to
neutralize developed acidity before milk is processed and to increase milk solids. Such a practice
is not permissible. This prevents milk from coagulation.
Apparatus: Test tube, pipette.
Chemicals: Dehydrated alcohol (95% ethanol), 1% rosalic acid solution (in 90% ethyl alcohol)
Procedure:
1. Draw about 5ml milk sample in a clean test tube
2. Add 5ml dehydrated alcohol (95%) into it and mix well.
3. Add 2-3 drops of 1% rosalic acid solution and mix
4. Note the change in color of milk
If a carbonate is present, a rose-red color appears whereas pure milk shows only a brownish
coloration.
62
5.15 Detection of Salt Adulteration in Raw Milk
Aim: To determine salt adulteration in raw milk.

Principle: Milk is adulterated with salt to increase milk solids. This test is generally applied in
the receiving station. The test is a modification of the usual argentometric titration for chloride
ions using potassium chromate as an internal indicator.
Normal milk contains chloride ions in the range 0.09 to 0.14%.
Apparatus: Test tubes, pipettes
Chemicals: 0.1N Silver nitrate solution (Dissolve 17 g silver nitrate in 1 liter water. Store in dark
bottle), 5% potassium chromate indicator (Dissolve 5g potassium chromate in distilled water to
make 100ml).
Procedure:
1. Take 5ml of 0.1N silver nitrate solution in a test tube
2. Add 2 drops of 5% potassium chromate indicator
3. The color of silver nitrate solution becomes brick red
4. Add 3ml milk and mix
5. Note the change in color of milk
Change of red color to yellow indicates milk having more than 0.14% chloride and it is
confirmed that milk is adulterated with salt. Normal milk gives a brownish red color.
Note: Milk from animals in very early or very late lactation can also give a positive chloride test.
[Source: A Textbook of Practical Dairy Chemistry – N. K. Roy, D. C. Sen: 1991]
63
5.16 Detection of Formaldehyde in Raw Milk (Hehner Test Method)
Aim: To determine formaldehyde in raw milk.

Principle: Formaldehyde is used to preserve milk and to increase shelf life of raw milk.
Formaldehyde is highly bactericidal which kills all contaminating microorganisms (in fact all
living tissues, including humans!). Formaldehyde gives characteristic violet color with ferric salts
and other oxidizing agents.
Apparatus: Test tubes, pipettes
Chemicals: 1% ferric chloride solution, concentrated sulfuric acid (sp. grav. 1.840)
Procedure:
1. Take 10ml milk in a test tube
2. Add 0.5ml of 1% ferric chloride
3. Add concentrated sulfuric acid so that it forms separate layer at the bottom without
mixing with milk. Drop the acid against the wall of the tube and not directly on the milk
surface.
4. Note the change in color
Appearance of violet or blue color at the junction of two liquids indicates the presence of
formaldehyde. The test is sensitive to one part in 10,000 parts.
Note: The test requires only traces of ferric chloride. It may be combined with the determination
of fat, by noting whether a violet color forms when sulfuric acid is added in the butyrometer.
64
5.17 Detection of Formaldehyde in Raw Milk (Chromotropic acid
Method)
Aim: To determine formaldehyde in raw milk by chromotropic acid test method

Principle: Formaldehyde is used to preserve milk and to increase shelf life of raw milk.
Formaldehyde is highly bactericidal which kills all contaminating microorganisms (in fact all
living tissues, including humans!). Formaldehyde gives characteristic violet color with ferric salts
and other oxidizing agents.
Apparatus: Test tube, water bath, steam distillation plant
Chemicals: Chromotropic acid (Prepare saturate solution of chromotropic acid by stirring 0.5g of
the acid in 100ml of 72% H2SO4 – the solution is pale yellow in color. Prepare 72% H2SO4 by
taking 150ml of conc H2SO4 and mixing with 100ml of ice-cold water), milk distillate (Dilute
100ml milk with 100ml water, acidify with a small quantity of H3PO4 and steam-distil the
mixture. Collect 50 of the distillate).
Procedure:
1. Take 5ml of chromotropic acid in a test tube and add with mixing 1ml of milk distillate
2. Place the mixture in boiling water bath for 15 min and observe during heating
Appearance of light to deep purple color indicates the presence of formaldehyde. The intensity of
color is proportional to the amount of formaldehyde present.
[Source: Official Methods of Analysis, Association of Official Analytical Chemists (AOAC),

USA, 14th edition, 1984]
65
5.18 Detection of Urea Adulteration in Raw Milk
Aim: To determine urea adulteration in raw milk.

Principle: Urea is added in milk to increase milk solids.
Apparatus: Pipettes, test tubes.
Chemicals: Sodium acetate-acetic acid buffer (1N, pH 4.75), trichloroacetic acid (TCA)-24%,
sodium hydroxide solution-2%, sodium hypochlorite solution-2% (Prepare fresh by passing
chlorine gas into 2% NaOH solution and standardize to 2% available chlorine by usual
iodometric titration), phenol solution – 5% (Prepare from almost colorless, reagent quality
redistilled phenol).
Procedure:
1. Take 5ml of milk in a 50ml Erlenmeyer flask, add 5ml sodium acetate buffer or TCA
solution and heat for 3 min in boiling water bath using a stop watch (no heating is
required in case of TCA being used)
2. Filter the precipitate in Whatman No. 42 filter paper or equivalent and collect 1ml of the
filtrate in a clean test tube
3. To the filtrate add 1ml of NaOH solution followed by 0.5ml sodium hypochlorite
solution, mix thoroughly and finally add 0.5ml phenol solution
Formation of a characteristic blue or bluish-green color indicates the presence of extraneous urea
in the milk sample. Filtrate from unadulterated milk remains colorless. The blue color remains
stable for at least 12 hrs. By this test, as low as 0.1% urea added to milk can be detected.
[Source: Textbook of Practical Dairy Chemistry – N. K. Roy and D. C. Sen, 1991]
66
5.19 Detection of Hydrogen Peroxide Preservative
Aim: To determine hydrogen peroxide in milk

Principle: Addition of hydrogen peroxide into milk increases the shelf-life of the milk by acting
as a preservative.
Apparatus: Test tube, pipette
Chemicals: 2% (w/v) paraphenylene diamine solution
Procedure:
1. Add 5ml of milk in a clean test tube
2. Add 5ml of sample raw milk
3. Add five drops of a 2% solution of paraphenylene diamine solution
Note: Hydrogen peroxide is decomposed when milk is heated or stored for a long interval
A blue color is developed in the presence of hydrogen peroxide.
[Source: Handbook of Food Analysis BIS, SP 18 (Part XI) - 1981
67
5.20 Detection of Neutralizer Caustic Soda
Aim: To determine neutralizer caustic soda

Principle: The highly developed acidity can be totally neutralized by the addition of caustic soda
(NaOH). NaOH forms salt to give residue of Na. Caustic soda invariably increases the ash
content and total alkalinity of the ash from a fixed quantity of milk. The detection of NaOH as
neutralizer is therefore based on preparation and ash solution and titration with HCl using
phenolphthalein as an indicator.
Apparatus: Test tubes, pipettes, burettes, porcelain dish
Chemicals: Standard hydrochloric acid (0.1N), phenolphthalein indicator
Procedure:
1. Measure 20ml of milk in a porcelain dish and ash it well
2. Disperse the ash in 10ml of water
3. Titrate with the standard 0.1N hydrochloric acid
The amount of standard hydrochloric acid required in the absence of neutralizers will not exceed
1.2ml. In titration, if hydrochloric acid consumed is more than 1.2ml it indicates the presence of
caustic soda in milk.
68
Chapter 6
PASTEURIZED MILK
69
70
6.1 Determination of Sensory Quality of Pasteurized Milk
Aim: To determine the sensory quality of pasteurized milk.

Principle: Judging the quality of milk by its taste and smell requires considerable skill, which
can pick out bad samples with a high degree of accuracy. The use of sensory technique is very
useful for preliminary assessment of milk quality and handling practice. Milk is smelled and
Apparatus: Plunger
In the case of large containers, a sample of at least 500ml should be taken. In the case of retail
packages and individual portions, an adequate number of packages should be made available.
During the sensory evaluation, the samples should have a temperature of 16±2°C. The sensory
evaluation liquid milk should be carried out in relation to appearance and flavor (odor and taste).
Procedure:
1. The evaluation of appearance is carried with separate scoring; involving the filling of
milk, color, visible purity, presence of foreign matters, spots of mold, and phase
separation. Examine in the opened container, if necessary by pouring out the milk from
the bulk container or the product from the package
2. The evaluation of consistency is made by feel, especially by muscles of the mouth
(mouth-feel)
3. The evaluation of flavor is made by smelling and tasting the liquid milk
4. Different categories of liquid milk should be evaluated in accordance with the table of
international standard.
- 70 -
Record the observation for all the samples in the data record sheet.
International Table of Liquid Milk Quality Terms
Untypical color Watery
Clotted Bitter
Curdy Cooked
Brown color Smoked
Foreign matter Burnt
Protein or fat flocs Chemical flavor
Protein or fat on the wall Feed flavor
Cream layer Foreign matter
Cream lumps Light induced flavor
Sedimentation Fruity
Ropy/String Malty
Separation of phases Metallic
Oily
Oxidized
Salty
Acid
Sour
Tallowy
Yeasty
Rancid
Unclean
Stale/Old
71
6.2 Determination of Fat in Milk
Aim: To determine the percentage of fat in milk by Gerber method

When sulfuric acid is added to milk, the proteins will be digested thus destroying the membranes
of the fat globules. The free fat is then released. The fat remains in liquid due to heat produced
and during centrifugation the fat being lighter will be separated on top of the solution. Addition
of amyl alcohol has the function of separation fat from other components.
amyl alcohol, 10.75ml pipette for milk at 27°C, butyrometer stoppers, butyrometer stand, key for
butyrometer stoppers.
colorless.
Sample preparation: If the sample is fresh, warm it to approximately 27°C and mix thoroughly
but do not shake it so vigorously as to cause frothing or churning of the fat. Pour the sample into
another clean dry vessel and back to the original. Repeat this process of pouring back and forth
until a homogeneous mixture is obtained. Allow the sample to stand for three or four minutes
after mixing to allow air bubble to rise; invert the sample bottle three or four times immediately
prior to taking milk for the test.
Procedure:
1. Measure 10ml of Gerber sulfuric acid into the butyrometer that should be clean and
absolutely free from fat residues
2. Pipette out 10.75ml of a well-mixed sample of milk and transfer it carefully to the
butyrometer without allowing it to mix with the acid. This is done by allowing the jet of
milk from the pipette to hit the inside wall of the butyrometer by holding the pipette in a
slanting manner and resitn the tip end on the mouth of the butyrometer.
3. Measure 1ml amyl alcohol into the butyrometer
4. Tighten the stopper and mix the content by shaking (stoppered end away for the body) the
butyrometer at a 45° angle until all the curd has been dissolved. Then invert the
butyrometer several times so that the contents are evenly distributed inside the tube.
5. Keep the butyrometer in the water bath at 65°C for 5 min
6. Place the butyrometer in the centrifuge and balance it. Centrifuge for 5 min (1100±100
rpm)
7. After centrifuging, temper the butyrometer in the water bath at 65°C for 5 min
8. Adjust the fat column within the scale on butyrometer and read off the top of the fat
column (bottom of meniscus, figure 7). Read to the nearest tenth of a percentage.
72
Record the fat percentage.
Note: For homogenized milk the procedure is above but centrifuge three times for 5 min each
time. Between each centrifugation, heat the butyrometer for 5 min in the 65°C water bath.
4
3.6
Figure 7: Method of taking butyrometer reading
73
6.3 Determination of Solids-Not-Fat Percentage in Milk
Aim: To determine the percentage of solid-not-fat in milk using a lactometer.

Principle: The main constituents of milk are water, fat, proteins, lactose and ash. The
constituents other than water are known as “total solids”. The total solids minus the fat is called
“solids-not-fat (SNF)”. The SNF is calculated using a formula that contains fat percentage and
corrected lactometer reading.
Apparatus: Lactometer (ISI certified/marked only), lactometer jar, thermometer (0-100°C),
apparatus for Gerber fat test.
Procedure:
1. Determine the fat percentage in milk as described in the section “Determination of Fat in
Milk”.
2. Adjust the temperature of the milk sample to 27°C.
3. Mix the contents of the sample bottle by inverting the bottle gently 5 times. Be careful in
order to avoid the formation of air bubbles and foam.
4. Invert the sample bottle gently two to three times. Pour enough milk into the lactometer
jar but avoid the formation of air bubbles. Insure that some milk overflows when the
lactometer is inserted.
5. Insert the lactometer gently to wet the stem not more than a short length about 3mm
beyond position of equilibrium. The lactometer should float freely and shall not touch the
sides of the jar.
6. Allow the lactometer to remain steady in the milk. Take the reading within 30 seconds.
See the result at level and take the reading at the point of the lactometer where the surface
of the milk cuts across the stem of the lactometer.
7. Note the temperature of the milk.
8. Obtain the corrected lactometer reading, CLR, by applying the correction factor shown in
the temperature correction chart (next page).
Calculate solids-not-fat (SNF) using the formula:
CLR
SNF% = + 0.25 × F + 0.5
4
Where, F = milk fat percentage
74
Correction to lactometer readings taken at temperatures other than 27°C
Temperature of observation, °C Correction
25.0 -0.7
25.5 -0.5
26.0 -0.3
26.5 -0.2
27.0 0
27.5 +0.2
28.0 +0.3
28.5 +0.5
29.0 +0.7
75
6.4 Determination of Acidity in Milk
Aim: To determine the acidity of milk

Principle: The pH value for fresh milk is normally about 6.6 at 25°C. this value indicates that
milk has a “natural acidity”. Acidity is determined by titration with alkali (sodium hydroxide) in
the presence of an indicator (phenolphthalein) and hence the term “titrable acidity”. The natural
acidity of milk is much more uniform. The higher the SNF in milk, the higher the natural acidity
and vice-versa. The titrable acidity of cow milk varies on an average from 0.13 to 0.14% and
buffalo milk form 0.14 to 0.15%. Developed or real acidity is due to lactic acid, formed as a
result of bacterial action on lactose of milk. Hence titrable acidity of stored milk is equal to the
sum of natural acidity and developed acidity. The titrable acidity is usually expressed as a
percentage of lactic acid. Colostrum has a high natural acidity in part because of its very high
protein content (Sukumar De).
Apparatus: Glass burette (50ml capacity with 0.1ml graduation), porcelain dish – white
bottomed – 100ml capacity, 1ml pipette, 10ml pipette, stirring rods.
Chemicals: 0.1N sodium hydroxide (NaOH) solution (Dissolve equal parts of sodium hydroxide,
sticks or pellets, in equal parts of water in a flask. Tightly stopper the flask with a rubber bung
and allow any insoluble sodium carbonate to settle down for 3-4 days. Use the clear supernatant
liquid for preparing the standard 0.1N solution. About 8ml of stock solution is required per liter
of distilled water. The solution should be accurately standardized against acid potassium
phthalate or oxalic acid), 1% phenolphthalein indicator.
Procedure:
5. Mix the sample avoiding incorporation of air bubbles and transfer 10ml with the pipette to
the porcelain dish.
6. Add 4 drops of 1% phenolphthalein indicator to the milk.
7. Titrate with 0.1N sodium hydroxide solution until slight pink color appears.
8. Note the volume of 0.1N sodium hydroxide solution used in the titration.
volume of 0.1N NaOH × 0.009
Percent acidity in milk = ×100%
volume of milk sample
0.009 is a conversion factor from milliliter of 0.1N NaOH to gram of lactic acid.
76
6.5 Determination of Phosphatase in Milk (BIS Method)
Aim: To assess the pasteurization effect.

Principle: When milk is pasteurized at 72°C for 15 sec, the phosphatase enzyme is denatured.
Presence of phosphatase is therefore indicating that pasteurization temperature has not been
reached or the pasteurized milk has been contaminated with raw milk.
The milk sample is diluted with a buffer at pH 10.2 and incubated at temperature of 37°C for 2
hours. Any alkaline phosphatase present in the sample will, under these circumstances, liberates
p-nitrophenol from added disodium p-nitrophenyl phosphate. The p-nitrophenol liberated is
measured by direct comparison with standard color discs in a simple comparator.
Apparatus: Water bath (maintained at 37±1°C, thermostatically controlled), comparator (with
special discs of standard color glasses calibrated in µg p-nitrophenol per ml of milk and 2×25
mm cells), test tubes (16×150mm and with rubber stoppers to fit), 1, 5, and 10ml pipettes, filter
paper (Whatman No. 2 or equivalent), litmus paper.
Chemicals:
Sodium carbonate-bicarbonate buffer: Dissolve 3.5g of anhydrous sodium carbonate and 1.5g
sodium bicarbonate in 1000ml of distilled water.
Buffer substrate: Dissolve 1.5g disodium p-nitrophenyl phosphate in 1000ml of sodium
carbonate-bicarbonate buffer.
Procedure:
1. Mix the milk sample carefully, if necessary with moderate heating. The temperature of
mixing should not exceed 35°C.
2. Pipette 5m of buffer substrate into a clean, dry test tube and add 1ml of the milk to be
tested
3. Stopper the tube; mix by inversion and place in the water bath
4. Prepare control tube with 5ml of the buffer substrate and 1ml of boiled milk from the
same sample. Place it in the same water bath
5. After 2 hours, remove the tubes from the bath, invert each and read the color developed
using the comparator and special disc. While reading place the control tube on the left of
the stand and the sample tube on the right
6. Record reading which lie between two standard color discs by adding a plus (+) or minus
(-) sigh to the figure of the nearest standard
77
All samples giving readings in excess of 10 µg is recorded as unsatisfactory or phosphatase-
positive.
Note:
The buffer substrate solution is stable if stored in a refrigerator at 4°C or less for one month. Any
instability is denoted by the formation of a yellow color. Whilst the is test always read against a
boiled product control containing the same buffer substrate solution, it is recommended that the
solution should not be used if it gives a color reading in excess of 10 µg when read in a 25mm
cell in the comparator using distilled water in the 5m cell.
[Source: Bureau of Indian Standards, IS: 8479 (Part I) – 1977]
78
6.6 Determination of Phosphatase in Milk (Funke Gerber Method)
Aim: To determine the pasteurization effect.

Presence of phosphatase is therefore indicating that pasteurization temperature has not been
reached or the pasteurized milk has been contaminated with raw milk.
Apparatus: 2 absolutely clean test tubes, incubator or water bath at 37°C, water bath with
boiling water, measuring spoon (Lactognost), Control chart (Lactognost).
Chemicals: Test kit: Lactognost consisting of Lactognost tablets I and II plus a bottle with
Lactognost III (after opening of the original bottle the content has to be used within 2months).
Store these chemicals in a refrigerator/cold store.
Procedure:
1. Two test tubes A (for analysis) and C (for control) are each filled with 10ml of distilled
water, 1 tablet Lactognost I, and 1 table Lactognost II.
2. After agitating, the tablets disintegrate; if necessary, crush with a glass rod
3. Measure 1ml each of milk into test tube A and test tube C. Heat test tube C to 85°C in
boiling water to destroy the phosphatase enzyme.
4. Incubate both the test tubes at 37°C for 1 hour
5. To both the test tubes, add one level measuring-spoonful of Lactognost III
6. After 10 min compare test tube A with test tube C. Match the blue color that develops
with the color chart.
Presence of phosphatase results in blue color. The color chart helps define when a very pale blue
should be called “phosphatase –negative” or “phosphatase-positive”
[Source: Funke Gerber]
79
Chapter 7
CREAM
80
81
7.1 Determination of Sensory Quality of Cream
Aim: To determine sensory quality of cream

Principle: Judging the quality of cream by its taste and smell requires considerable skill, which
useful for preliminary assessment of milk product quality and handling practice. Cream is
smelled and observed visually to see if there are any sensory defects.
Apparatus: 100ml beaker, spatula
Preparation of sample:
In case of large containers, a sample of at least 500g should be taken. In the case of retail
Before evaluation it is recommended that the samples be kept at the temperature mentioned on
the packages or laid down in national legislation. If there is no indication, a temperature of 4±2°C
is recommended. For the evaluation of appearance, the samples should if possible be presented in
the original packages. For the evaluation of flavor, individual portions of at leas 50-00g should be
available for each assessor. During the evaluation the samples should have a temperature of
16±2°C.
Procedure:
The sensory evaluation of cream should be carried out in relation to appearance, consistency and
flavor
1. The evaluation of appearance can be carried out simultaneously by the whole panel with
separate scoring; involving the filling and surface of the cream, color, visible purity,
presence of foreign matter, spots of mold, seepage of whey and phase separation. The
evaluation is made by examination in the opened package, if necessary by pouring out the
product from the package.
2. The evaluation of consistency involves thickness, stickiness and coarseness. Evaluation
can be made by blending the product with a spoon before spreading the sample in the
mouth. The defects mentioned in this method are applicable to fluid cream as well as
cream products with high viscosity.
3. Different categories of cream should be evaluated in accordance with the international
standard.
- 81 -
International Table of Cream Quality Terms
Overfilled Watery
Underfilled Flat
Shrunken Bitter
Heterogeneous surface Cooked
Untypical color Burnt
Non-uniform color Smoked
Marbled Fatty/Oily
Foreign matter Feed flavor
Separation of whey Foreign flavor
Spot of mold Light induced flavor
Cream layer Cheesy
Cream plug Malty
Sedimentation Metallic
Foaming Musty
Oxidized
2. Consistency Salty
Setting Fermented
Lumps or flakes Acid
Dripping Sharp
Uneven Harsh
Gritty Sour
Sticky Tallowy
Too fluid Yeasty
Ropy/stringy Rancid
Dried surface Astringent

Gelatinous Unclean
Stale/Old
Too sweet
Too salty
[Source: International Dairy Federation IDF Standard 99C: 1971]
- 82 -
7.2 Determination of Fat in Cream (Roeder Method)
Aim: To determine the percentage of fat in cream by Roeder method

Principle: When sulfuric acid is added to cream, the proteins will be digested thus destroying the
Addition of amyl alcohol has the function of separation fat from other components
Apparatus: Gerber centrifuge (1100±100 rpm), cream butyrometer (according to Roeder), water
bath that can be maintained at 70°C, safety pipettes or tilt measures for H2SO4 and amyl alcohol,
precision scale with at least two decimals, hot water bath maintained at 65±2°C.
Chemicals: Sulfuring acid of density 1.525 at 20°C (pour 670ml Gerber H2SO4 in 330ml of
distilled water), amyl alcohol.
Sample preparation: The sample must be inverted gently at least 5 times to distribute the fat. If
the sample has been chilled, it must be slowly warmed to 40°C and then cooled to room
temperature with periodic inversions.
Procedure:
1. Weigh 5g of cream into the beaker linked to the stopper of the Roeder butyrometer and
introduce into the butyrometer
2. Add sulfuric acid through the upper opening of the butyrometer so as to cover the top
edge of the beaker
3. Fix the stopper and place the butyrometer in the 70°C water bath. Repeat shaking until the
protein has dissolved completely
4. Ass sulfuric acid up to the beginning of the scale and then add 1ml of amyl alcohol
5. Seal the butyrometer with the stopper, shake it and keep it for 5 min in the 70°C water
bath
6. Centrifuge the butyrometer in the Gerber centrifuge for 5 min
7. After centrifuging, temper the butyrometer in the water bath at 65°C for 5 min
8. Adjust the fat column to the zero point and read off the top of the fat column (bottom of
the meniscus).
Record the fat percentage with either 0 or 5 as first decimal. Difference between double
determinations on the same sample should not exceed 0.5%
[Source: Quality Handbook, Dairy Development Corporation]
- 83 -
7.3 Determination of Fat in Cream (BIS method)
Aim: To determine the percentage of fat in cream by BIS method.

Principle: When sulfuric acid is added to cream, the proteins will be digested thus destroying the
Apparatus: Gerber centrifuge (1100±100 rpm), cream butyrometer (70% standard), water bath
that can be maintained at 70°C, safety pipettes or tilt measures for H2SO4 and amyl alcohol,
precision scale with at least two decimals, hot water bath maintained at 65±2°C.
Chemicals: Sulfuring acid of density 1.525 at 20°C (pour 670ml Gerber H2SO4 in 330ml of
distilled water), amyl alcohol.
Sample preparation: The sample must be inverted gently at least 5 times to distribute the fat. If
the sample has been chilled, it must be slowly warmed to 40°C and then cooled to room
temperature with periodic inversions.
Procedure:
1. Transfer 10ml of sulfuric acid into the butyrometer by means of the 10ml pipette for
sulfuring acid or the automatic tilt measure taking care not to wet the neck of the
butyrometer with the sulfuric acid.
2. Weigh 5±0.01g of sample into the butyrometer without soiling the neck, using any
suitable form of support for the butyrometer on the balance
3. Add about 6ml of hot water (70°C) to the butyrometer
4. Measure 1ml of amyl alcohol into the butyrometer by means of the alcohol pipette or
automatic measure
5. Adjust the level of the contents to about 5mm below the shoulder by further additions of
hot water
6. Seal the butyrometer with the stopper, shake it and keep it for 5 min in the 65°C water
bath
7. Centrifuge the butyrometer in the Gerber centrifuge at 1100±100 rpm for 5 min
8. After centrifugation, temper the butyrometer in the water bath at 65°C for 5 min
9. Adjust the fat column to the zero point and read off the top of the fat column (bottom of
the meniscus)
84
Record the fat percentage with either 0 or 5 as first decimal. Difference between double
determinations on the same sample should not exceed 0.5%.
[Source: Bureau of Indian Standards IS: 1224 (Part II) – 1971]
85
7.4 Determination of Acidity in Cream
Aim: To determine the acidity of cream.

Principle: The titrable acidity of all fresh cream should be consistent with the fat percentage of
the cream. There exists an inverse relationship between the fat percent and titrable acidity
percent. The titrable acidity percent of freshly separated cream is always lower than that of the
milk from which it was separated.
Apparatus: Burette with soda-lime guard tube, porcelain dishes (white hemispherical of
approximately 60ml capacity), stirring rods of glass (flattened at one end).
Chemicals:
0.1N sodium hydroxide, phenolphthalein indicator: Dissolve 1g of phenolphthalein in 110ml
rectified spirit. Add 0.1N sodium hydroxide solution until one drop gives a faint pink coloration.
Dilute with distilled water to 200ml.
Rosaniline acetate stock and bench solution: Dissolve 0.12g of rosaniline acetate in
approximately 50ml rectified spirit containing 0.5ml of glacial acetic acid. Make up to 100ml
with rectified spirit for stock solution. For bench solution dilute 1ml of the stock solution to
500ml with a mixture of rectified spirit and distilled water in equal proportions by volume.
Note: the stock solution and the bench solution should be stored in dark brown bottles securely
stoppered with rubber bungs.
Procedure:
1. Weigh 10g±0.1mg cream into each of two white porcelain dishes
2. Add 10ml of water to both and stir to disperse the cream
3. Prepare color control by adding and stirring 2ml dilute rosaniline acetate solution to one
of the porcelain basins
4. Stir 2ml phenolphthalein solution into the other porcelain dish
5. Titrate the latter one by adding sodium hydroxide solution, from a 10ml burette fitted
with a soda-lime guard tube, until the color matches the pink color of the control
Expression of the Result:
V × N ×9
Titrable acidity in terms of lactic acid, g/100g of cream =
M
Where, V = volume in ml of the standard NaOH solution
N = normality of the standard NaOH
M = mass in g of cream taken for the test
[Source: Bureau of Indian Standards, IS: 3509-1966]
86
Chapter 8
BUTTER
87
88
8.1 Determination Sensory Quality of Butter
Aim: To determine sensory quality of butter

Principle: Judging the quality of butter by its taste and smell requires considerable skill, which
useful for preliminary assessment of milk product quality and handling practice. Butter is smelled
and observed visually to see if there are any sensory defects.
In the case of bulk butter, a sample taken with a butter trier should be made available for sensory
evaluation. In the case of retail packaging, an adequate number of packages should be made
available. Before evaluation it is recommended that the samples be kept at the temperature
mentioned on the packages or laid down in national legislation. During the evaluation, the butter
should have a temperature of 14±1°C. A significant deviation from this temperature prevents a
reliable evaluation, since the temperature of paramount importance.
Procedure:
The sensory evaluation should be carried out in relation to appearance (exterior and interior),
consistency and flavor.
1. Appearance involves the following main features: color, visible purity, mold growth and
water dispersion
2. Consistency involves the following main features: firmness and spreadability
3. Flavor involves the following features: taste and odor
4. Different categories of butter should be evaluated in accordance with International
Standard.
- 88 -
International Table of Butter Quality Terms
loose (free) moisture Flat
Uneven color Unclean
Streaky Foreign flavor
Marbled Stale
Specked-mottled Cheesy
Oil separation Acid
Over-colored Yeasty
Weak (open texture) Cooked
Foreign matter Moldy
Moldy Rancid
Undissolved salt Oily
Layered Oxidized
Holes Metallic
Bleached surface Fishy
Cardboardy
2. Consistency Paper
Short Burnt
Greasy Feed flavor
Sticky Bitter
Hard Over-salted
Soft Putrid
Brittle Malty
Crumbly Chemical flavor
Doughy Soapy
Uncharacteristic
Harsh
- 89 -
8.2 Determination of Fat Content in Butter by Gerber Method
Aim: To determine the fat content of butter

Principle: When sulfuric acid is added to butter, the proteins will be digested thus destroying the
Apparatus: Butter butyrometer (70-90% range) with stopper funnel, balance, Gerber centrifuge
(1100±100 rpm), water bath (65±2°C)
Chemicals: Gerber sulfuric acid and amyl alcohol (as in determination of fat in milk)
Procedure:
1. Weigh 5g well-mixed butter sample into the stopper funnel and fix the funnel to butter
butyrometer
2. Add 10ml sulfuric acid through the upper opening of the butyrometer
3. Add 1ml amyl alcohol to the butyrometer and adjust the level p to the top graduated scale
mark by adding distilled water
4. Close the butyrometer with a rubber stopper and mix the content thoroughly by inverting
the butyrometer at least 10 times
5. Centrifuge for 5 min and place the butyrometer in the water bath at 65°C
6. Expression of Result:
7. Adjust the fat column and read the fat percentage
Note:
This method is not very accurate. A more accurate estimate for fat content in butter is found by
subtracting moisture %, salt % and milk solids-not-fat% from 100%. A good estimate for milk-
solids-not fat in washed butter is 0.5%.
90
8.3 Determination of Fat Content in Butter by Gerber Method
Aim: To determine the fat content of butter

Principle: When sulfuric acid is added to butter, the proteins will be digested thus destroying the
Apparatus: Butter butyrometer (70-90% range) with stopper funnel, balance, Gerber centrifuge
(1100±100 rpm), water bath (65±2°C)
Chemicals: Gerber sulfuric acid and amyl alcohol (as in determination of fat in milk)
Procedure:
1. Weigh 2.5g±1mg of butter sample in a 25ml beaker and mix it with small portions of 1:1
sulfuric acid. Transfer the contents into the butyrometer with the help of a small funnel
2. Rinse the beaker about six times with small quantities of dilute sulfuric acid to make sure
that all butter has been transferred
3. Mix with 10ml of water and 10ml of sulfuric acid in a small beaker
4. Add between 15 to 20ml of the mixture to the butyrometer depending on its size
5. Add 1ml of amyl alcohol
6. Close the butyrometer with a rubber stopper and mix the content thoroughly by inverting
the butyrometer at least 10 times
7. Centrifuge at 1100±100 rpm for 5 min and place the butyrometer in the water bath at
65°C
Adjust the fat column and read the fat percentage
[Source: Bureau of Indian Standards IS: 3507-1966]
91
8.4 Determination of Fat by Soxhlet Extraction Method
Aim: To determine fat in butter by soxhlet extraction method

Principle: Fat-soluble material in a food is extracted from an oven dried sample using a soxhlet
extraction apparatus. The ether is evaporated and the residue weighed.
The ether extract or crude fat of a food represents, besides the true fat (triglycerides), other
materials such as phospholipids, sterols, essential oils, fat-soluble pigments, etc., extractable with
ether. Water-soluble materials are not extracted since the sample has been thoroughly dried prior
to extraction with anhydrous ether or petroleum ether.
Apparatus: Thimble, cotton plug, fat extraction tube, soxhlet apparatus, soxhlet flask,
condenser, water bath, beaker, funnel.
Chemicals: Anhydrous ether
Extraction of fat from food materials is ordinarily done with anhydrous ethyl ether or petroleum
ether (b.p. 35-45°C). Of these two, the petroleum ether is cheaper and requires no special
preparation beyond a possible fractionation to secure material boiling within the desired limits. It
has also the advantage that it is not affected by traces of moisture present in the material to be
extracted and does not take up moisture during the extraction. Ethyl ether though a better solvent
for the fat than the petroleum ether moist ether will dissolve sugar and other materials that should
not be included in the true ether extract.
Procedure:
1. Transfer the dried sample remaining after moisture determination to a thimble and plug
the top of the thimble with a wad of fat-free cotton.
2. Drop the thimble into the at extraction tube of a soxhlet apparatus
3. Attach the bottom of the extraction tube to the soxhlet flask
4. Pour approximately 75ml or more of anhydrous ether through the sample in the tube into
the flask
5. Attach the top of the fat extraction tube to the condenser
6. Extract the sample for 16hr or longer on a water bath
7. The water bath should be regulated so that the ether, which volatilizes condenses and
drops continuously upon the sample without any appreciable loss
8. At the end of the extraction period, remove the thimble from the apparatus and distill off
most of the ether by allowing it to collect in the soxhlet tube
9. Pour off the ether when the tube is nearly full
10. When the ether has reached a small volume, pour it into a small, dry (previously weighed)
beaker through a small funnel containing a plug of cotton
11. Rinse the flask and filter thoroughly, using several small portions of ether
12. Evaporate the ether on a steam bath at low heat, preferable under a current of air
92
13. Dry at 100°C for 1 hr, cool, and weigh
14. The difference in the weights gives the ether-soluble material present in the sample.
Calculation
Wt. of ether-soluble material ×100
% Crude fat =
Wt. of sample
[Source: Manual of Analysis of Fruit and Vegetable Products – S. Ranganna, 1977]
93
8.5 Determination of Moisture in Butter (Routine Method)
Aim: To determine moisture content of butter using routine method.

Principle: The moisture content of butter is measured by drying off water above 100°C.
Apparatus: Flat-bottomed aluminum dish, tongs, balance, electric hot plate, desiccator, gas
burner.
Procedure:
1. Clean the aluminum dish and dry in an oven
2. Allow to cool to the room temperature in a desiccator and weigh the dish. Weight =
W±10.1mg
3. Accurately weigh into the dish 10g of the sample by difference. Weight of dish + sample
= W1.
4. Place the dish over the hot plate and heat it agitation continued by swirling the dish using
tongs. A glass rod may also be used for stirring but the rod must be weighed along with
the dish previously. Control the heating and agitation so that losses by spattering and
frothing are avoided
5. Continue heating of sample, until frothing stops and foaming breaks. The color of the
non-fat solids changes from creamy white to yellow brown. Note that a whitish, yellow
color indicates insufficient heating which results in low values. On the other hand a dark
brown color or black curd indicates over heating, which results in high values.
6. Allow the dish to cool in desiccator. When cooled, place the dish on the balance. Record
the weight. Weight = W2
100 ( W1-W2 )
Moisture percent by weight =
( W1-W )
Where,
W1 = weight in g of the dish with the material before heating to constant weight
W2 = Weight in g of the dish with the material after heating to constant weight
W = Weight in g of the empty dish (if the stirring rod is to be used, this has to be weighed
along with the dish)
[Source: Bureau of Indian Standards IS: 3507-1966,

International Dairy Federation IDF standard – 137: 1986]
94
8.6 Determination of Moisture in Butter (Reference Method)
Aim: To determine moisture content of butter using reference method.

Principle: The moisture content of butter is measured by drying off water above 100°C.
Apparatus: Flat-bottomed aluminum dish, drying oven – maintained at 100±1°C, hot plate or
gas burner or spirit burner, desiccator, glass rods, water bathe – with rings to take dishes of 7.5cm
diameter, clay pipe triangles and balance of 0.001g sensitivity.
Procedure:
1. Clean the dish and the glass rod and dry in the oven maintained at 100±1°C for at least 1
hr
2. Allow to cool to the room temperature in a desiccator and weigh the dish
3. Accurately weigh into the dish 3 to 4g of the prepared butter sample
4. Place the dish on a steam bath supported on a clay pipe triangle for at least 20 min,
stirring at frequent intervals until no moisture can be seen at the bottom of the dish
5. Wipe the bottom of the dish and transfer it to the oven maintained at 100±1°C and keep it
for 90 min
6. Allow the dish to cool in the desiccator as before and weigh
7. Heat the dish again in the oven for 30 min
8. Repeat the process of heating, cooling and weighing until the difference between two
consecutive weights does not exceed 0.1mg
9. Record the lowest weight
Expression Result:
100 ( W1-W2 )
Moisture and volatile mater content in butter, percent by weight =
( W1-W )
Where,
W1 = weight in g of the dish with the material before heating to constant weight
W2 = Weight in g of the dish with the material after heating to constant weight
W = Weight in g of the empty dish (if the stirring rod is to be used, this has to be weighed
along with the dish)
95
8.7 Determination of Water, Solids-Not-Fat and Fat Contents on the
Same Test Portion
Aim: To determine water, solids-not-fat and fat contents on the same test portion.
Principle:
Determination of water content: Drying of a known mass of butter at 102±2°C and weighing to
determine the loss of mass. The loss of mass, expressed as percentage.
Determination of solids-not-fat content: Extraction of the fat from the dried butter is done with
light petroleum or n-hexane and the weight of the residue taken. The percentage by mass of
substances is determined as solids-not-fat.
Determination of fat content: the percentage by mass obtained by subtracting the water content
and solids-not-fat content from 100
Apparatus: Analytical balance, drying oven (at 102±2°C), dishes (of glass, porcelain or metal,
25mm high and at least 50mm in diameter), filter crucible (sintered glass, porosity grade P 40,
pore diameter 16-40µm, with suction flask), stirrer, desiccator.
Chemicals: n-hexane or light petroleum (petroleum spirit) with any boiling range between 30
and 60°C. the reagent shall not leave more than 1mg of residue after evaporation of 100ml.
Procedure:
Preparation of the test sample:
Warm the laboratory sample in the original unopened container, which should be from one-half
to two thirds full, to a temperature at which the sample will be soft enough to facilitate a
thorough mixing to a homogeneous state (either by a mechanical shaker or by hand) without any
rupture of emulsion. The temperature of mixing should normally not exceed 35°C.
Cool the sample to ambient temperature, continuing to mix until cooling is complete. As soon as
possible after cooling, open the sample container and stir briefly (not longer than 10sec) with a
suitable device, for example a spoon or spatula, before weighing.
A: Determination of water content
Dry a dish in the oven at 102±2°C for at least 1hr
1. Allow the dish to cool in the desiccator to the temperature of the balance room and weigh
to the nearest 0.1mg
2. Weigh in the dish, to the nearest 1mg, a test portion of between 2 and 6 g of the test
sample. For unsalted butter, the test portion shall be between 5 and 6g.
3. Place the dish in the oven at 102±2°C and leave it for 2h
4. Allow the dish to cool in the desiccator to the temperature of the balance room and weigh
to the nearest 0.1mg
96
5. Repeat the drying process, for 1 hr and then for additional 30 min periods, cooling and
weighing each time, until constant mass (mass change not exceeding 0.5mg) is reached.
In the event of an increase in mass, take for the calculation the lowest mass recorded.
B: Determination of solids-not-fat
1. Dry a filter crucible in the oven at 102±2°C for at least 1 hr
2. Allow the crucible to cool in the desiccator to the temperature of the balance room and
weigh to the nearest 0.1mg
3. Add 10-15ml of warm (see note) n-hexane or light petroleum to the dish containing the
dry matter left from the water determination, to dissolve fat.
4. Note – In the case of n-hexane of if light petroleum having an initial boiling point of 40°C
– or above, use a temperature of 35°C; in the case of light petroleum having an initial
boiling point below 40°C, use a temperature of 25°C
5. Detach as much as possible of the sediment adhering to the dish by using the stirrer, and
transfer the contents quantitatively into the weighed crucible with the aid of the stirrer tip
6. Repeat this operation 3 to 5 times
7. Wash the sediment in the crucible with 25ml of warm (see note in 3) n-hexane or light
petroleum
8. Dry the dish and crucible in the oven at 102±2°C for 30 min
9. Allow the dish and crucible to cool in the desiccator to the temperature of the balance
room and weigh to the nearest 0.1mg
10. Repeat operations 7 and 8 until constant mass (mass change not exceeding 0.5mg) is
reached
Number of determinations
Carry out the procedure specified in A and b on duplicate test portions taken from the same
prepared test sample.
Expression of Results
Method of calculation of water content
For each of the duplicate test portions, calculate the water content, E, as percentage by mass,
using the following formula:
m1 − m 2
E= × 100
m1 − m 0
Where,
m0 = mass in g of the empty dish
m1 = mass in g of the test portion and dish before drying
m2 = mass in g of the test portion and dish after drying
97
Method of calculation of solids-not-fat content
For each of the duplicate test portions, calculate the solids-not-fat content, S, as a percentage by
mass, using the following formula:
(m 4 − m3 ) + (m5 − m 0 )
S= ×100
(m1 − m 0 )
Where,
m0 and m1 defined earlier = mass in g of the empty dish
m3 = mass in g of the empty crucible
m4 = mass in g of the crucible containing the sediment
m5 = final mass in g of the dish
Method of calculation of fat content
The percentage, by mass, of fat is equal to:
100-(E+S)
Where,
E = percentage, by mass, of water
S = percentage, by mass, of solids-not-fat
Express the result to the first decimal place
[Source: International Dairy Federation IDF Standard 80: 1977]
98
8.8 Determination of Salt Content in Butter
Aim: To determine salt content in butter by Mohr method.

Principle: Salt content of butter, the chloride content as determined by the specified method, is
also expressed as the equivalent content of sodium chloride. Melting of butter by adding boiling
water and titration of the extracted chlorides in the mixture with a solution of silver nitrate by
using potassium chromate as indicator is a commonly used method referred to as Mohr method.
Apparatus: Analytical balance, titration vessel of glass, eγ, a conical flask or beaker of capacity
250ml, graduated measuring cylinder of capacity 100ml, pipette of 2ml, burette of capacity 50ml.
Chemicals: 5% potassium chromate indicator solution (50g of K2CrO4) in 1000ml of water),
silver nitrate standard (0.08 to 0.1Mole/liter)
Note: If a solution containing 14.53g/liter (0.0885 mol/liter) of silver nitrate is used, 1ml of this
solution is equivalent to 5mg of sodium chloride, thus making the calculation of the salt content
of butter easier.
Procedure:
1. Weigh 5g±1mg of butter either directly into the titration vessel or on a piece of grease-
proof paper or plastic film that is transferred with the butter to the titration vessel
2. Add 100ml of boiling water or 100ml of cold water and heat to boiling
3. Mix the contents of the vessel and add 2.0ml of potassium chromate indicator solution
4. Titrate the contents with the silver nitrate solution until an orange tint, which persists for
30 sec is obtained
5. Record the volume of silver nitrate used in millimeters
6. Carry out a blank test using all reagents but omitting the test portion
Calculation
5.84 × V × C
The salt content expressed as a percentage by mass is equal to
M
Where, V = the volume in ml of silver nitrate solution
C = the exact concentration of the silver nitrate solution in moles per liter
M = the mass in g of the test portion
99
If a solution containing 14.33g/liter is used and the sample mass is 5g, the calculation of the salt
content of the butter may be simplified such that it is equal to
V
10
Where,
V = the volume in milliliter of silver nitrate solution used. In either case report the result
to one decimal place.
If a blank value is obtained the volume of silver nitrate solution, in milliliters, obtained should be
subtracted from V.
[Source: International Dairy Federation IDF Standard – 12B: 1988]
100
8.9 Determination of Salt Content in Butter (Cornell Method)
Aim: To determine salt content in butter by Cornell Method.

Principle: Cornell method gives a fairly accurate result with minimum consumption of costly
silver nitrate. In this method, the butter sample is mixed with hot water to dissolve the salt and
the latter estimated by titrating against standard silver nitrate solution.
Apparatus: Glass beaker, hot plate, burette, pipettes, and float bottom aluminum beaker.
Chemicals: N/10 silver nitrate solution, potassium chromate (5%).
Procedure:
1. Weigh 10g of the prepared butter in the aluminum beaker
2. Measure out exactly 300ml of water with a temperature around 78°C and add a portion of
this water to the beaker containing the 10g±1mg of butter sample
3. Repeat this operation of adding hot water, rinsing and transferring to the beaker until the
moisture cup is cleared or its content and then add the remaining of the 300ml water
4. Shake the content of the beaker throughout so as to distribute the salt evenly throughout
the solution and let it stand 2-3 min for the fat to rise to the surface
5. Measure out a Babcock pipette full (17.6ml) of the clear brine into a porcelain basin and
add 4-5 drops of a 5% potassium chromate indicator
6. Titrate with N/10 silver nitrate solution. Add the solution stirring constantly until brine
turns to a light brown color
7. Read the millimeter of N/10 silver nitrate solution used in the titration
Each milliliter of N/10 silver nitrate solution represents one percent of salt
101
8.10 Determination of Solid-Not-Fat Content in Butter
Aim: To determine solid-not-fat content in butter.

Principle: Butter contains small amount of milk proteins. The quantity varies with the method of
manufacture and the number of washing given after churning is over. In order to ensure good
storage quality, butter should contain as little of the protein residue as possible. Normally, protein
residue should not exceed 1.5%. It is estimated from the fat free residue obtained after moisture
determination. The residue is dried and weighed.
Apparatus: Gooch crucible or sintered funnel (with filter flask with adapter), glass funnel, flat
bottom flask (250ml capacity), desiccator (with efficient desiccant), asbestos, glass funnel (with
folded 12.5cm Whatman No.1 or its equivalent filter paper).
Chemicals: Petroleum hydrocarbon solvent (boiling range 40-60°C)
Procedure:
1. Prepare as asbestos mat in a Gooch crucible or sintered funnel, dry in the oven maintained
at 100±1°C, cool in the desiccator and weigh
2. Alternatively dry, cool and weigh ordinary glass funnel with folded 12.5cm filter paper
3. Melt the residue in the moisture dish or cup from moisture determination
4. Add 25ml to 50ml of petroleum solvent and mix well
5. Fit the crucible to the filter flask or place the funnel with filter paper on a filter stand
6. Wet the asbestos mat or the filter paper with petroleum solvent and decant the fatty
solution from the dish into the asbestos or the filter paper, leaving the sediment in the dish
7. Macerate the sediment twice with 20 to 25ml of petroleum solvent and decant again the
fatty solution into the asbestos or the filter paper
8. Filter the solution and collect the filtrate in a clean, dried, tared 250ml flat bottom flask
containing glass bead
9. With the aid of a wash bottle sediment from the dish into the crucible or the filter paper
10. Finally wash the crucible or the filter paper until free from fat, collecting all the filtrate in
the flask
11. Dry the crucible of filter funnel in the oven maintained at 100±1°C for at least 30 min
12. Cool in the desiccator and weigh
13. Repeat drying, cooling and weighing until the loss of weight between the consecutive
weighing does not exceed 0.1mg. Preserve the residue for the determination of salt.
102
Expression Result
Calculation
W1 − W2
Solids-not-fat and salt % = ×100
W
Where,
W1 = Weight in g of the filter paper with residue
W2 = Weight in g of the filter paper alone
W = Weight in g of the sample
Solid not fat % by weight = Solid not fat and salt % - salt % by weight in salt test
103
8.11 Determination of Coloring Matter in Butter
Aim: To determine coloring matter in butter.

Principle: Several dyes and colors like azo-dyes, annatto or other vegetable color, and gallate
antioxidants in butter give characteristic color upon treatment with alkali or acid.
Apparatus: Balance, test tubes, pipettes
Chemicals: Ethyl ether, hydrochloric acid (1:1), 10% sodium hydroxide solution, 2% sodium
hydroxide, stannous chloride (40% containing sufficient hydrochloric acid to make the solution
acidic and a small piece of tin to keep it reduced), filter paper.
Procedure:
1. Pour 2g of filtered butter, dissolved in ether into each of two test tubes
2. To one tube add 1-2ml of hydrochloric acid (1:1), and to the other about same volume of
10% sodium hydroxide solution
3. Shake the tubes well and allow them to stand
4. In the presence of some azo dyes, acid solution shows pink to dark red color, while the
alkaline solution in another tube shows no color
5. If on the other hand annatto or other vegetable color is present, alkali solution is colored
yellow, while no color is apparent in acid solution (red color changing to yellow,
especially on warming in alkaline solution may be due to presence of gallate antioxidants)
6. Pour on moistened filter paper alkaline solution of color obtained by shaking clear butter
with warm 2% sodium hydroxide solution
7. If annatto is present, paper absorbs color, so that when washed with a gentle stream of
water it remains dyed straw color
8. Dry the filter and add one drop of 40% stannous chloride solution
9. Again dry carefully and observe the color
Expression of Results:
If the color turns purple, presence of annatto is confirmed.
104
8.12 Determination of Titrable Acidity in Butter
Aim: To determine titrable acidity in butter.

Principle: The butter is melted in hot water and the hot solution is titrated with standard alkali
till neutral to phenolphthalein.
Apparatus: Burette with soda-lime guard tube, conical flask (250ml capacity)
Chemicals:
0.02N sodium hydroxide: Prepare a concentrated stock solution of sodium hydroxide in water
(1+1). Tightly stopper the flask with a rubber bung and allow any insoluble sodium carbonate to
settle down for 3 to 4 days. About 1.6 ml of stock solution is required per liter of distilled water
to make 0.02N solution. The solution is accurately standardized with acid potassium phthalate
(A. R.) or oxalic acid (A. R.).
Phenolphthalein indicator solution: Dissolve 1g of phenolphthalein in 110ml rectified spirit. Add
0.1N sodium hydroxide solution until one drop gives a faint pink coloration. Dilute with distilled
water to 200ml.
Procedure:
1. Weigh accurately about 20g±1mg of the butter sample in a dry 250-ml conical flask
2. Add 90ml of hot, previously boiled water and shake the contents
3. While still hot, titrate with 0.02N sodium hydroxide, using 1ml of the phenolphthalein
indicator
9× N× V
Titrable acidity (as lactic acid) % by weight =
W
Where,
N = Normality of sodium hydroxide
V = Volume of sodium hydroxide
W = Weight in g of the sample
105
8.13 Determination of Rancidity (Peroxide Value) in Butter
Aim: To determine rancidity (peroxide value) in butter.

Principle: In oxidative rancidity, oxidation of fat due to the combination of oxygen with
unsaturated fatty acids takes place and results in the formation of compounds with a peroxide
structure. These are detected by the liberation of iodine from an acid solution of potassium
iodide. The peroxide value is a measure of the oxidative rancidity in butter and is expressed as
milliliter of 0.002N sodium thiosulfate per gram or as milliequivalents of peroxide oxygen per kg
of sample.
Apparatus: Graduated pipette (1ml capacity), Erlenmeyer flask (glass stoppered 250ml
capacity), measuring cylinder.
Chemicals:
Acetic acid-chloroform solution: Mix 3 parts by volume of glacial acetic acid with 2 parts by
volume of chloroform.
Saturated potassium iodide solution: Prepare aqueous solution.
Sodium thiosulfate, 0.01N: Dilute 10ml of 0.1N sodium thiosulfate solution in distilled water to
give 100ml (in a volumetric flask)
Starch indicator: Prepare 1% solution in water.
Procedure:
1. Weigh 5g±1mg of sample into a 250-ml glass-stoppered Erlenmeyer flask and then add
30ml acetic acid-chloroform solution
2. Swirl the flask until the sample is dissolved in the solution
3. Add 0.5ml of saturated potassium iodide solution using a pipette
4. Allow the solution to stand with occasional shaking for exactly 1 min.
5. Add 30ml of distilled water and mix
6. Titrate against 0.1N sodium thiosulfate adding it gradually and with constant vigorous
shaking
7. Continue titration until yellow color has almost disappeared
8. Add about 0.5ml starch indicator
9. Continue titration shaking the flask vigorously near the end point to liberate all the iodine
from the chloroform layer
10. Add the thiosulfate dropwise until blue color has just disappeared
Note: If titration is less than 0.5ml with 0.1N sodium thiosulfate, repeat the determination using
0.01N sodium thiosulfate solution. The blank determination must not exceed 0.1ml of the 0.1N
sodium thiosulfate.
106
Interpretation of result:
Titration value × N ×1000
Peroxide value (m. eq/kg of fat) =
Weight of sample
Where,
N = Normality of Na2S2O3 solution
m. eq = milliequivalent
[Source: Association of Official Analytical Chemists, (AOAC), USA]
107
Chapter 9
GHEE
108
109
9.1 Determination of Sensory Quality of Ghee
Aim: To determine sensory quality of ghee

Principle: A consumer judges the quality of ghee by its taste and aroma and accepts it on this
basis. Though preference for ghee flavor varies considerable from region to region the main
characteristics for its sensory evaluation, namely flavor, texture, color, and clarity remains the
same.
Procedure:
1. 50ml ghee should be available for evaluation
2. Sensory evaluation should always start with the visual observation of the sample
3. Color should be judged first followed by the texture, odor, taste and aroma
4. Suspended impurities should be judged after melting the ghee (if already solidified)
The characteristics compared are as follows:
1. Flavor
Rancid
Curdy: This flavor is an antithesis flavor and is a result of under clarification during ghee
making. The flavor reminiscent of curd left over during refining
Burnt: This is the same as brown burnt
Smoky: A characteristic in ghee heated over cow dung/firewood using improperly designed
fireplace where smoke directly comes in contact with butterfat during its clarification
2. Texture
Greasy: this is associated with undesirable texture in grains of ghee and is a result of
oxidation affecting the firmness of grains
Hard
3. Color
Brown burnt: This refers to overheated flavor in ghee, which results when butter is clarified
at 130°C and above. This is usually accompanied by a brownish to dark discoloration
4. Freedom from suspended impurities
Ghee residue
[Source: Bureau of Indian Standard IS: 7770-1975]
- 109 -
9.2 Determination of Moisture in Ghee
Aim: To determine moisture content in ghee

Principle: The moisture content in ghee is measured by drying off by drying off water.
Apparatus: Flat-bottomed aluminum dish (3” diameter and 12mm deep), desiccator, hot plate
heater, balance (3 decimals).
Procedure:
1. Weigh a clean, dry and flat-bottomed aluminum dish. Weight Wg±0.1mg
2. Weigh accurately about 10g of ghee into dish. Weight W1
3. Put the dish with the weighed ghee on the hot plate heater. Slowly increase the heat. Stir
the sample clamping by tongs to distribute heat in all sample area
4. Continue the heating until it stops foaming and fuming of moisture
5. The temperature of ghee should not be reached above 130°C at the end of the heating
6. Let the sample be up to little brown in appearance but do not let it became dark brown
7. Cool in desiccator and weigh. Note the weight, W2
(W1 − W2)
Moisture % in ghee: ×100%
(W1 − W)
[Source: Nepal Bureau of Standard and Metrology NS-2-2035]
110
9.3 Determination of Moisture in Ghee (Reference Method)
Aim: To determine moisture content in ghee

Principle: The moisture content in ghee is measured by drying off by drying off water at 100°C.
Apparatus: Flat-bottomed aluminum dish (3” diameter and 12mm deep), desiccator, hot air oven
(98-100°C), balance (at least two decimal places).
Procedure:
1. Weigh a clean, dry and flat bottomed aluminum dish. Weight Wg±1mg
2. Weigh accurately about 10g of ghee into the dish. Weight W1
3. Put the dish in the hot air oven at 100°C and dry for 90 min
4. Cool in desiccator and weigh. Note the weight W2
5. Repeat heating in the oven for 30 min plus cooling and weighing until further loss in
weight is less than 0.001g. Final weight W2
(W1 − W2)
Moisture % in ghee = ×100%
(W1 − W)
[Source: Handbook Dairy Development Corporation]
111
9.4 Determination of Free Fatty Acid in Ghee
Aim: To determine free fatty acid in ghee

Principle: An added alcohol dissolves free fatty acids present in the ghee. This acid is titrated
against a standard alkali and the result is expressed as percent oleic acid.
Apparatus: Glass burette (50ml capacity with 0.1ml graduation), porcelain dish (white
bottomed, 100ml capacity), conical flask – 250ml, 50ml pipette.
Chemicals: Neutralized 95% alcohol, sodium hydroxide (0.1N), phenolphthalein indicator (1%).
Procedure:
1. Weigh a clean, dry conical flask, weight Wg±1mg
2. Weigh accurately about 10g of ghee into the flask. Weight W1
3. Add 50ml of neutralized 95% alcohol
4. Heat to boiling by immersing the flask into a water bath while shaking
5. Shake the flask thoroughly to dissolve the free fatty acids
6. Add 4 drops of phenolphthalein indicator and titrate with 0.1N sodium hydroxide solution
until slight pink color appears. Volume of 0.1N NaOH used = Vml.
V × 2.82
Percentage of free fatty acids expresses as oleic acid = ; 2.82 is a conversion factor
(W1 − W)
[Source: Nepal Bureau of Standards and Metrology (NS-2-2035)]
112
9.5 Determination of Refractive Index of Ghee
Aim: To determine refractive index of ghee

Principle: Refractive index is the ratio of velocity of light in vacuum to the velocity in the
sample medium. More generally, it is expressed as the ratio between the sine of the angle of
incidence to the sine of the angle of refraction when a ray of light of a definite known wavelength
(usually 589.3µm the mean of the D-line of sodium) passes from air into ghee. Refractive index
of ghee is measured at 40°C to ensure that the sample is completely melted.
Accurate results are obtained by using monochromatic light of a wavelength of 589.3µm.
Diffused white light may be used provided the instrument is fitted with a suitable compensator.
Reading with white light is only accurate when a perfectly colorless and sharp line of
demarcation is obtained between the dark and light shades.
The refractive index should be read on an Abbe refractometer, which gives the true refractive
index, or on a butyro-refractometer, which reads on an arbitrary scale at constant temperature as
near 40°C as possible.
Apparatus: Precision refractometer or butyro-refractometer (fitted with an accurate thermometer
reading from 40-50°C), hot water circulating device (to maintain the temperature of the prism
constant at 40±1°C), sodium lamp (day light can also be used if the refractometer has an
achromatic compensator).
Chemicals: Standard fluid for checking the accuracy of the instrument
Procedure:
1. The sample shall be rendered optically clear and free from water and other suspended
impurities
2. Charge the instrument, open double prism by means of screw head
3. Put a few drops of sample into a funnel-shaped aperture between prisms
4. Close prisms firmly by few minutes before reading is taken so that temperature of sample
and instrument will be same
113
For conversion of refractive index values into butyro-refractometer reading and vice-versa use
table below:
B. R. Reading Refractive B. R. Reading Refractive B. R. Reading Refractive
Index Index Index
1 2 1 2 1 2
35.0 1.4488 40.5 1.4527 46.0 1.4565
35.5 1.4491 41.0 1.4531 46.5 1.4569
36.0 1.4495 41.5 1.4534 47.0 1.4572
36.5 1.4499 42.0 1.4538 47.5 1.4576
37.0 1.4502 42.5 1.4541 48.0 1.4579
37.5 1.4506 43.0 1.4545 48.5 1.4583
38.0 1.4509 43.5 1.4548 49.0 1.4586
38.5 1.4513 44.0 1.4552 49.5 1.4590
39.0 1.4517 44.5 1.4555 50.0 1.4593
39.5 1.4520 45.0 1.4558
40.0 1.4524 45.5 1.4562
The refractive index decreases with a rise and increases with a fall in temperature. If the
temperature is not exactly at 40°C, X is added to the observed reading for each degree above or
subtracted for each degree below 40°C pro data where
X for butyro-refractometer = 0.55
Normally the temperature of observation shall not deviate by more than ±2°C
Accuracy of the Method: The maximum difference between duplicate determinations shall not
exceed 0.002 unit for the refractive index and 0.1 for the butyro-refractometer reading.
[Source: Bureau of Indian Standards IS 3508-1966,

Nepal Bureau of Standards and Metrology NS 2-2035]
114
9.6 Determination of RM Value (Reichert-Meissl) in Ghee
Aim: To determine RM value of ghee

Principle: RM value is the number of 0.1N aqueous alkali solution required to neutralize the
steam-volatile, water soluble free fatty acids distilled from 5g of ghee, under the precise
condition specified in the method.
Apparatus: The assembly of the apparatus contains condenser, still head, receiving flask (110ml
capacity), Polenske flask, stand for condenser, graduated cylinders (10ml, 25ml), gas burner,
glass funnel.
Chemicals: Glycerol 98% (w/w), dilute sulfuric acid (25ml acid diluted to 1 liter),
phenolphthalein indicator (0.5% in 95% v/v ethyl alcohol or rectified spirit), glass beads, sodium
hydroxide solution (0.1N aqueous), sodium hydroxide (50% saturated solution: dissolve 1:1 by
weight in water, store protected for carbon dioxide, use only the clear portion), filter paper
(Whatman No. 4).
Procedure:
1. Weigh accurately 5g of ghee sample in a clean and dry 300ml Polenske flask. Add 20ml
of 98% pure glycerol and 2ml of 50% sodium hydroxide. Put 4-5 glass beads.
2. Heat it over burner and keep on stirring the flask. Let completely saponify the milk fat.
The solution will become transparent. Avoid overheating, such overheating could be
indicated by darkening of the solution
3. Add 93ml of boiling distilled water to flask very slowly
4. Add 50ml of dilute sulfuric acid to the flask. The color will become milky white
5. Fix the still head to the flask and condenser; connect the one end of the condenser to the
tap water. Now open the tap water
6. Before heating the flask place 110ml flat bottom receiving flask at the lower side of
condenser
7. Start heating the flask with the help of burner
8. Heat it up to 15-19 min (in this time we should collect 110ml distillate)
9. Now cool it under refrigerated condition to about 20°C
10. Filter the content (distillate) through Whatman filter paper No 4
11. Now take 100ml of filtrate in a clean and dry conical flask
12. Add 2-3 drops of phenolphthalein indicator and titrate against 0.1N sodium hydroxide.
Note the titer value
13. Similarly carry on the blank as above stated method
115
RM value = 1.10×(T-B)
Where,
T = Titer value (volume in ml of 0.1N NaOH solution used for titration)
B = Blank titer
[Source: Bureau of Indian Standards, IS: 3508-1966]
116
9.7 Determination of Insoluble Impurities in Ghee
Aim: To determine insoluble impurities in ghee

Principle: Insoluble impurities represent foreign matter exclusive of moisture, which are not
dissolved by light petroleum.
Apparatus: Conical flask of 250ml capacity provided with stopper
Chemicals: Light petroleum of boiling range 40 to 60°C
Procedure:
1. Weigh accurately about 20g±1mg of the moisture-free sample into a conical flask.
Wg±0.1mg
2. Add 200ml of light petroleum
3. Stopper the flask and shake it well
4. Allow standing at room temperature for 30 min
5. Filter the solution through a previously dried and tared 12-cm diameter filter paper.
Weight = W1
6. Wash the filter paper and the flask with light petroleum till fat-free
7. Remove the filter paper, allow the solvent to evaporate and dry in an oven at 98 to 100°C
for 1 hr
8. Weigh the filter paper. W2g±0.1mg
9. Repeat drying and weighing until the loss of weight between successive weighings do not
exceed W1g±0.1mg
Calculation
100(W2 − W1)
Insoluble impurities, percent by weight =
W
Where,
W2 = Weight on g of the filter paper and impurities
W1 = Weight in g of the dry filter paper
W = Weight in g of the sample taken
[Source: bureau of Indian Standards IS: 3508-1966]
117
9.8 Determination of Melting Point of Ghee
Aim: To determine melting point of ghee

Principle: Ghee being a heterogeneous mixture of glycerides does not have a sharp melting point
but for control purposes it is possible to obtain useful comparison of samples by determining the
temperature at which a column of fat of fixed length rises in a open capillary tube. It is essential,
however, that the comparison should be carried out under exactly the same conditions and the
suitable conditions are described in the procedure.
Apparatus: Melting point tubes (thin walled, uniformly bored capillary glass tubes open at both
ends), thermometer, melting point apparatus (beaker with a side tube heating arrangement), heat
source.
Procedure:
1. Melt the sample and filter it through a filter paper to remove any impurities and the last
traces of moisture and let the sample to be absolutely dry
2. Mix the sample thoroughly and insert a clean melting point tube into the molten sample
product so that a column of material, about 10mm long, is forced into the tube
3. Chill the sample in the tube at once by placing the end of the tube containing the sample
against a piece of ice until the fat gets solidified
4. Place the melting point tube in a test tube and keep it for one hour either in a refrigerator
or in water maintained at 4-10°C
5. Remove the melting point tube and attach with a rubber band or any other suitable means
to the thermometer so that the lower end of the melting point of the tube is even with the
bottom of the bulb of the thermometer
6. Pour water at about 10°C into the beaker or the thiele tube and suspend the thermometer
in the center of the apparatus, so that the lower end of the sample column is about 30mm
below the surface of water
7. Heat the side tube of the apparatus gently, so that the temperature of the water rises
slowly at the rate of 2°C per min till the temperature reaches 25°C and thereafter at the
rate of 0.5°C per min
8. Note the temperature of the water when the sample column commences to rise in the
melting point tube
118
Expression of Result
The average of two such separate determinations as the melting point, provided that the readings
do not differ by more than 0.5°C
[Source: Nepal Bureau of Standards and Metrology NS: 2-2035]
119
9.9 Determination of Rancidity (Peroxide Value) of Ghee
Aim: To determine rancidity (peroxide value) of ghee

Principle: In oxidative rancidity, oxidation of fat due to the combination of oxygen with
unsaturated fatty acids takes place and results in the formation of compounds with a peroxide
structure. These are detected by the liberation of iodine from an acid solution of potassium
iodide. The peroxide value is a measure of the oxidative rancidity in ghee and is expressed as
milliliter of 0.002N sodium thiosulfate per gram or as milliequivalents of peroxide oxygen per kg
of sample.
Apparatus: Graduated pipette (1ml capacity), Erlenmeyer flask (glass stoppered 250ml
capacity), measuring cylinder.
Chemicals:
• Acetic acid-chloroform solution: Mix 3 parts by volume of glacial acetic acid with 2 parts
by volume of chloroform.
• Saturated potassium iodide solution: Prepare aqueous solution.
• Sodium thiosulfate, 0.01N: Dilute 10ml of 0.1N sodium thiosulfate solution in distilled
water to give 100ml (in a volumetric flask)
• Starch indicator: Prepare 1% solution in water.
Procedure:
1. Weigh 5g±1mg of sample into a 250-ml glass-stoppered Erlenmeyer flask and then add
30ml acetic acid-chloroform solution
2. Swirl the flask until the sample is dissolved in the solution
3. Add 0.5ml of saturated potassium iodide solution using a pipette
4. Allow the solution to stand with occasional shaking for exactly 1 min.
5. Add 30ml of distilled water and mix
6. Titrate against 0.1N sodium thiosulfate adding it gradually and with constant vigorous
shaking
7. Continue titration until yellow color has almost disappeared
8. Add about 0.5ml starch indicator
9. Continue titration shaking the flask vigorously near the end point to liberate all the iodine
from the chloroform layer
10. Add the thiosulfate dropwise until blue color has just disappeared
Note: If titration is less than 0.5ml with 0.1N sodium thiosulfate, repeat the determination using
0.01N sodium thiosulfate solution. The blank determination must not exceed 0.1ml of the 0.1N
sodium thiosulfate.
120
Interpretation of result:
Titration value × N ×1000
Peroxide value (m. eq/kg of fat) =
Weight of sample
Where,
N = Normality of Na2S2O3 solution
m. eq = milliequivalent
[Source: Association of Official Analytical Chemists, (AOAC), USA]
121
9.10 Determination of Vegetable Fat in Ghee by the Phytosteryl
Acetate test
Aim: To determine vegetable fat in butter ghee by the phytosteryl acetate test
Principle: The sensitivity of the test depends upon the character of the vegetable fat used for
admixture. The sterol content is determined gravimetrically after saponification of the fat and
precipitation of the sterols by adding an alcoholic digitonine solution to the soap solution. The
melting point of the sterol acetate is determined after acetylating the sterol digitonide by acetic
anhydride. The crystal form of the sterols is microscopically examined after converting the sterol
acetates into the steroids by saponification with an alcoholic potassium hydroxide solution.
Apparatus: Conical flask 250ml, microfiltering device, melting point apparatus, melting point
tubes, microscope slides and coverslips, microscope, thermometer.
Chemicals: Potassium hydroxide analytical grade solution (dissolve 400g of potassium
hydroxide in 600g of distilled water), digitonine analytical grade (dissolve 10g digitonine in 1
liter of 96% ethanol v/v), ethanol (redistilled 95-96% v/v and 80% v/v), diethyl ether, acetic
anhydride.
Procedure:
A. Determination of the sterol content:
1. Weigh accurately 100g of fat in a conical flask of 250ml capacity
2. Add 10ml of potassium hydroxide solution and 20ml of 95-96% ethanol v/v and add 20
glass beads
3. Place the air cooled condenser on the flask, heat on a boiling water bath until the solution
has become clear and continue boiling for half an hour
4. Add 60ml of water and then 180ml of 96% ethanol v/v and raise the temperature to about
40°C
5. Add 30ml of the alcoholic 1% digitonine solution, shake, and allow to cool
6. Place the flask in a refrigerator at about 5°C for about 12 hrs
7. Collect the precipitate of sterol digitonide by filtering through a filter paper (Whatman
No. 1 or equivalent) in a Buchner funnel (8mm dia)
8. Wash out the precipitate with water at about 5°C until the filtrate stops foaming, then one
once with 25 to 50ml of 96% ethanol v/v and at last once with 25-50ml diethyl ether
9. Dry the filter paper with precipitate on a watch glass in a drying oven at 102±2°C for
about 10-15 min
10. Fold the filter paper into two, allowing the precipitate to come off as a pellicle, transfer
the precipitate into a weighing bottle and weigh
B. Preparation of steryl acetate and determination of the melting point
1. Transfer 100g±5mg of the sterol digitonide to a test tube, add 1ml of acetic anhydride,
and heat the tube in a glycerol bath at 130-145°C until precipitate has dissolved
122
2. Continue heating for 2 min and allow cooling at about 80°C
3. Add 4ml of 96% ethanol v/v, mix and heat slightly to dissolve any steryl acetate which
may tend to crystallize out
4. Filter the still warm solution through a small medium speed filter paper impregnated with
ethanol and collect the filtrate in another test tube
5. Heat the filtrate in the latter test tube and carefully bring to gentle boiling. While still
boiling add carefully drop by drop from a pipette 1 to 1.5ml of water until the steryl
acetate is just about to precipitate but still remain in solution. Avoid superheating
6. Add a few drops of 95-96% ethanol v/v to dissolve again any precipitated steryl acetate.
Allow cooling in air for 2 hours and finally in ice water for half an hour
7. Filter the crystallized steryl acetate on a small disc of fast speed filter paper by suction in
a glass microfiltration device and rinse the crystals with 1ml of 80% ethanol v/v
8. Redissolve the crystal cake by heating on a microburner in 1ml ethanol 96% in a short
heat-resistant glass test tube (12mm dia, 35mm length)
9. Allow to cool first in air for 15 min and then in ice water for 5 min and filter crystallized
sterol acetate
10. Repeat dissolving crystallization and filtration to obtain the third, occasionally the fourth
or fifth recrystallization
11. Dry the crystal cake on the paper first at about 30°C in a drying oven and then at 102°C in
drying oven for 10-15 min
12. Grind the crystal cake in a small agate mortar to make a finely divided powder and fill a
melting point tube to a height of 3mm
13. Determine the melting point in the melting point apparatus raising the temperature very
slowly in the last phase of the melting process at a rate of 0.5°C per min
14. Take a reading on the thermometer at the moment that the last crystal grain has just
disappeared, as the melting point
C. Microscopic examination of the sterols
1. Dissolve about 10mg of the sterol acetate in a small test tube in 1ml of 96% ethanol v/v
and add 1-2 drops of potassium hydroxide solution
2. Heat on a boiling water bath until boiling begins and the steryl acetate has dissolved
3. Add 10ml distilled water; transfer the solution to a 125ml separating funnel and shake
with 25ml of diethyl ether. After separation drain and discard the aqueous layer
123
4. Wash the ether layer with three 5ml portion of distilled water. Transfer the ether layer to a
50ml beaker and evaporate to dryness
5. Dissolve the residue in 10ml of 80% ethanol v/v. Place a drop of the clear solution on a
microscope cover slip, wait until crystallization starts on the periphery of the drop, then
invert the cover slip and lay it on a microscope slide
6. Examine the crystals under the microscope at 200× linear magnification
Calculation
0.25 × B
Total sterol content, % = × 100
A
Where,
A = weight in g of the ghee sample
B = weight in g of the sterol digitonide
124
Expression of the Result:
Melting point
If the melting point of the steryl acetate is found to be between 114 and 115°C, the ghee sample
is considered to be free from vegetable fat
If the melting point of the sterol acetate is found to be higher that 117°C, the fat sample is
considered to contain vegetable fat
If the melting point of the sterol acetate is found to be lower than 117°C and higher than 115°C
the fat sample is only considered to contain vegetable fat if the melting point is increased after
replicate recrystallization
Microscopic crystal shapes
If under the microscope the sterol crystals only show the form of a parallelogram with an obtuse
angle of 100°, which is characteristics for cholesterol, the fat sample is considered to be free from
vegetable fat.
If under the microscope the sterol crystals also show the elongated hexagonal from with an apical
angle of 108°, which is characteristic of phytosterols, or if some of the sterol crystals have a
reentry angle (swallow’s tail), which is characteristic for mixtures of cholesterols and
phytosterols, the fat sample is considered to contain vegetable fat.
[Source: Bureau of Indian Standards IS: 3508-1966,

International Dairy Federation IDF Standard 32: 1965]
125
126
Chapter 10
ICE CREAM
127
128
10.1 Determination of Sensory Quality of Ice Cream
Aim: To determine sensory quality of ice cream

Principle: Judging the quality of ice cream by its taste and smell requires considerable skill,
which could only be acquired by practice. Sensory tests are used in all dairies and an experienced
person can pick out bad samples with a high degree of accuracy. The use of sensory technique is
very useful for preliminary assessment of milk product quality and handling practice. Ice cream is
Preparation of sample
In the case of large containers, a sample of at least 500g should be taken. In the case of retail
During the evaluation the ice cream should have a temperature of -13±2°C.
Procedure:
The sensory evaluation of ice cream should be carried out in relation to appearance, body/texture,
flavor and melting properties.
The evaluation of appearance involves the filling and the surface of the ice cream, color, visible
purity, and the amount and uniformity of ingredients/flavoring. The evaluation is made by
examining the sample and it cut surfaces
The evaluation of body and texture involves smoothness, uniformity, coarseness, stickiness, the
presence or absence of sandiness and the relative size of the ice crystals. The evaluation is made
when cutting the sample with a spoon and chewing the sample, letting it melt in the mouth.
The evaluation of flavor is made by letting a sample melt in the mouth, observing the taste and
smell
The evaluation of melting properties involves the following: how the sample has retained its form
and approximate size, whether free liquid has leaked out, whether the liquid appears
homogeneous and creamy, curdled, foamy or watery. The melting properties are observed by
visual examination of the samples kept at a temperature of 22±2°C. It is essential that the same
time interval and the same size of sample are used for the same type of ice cream
Different categories of ice cream should be evaluated in accordance with the International
Standard.
- 127 -
International Table of Ice Cream Quality Terms
Underfilled Lack of flavor
Overfilled Too intense flavor
Unevenly filled Uncharacteristic
Shrunken Unclean
Melted Stale/Old
Ice crystals Foreing flavor
Defective shape Light-induced flavor
Broken stick Acid
Too short stick Sour
Too long stick Salty
Non-uniform color Malty
Air bubbles Cooked
Foreign matter Rancid
Lack of, or poor distribution of ingredients Oxidized
Metallic
2. Body/Texture Caardboardy
Uneven Fatty/Oily
Grainy Tallowy
Brittle (short) Bitter
Crumbly Too sweet
Coarse (icy) Lacks sweetness
Fluffy/foamy Whey flavor
Gummy (pasty, sticky) Milk powder flavor
Heavy (pudding-like) Chemical flavor
Weak (watery) Defective aromatization
Snowy Defective ingredients
Spongy Yeasty, fermented
Fatty
Cold mouthfeel 4. Melting properties
Gritty/Sandy Too quick melting
Soggy wafer or cracker Too slow melting
Hard, firm Foamy
Ice crystals Watery
Free whey
Curdy melt down
Spongy
[Source: International Dairy Federation IDF Standard, 99C: 1977]
- 128 -
10.2 Determination of Fat Content in Ice Cream
Aim: To determine fat content of ice cream by Gerber method

Principle: When sulfuric acid and amyl alcohol are added to ice cream, the proteins will be
digested thus destroying the membranes of the fat globules. The free fat is then released. The fat
remains in liquid due to heat produced and during centrifugation the fat being lighter will be
separated on top of the solution. Addition of amyl alcohol has the function of separation fat from
other components.
Apparatus: Ice cream butyrometer (0-12 or 0-10% range), 1ml pipettes, 10ml pipette, water bath
(65±2°C), electronic balance, centrifuge with 1100-1200 rpm.
Chemicals: Sulfuric acid (sp. gr. 1.807-1.812), amyl alcohol (95%)
Procedure:
1. Pour 10ml sulfuric acid into ice cream butyrometer with the help of 10ml tilt measure and
take care not to wet the stem of the butyrometer
2. Take 5ml melted ice cream sample and pour slowly into butyrometer above sulfuric acid
layer taking care not to wet the stem of butyrometer
3. Shake gently until the contents mix well and then temper it in water bath at 65°C for 5-10
min
4. Centrifuge the butyrometer at 1100±100 rpm for 5 min and temper it in water bath again
for 5 min
5. Adjust the lower level of fat at zero and read the fat content
The total reading of fat seen in butyrometer gives directly the percentage of fat.
[Source: Nepal Bureau of Standards and Metrology NS: 36-2040]
129
10.3 Determination of Fat Content in Ice Cream (Funke Gerber Method)
Aim: To determine fat content of ice cream by Funke Gerber method.

Principle: When sulfuric acid and amyl alcohol are added to ice cream, the proteins will be
digested thus destroying the membranes of the fat globules. The free fat is then released. The fat
remains in liquid due to heat produced and during centrifugation the fat being lighter will be
separated on top of the solution. Addition of amyl alcohol has the function of separation fat from
other components.
Ice cream contains a high amount of sugar and in order to avoid its charring, Gerber sulfuric acid
is diluted further.
Apparatus: Ice cream butyrometer (0-12 or 0-10% range), 1ml pipettes, 10ml pipette, water bath
(65±2°C), electronic balance, centrifuge with 1100-1200 rpm.
Chemicals: Sulfuric acid (mix 87 volumes of conc. H2SO4 of sp. gr. 1.820 with 13 volumes of
water; remember to pour sulfuric acid into water), amyl alcohol (sp. gr. 0.815-0.818).
Procedure:
REMEMBER TO USE THE DILUTED SULFURIC ACID
1. Weigh accurately 5g of melted sample into the ice cream butyrometer
2. All 6ml of hot water for dilution and for washing
3. Take 10ml of sulfuric acid into the butyrometer and add 1ml of amyl alcohol
4. Insert the stopper. Shake, invert for 5 times and centrifuge for 5 min at 1100-1200 rpm
Temper the butyrometer in the water bath for 5 min as for milk by immersing butyrometer stem
upwards and read the fat content.
[Source: Funke Gerber]
130
10.4 Determination of Total Solids Content in Ice Cream (NS Method)
Aim: To determine total solids in ice cream.

Principle: The total solid content of ice cream is determined by drying off its moisture content at
100°C.
Apparatus: Flat-bottomed dishes (made of nickel or other metal which is not of rusting type, 7-
8cm dia, 2.5cm depth and with one glass rod), sand (digested by conc. hydrochloric acid, washed
with water, and dried and heated in flame), well-ventilated oven.
Procedure:
1. Take about 20g of sand in dish with glass rod and heat in oven for 1 hr
2. Cool the dish into desiccator up to room temperature
3. Take 3g of ice cream sample in the dish and add few drops of pasteurized water in sand
which contain the sample and mix the sample and wet sand with glass rod
4. Spread the sample and wet sand uniformly on the dish surface
5. Heat the dish over water bath for 20-30 min
6. Place the dish in hot air oven at 102±1°C and heat for about 4 hrs
7. Cool the dish in desiccator up to room temperature
8. Weigh the dish
9. Continue the process of heating, cooling and weighing until the value of two consecutive
weights do not deviate by 0.5mg
100 − A
Total solids (%) in mass =
B
Where,
A = Weight of residue after heating (g)
B = Weight of sample taken (g)
[Source: Nepal Bureau of Standards and Metrology NS 36-2040]
131
10.5 Determination of Total Solids Content in Ice Cream (Reference
Method)
Aim: To determine total solids in ice cream.

Principle: A known quantity of the sample is dried with sand at a constant temperature. The
mass after drying represents the mass of the total solids.
Apparatus: Analytical balance (sensitivity 0.1mg), desiccator containing an efficient drying
agent, drying oven (well ventilated and thermostatically controlled, adjusted to operate at
102±2°C), non-corrodible flat dishes (about 2.5cm deep and about 7.5cm in diameter with well-
fitting lids), boiling water bath, flat-ended glass rod, quartz sand or sea sand.
Procedure:
1. Put in the dish about 25g of the prepared sand and place the flat-ended glass rod on the lid
2. Transfer the dish to the oven at 102±2°C and place the lid containing the rod next to the
dish. Leave in the oven for about 2 hrs
3. Place the lid and rod on the dish and transfer to a desiccator. Allow to cool to room
temperature and weigh
4. Tilt the dish until the sand moves to one side, then place in the open space 3 to 4g of the
melted and well-mixed sample. Weigh the sample in grams
5. Add about 3ml of water, mix with sample by means of the rod, and then mix the diluted
ice cream thoroughly with the sand. Leave the stirring end of the rod in the mixture, the
other end resting on the side of the dish
6. Place the dish on the boiling water bath for about 30 min, carefully stirring during the
early part of this period, so that the mass when dry shall not form a cake, but shall be well
aerated and in the form of a crumbly mixture. Lay the rod flat in the dish
7. Transfer the dish, with its lid alongside to the oven at 102±2°C. leave for about 2 hrs
8. At the end of this time, place the lid on the dish, transfer it to a desiccator, allow cooling
to room temperature as before and weigh
9. Repeat the heating for periods of 1 hr, cooling and weighing until the loss of weight
between successive weighings does not exceed 1mg.
132
M2 − m
The total solids content expressed as a percentage by mass = ×100
M1 − m
Where,
M2 = The mass in g of the sample after drying
m = The mass in g obtained of the dish with sand
M1 = the total mass in g obtained after adding sample into sand in dish
[Source: International Dairy Federation IDF Standard 70: 1972]
133
10.6 Determination of Total Solids Content in Ice Cream (Routine
Method)
Aim: To determine total solids content of ice cream using gravimetric method.
Principle: The total solids content of ice cream is determined by drying off its moisture at
100°C.
Apparatus: Porcelain dish, hot air oven, balance (sensitivity 0.1mg)
Procedure:
1. Weigh a clean, dry and empty porcelain dish. Weight W±0.1mg
2. Weigh 2 to 4g of the mixed sample of ice cream into the dish. Weight W1±0.1mg
3. Place the dish uncovered on a boiling water bath at least for 30min until it appears dry
4. Remove the dish from the water bath, wipe the bottom and keep the dish in the hot air
oven over a silica triangle and heat at 98-100°C for about 3 hrs
5. After 3 hrs, transfer the dish to a desiccator; allow it to cool for about 30 min
6. Weigh the dish. Weight W2±0.1mg
7. Return the dish to the oven and heat for 1 hr
8. Remove it to the desiccator, cool and weigh as before. Repeat if necessary until the loss of
weight between successive weighing does not exceed 0.5mg
(W2 − W)
% Total Solids in Ice Cream = ×100
(W1 − W)
134
10.7 Determination of Titrable Acidity in Ice Cream
Aim: To determine titrable acidity in ice cream.

Principle: Titrable acidity shall be determined before adding coloring agent into ice cream.
Apparatus: Burette, physical balance, porcelain basin, glass rod.
Chemicals: Sodium hydroxide (0.1N), phenolphthalein indicator (0.5%).
Procedure:
1. Take 20g of ice cream sample in a clean porcelain basin
2. Add 50ml water (boiled and cooled to room temperature)
3. Mix well with glass rod
4. Add few drops of phenolphthalein indicator and mix well with glass rod
5. Now titrate it against sodium hydroxide solution adding drop by drop
6. Mix well the sample while titrating. Continue the titration until it turns light pink
7. Note down the volume of sodium hydroxide consumed to titrate
Calculation
9V × N
Titrable acidity (as lactic acid) =
M
Where,
V = volume of sodium hydroxide consumed
N = Normality of sodium hydroxide
M = mass of sample taken
[Source: Nepal Bureau of Standard and Metrology NS 36-2040]
135
10.8 Determination of Overrun in Ice Cream
Aim: To determine weight per liter and overrun of ice cream.

Principle: Overrun is defined as the volume of ice cream obtained in excess of the volume of the
mix. It is usually expressed as a percentage. This increased volume is composed mainly of the air
incorporated during the freezing process. The amount of air which is incorporated depends upon
the composition of the mix and the way it is processed and is regulated so as to give the
percentage of overrun or yield which will give the proper body and texture necessary for a quality
product. Too much air will produce a snowy and fluffy ice cream while too little, a soggy, heavy
product.
The determination of overrun in frozen and hardened ice cream is a somewhat complex problem
to solve due to the fact that the weight of mix is unknown and has to be determined before
calculation can be made.
The overrun in ice cream depends upon the amount of air whipped into the mix during the
freezing process. In this test, the volume of water and alcohol used corresponds with the volume
of air originally contained in the ice cream and the difference between the sum of these two and
the capacity of the flask is equivalent to the volume occupied by the sample.
Apparatus: Analytical balance (accuracy 0.001g), beaker, volumetric flask, glass funnel.
Chemicals: n-amyl alcohol sp.gr. 0.817
Procedure:
1. Weigh a unit of ice cream and from it calculate the weight of the ice cream per liter. For
example, 200ml ice cream cup can be obtained, the ice cream carefully removed and the
empty dry cup weighed
2. The difference in weights between the cups when filled and when empty is, therefore, th
weight of 200ml of frozen ice cream
3. Five times this weight would then equal the weight of a liter
4. Weigh and record the exact weight of a clean, dry 400ml beaker
5. Into the beaker weigh exactly 130g of frozen ice cream
6. Place the beaker in water bath warmed to 49°C and melt
7. Weigh and record the exact weight of 250ml volumetric flask
8. Using a glass funnel, transfer 130g of melted ice cream into the 250ml flask
9. Add exactly 10g of n-amyl alcohol to the flask and mix to break the surface tension of the
melted ice cream and release the incorporated air. Ten grams of n-amyl alcohol occupy a
volume of 12-24ml
10. Cool the flasks with contents to 15.5°C using a cold water or ice water bath
11. Rinse the beaker containing melted mix with several small rinsing of distilled water,
adding each rinse to the 250ml flask
136
12. Again cool the flask with contents to 15.5°C and using the final rinse water, bring the
volume to 250ml mark
13. The bottom of the meniscus should correspond with the mark when temperature is exactly
15.5°C
14. Dry the outside of the flask and reweigh
1. Calculate the weight in g of the contents
2. Calculate the weight in g of water added to the flask
3. Calculate the volume in ml occupied by the sample of ice cream
4. Determine the specific gravity of the mix by dividing its weight (130g) by the volume in
ml which is occupied
5. Determine the weight in g per liter of mix by multiplying by the specific gravity
[Source: Nepal bureau of Standard and Metrology NS 36-2040,

Handbook of Food Analysis BIS, SP: 18 (Part XI)-1981]
137
10.9 Determination of Percentage Overrun in Ice Cream
Aim: To determine the percentage overrun in ice cream making by a gravimetric method.
Apparatus: Weighing balance (at least two decimals) and an ice cream cup of specific known
volume.
Procedure:
1. Take the accurate weight of the ice cream cup. Weight W
2. Fill up the cup with ice cream mix and weigh again. Weight W1
3. Empty the cup and fill the same with ice cream after freezing and weigh again. Weight
W2
1. From: Quality Handbook, DDC:
W1 − W2
% Overrun = × 100
W2 − W2
2. From: International Dairy Federation IDF- 46:1 969:
Overrun (ratio, volume/mass):
It is generally recognized that, at present, there is no fully reliable method for controlling
overrun (ratio, volume/mass) as below:
Volume of finished product in liters
The ratio = should not exceed 2.
Mass of finished product in kg
However, this ratio is increased to 2.25 (max) in the case of ice cream and milk ices with total
solids content of 35% or higher.
[Source: Quality Handbook Dairy Development Corporation,

International Dairy Federation IDF-46: 1969]
138
10.8 Determination of Phosphatase in Ice Cream
Aim: To determine phosphatase in milk.

Presence of phosphatase enzyme is therefore indicates that a correct pasteurization temperature
has not been reached or the pasteurized milk has been contaminated with raw milk.
In ice cream phosphatase test is performed to check whether the ice cream mix is sufficiently
pasteurized before ice cream freezing or not.
Apparatus: Lovibond all purpose comparator and its standard, standard disc, rubber stoppers,
test tubes.
Chemicals: Buffer solution (3.5g sodium carbonate and 1.5g sodium bicarbonate mixed in 1 liter
distilled water), substrate (95% pure disodium p-phenyl phosphate), buffer substrate (0.15g
substrate and 100ml buffer solution mixed to make buffer substrate).
Procedure:
1. Take 10ml buffer substrate solution in a sterile test tube
2. Warm the content in water bath up to 37-38°C
3. Add 2ml sample into the test tube and stopper it by sterile rubber stopper and mix it well
4. Make another blank sample tube with boiled milk
5. Incubate both tubes in water bathe at 37-38°C
6. After 30 min take out the tubes and note the color of the solution and again incubate it
7. Incubate it for 90 min and note the second reading of the color developed
8. The color of solution of sample and blank are compared with standard color of Lovibond
comparator
9. Note the nearest equal color of the standard by rotating the color disc
Disc reading after 30 min incubation
0 – Pasteurized
6 – Suspicious
Above 10 – Not pasteurized
Disc reading after 2 hrs incubation
0 to 10 – Pasteurized
Above 10 – Not pasteurized
[Source: Nepal Bureau of Standard and Metrology NS: 36-2040]
139
10.8 Determination of Sugar (Sucrose) Content in Ice Cream
Aim: To determine sugar (sucrose) content in ice cream.

Principle: Sucrose content in ice cream is determined using Fehling solution. First, reducing
sugars are determined.
Apparatus: Conical flask, filter paper, balance (sensitivity 0.1mg).
Chemicals:
Copper sulfate solution (solution A): Dissolve 34.639g of copper sulfate (CuSO4.5H2O) in water,
dilute to 500ml and filter if necessary.
Potassium sodium tartrate (solution B): Dissolve 173g of potassium sodium tartrate and 50g of
sodium hydroxide in water, dilute to 500ml and filter.
Fehling solution: Prepared by mixing equal volumes of solution A and solution B just before
using. 20ml is approximately equal to 40ml of 0.25% invert sugar solution.
Hydrochloric acid: (density 1.18, approximately 12N).
Hydrochloric acid: 5.34N
Alumina cream: To a cold saturated solution of aluminum potassium sulfate in water add
sufficient aqueous ammonia of density 0.880, stirring continuously to make the mixing alkaline
to litmus paper. Wash with water by decantation, allowing the gelatinous precipitate to settle
thoroughly between each washing, until only a trace of sulfate remains in the washings.
Neutral lead acetate solution: Prepare a concentrated solution of lead acetate in cold water,
neutralize, if necessary to litmus paper by adding acetic acid or sodium hydroxide. Dilute to a
density of approximately 1.25 at 20°C and filter. This requires the solution of about 41g of lead
acetate diluted to a final volume of 100ml after neutralization.
Sodium oxalate solution: Saturated solution in water.
Standard invert sugar solution: Transfer 23.75g of sucrose, dried overnight in a vacuum
desiccator to a one liter graduated flask with the aid of about 100ml of water. Add 10mlof conc.
hydrochloric acid, mix thoroughly and allow to stand for at least three days at a temperature
approximately 20°C. Dilute to 1 liter. This stock solution contains 2.5g of invert sugar per 100ml
and may be kept for up to four weeks without changing its concentration.
Standard dilute invert sugar solution: Transfer 50ml of the stock solution, make just neutral to
litmus paper with 1N sodium hydroxide solution and dilute to 500ml just before use (1ml =
2.5mg invert sugar).
Procedure:
Determination of original reducing sugars
1. Place about 10g of the prepared sample of ice cream weighed into a 250ml conical flask
and dilute with 150ml of water
2. Mix thoroughly the contents of the flask
140
3. Add neutral lead acetate drop by drop, mixing by rotating the flask until no further
precipitate is formed
4. Add one drop of alumina cream, again mix and allow to stand for a few minutes
5. Rotate the flask at intervals to ensure complete precipitation of protein
6. Add just sufficient sodium oxalate solution to precipitate any excess lead
7. Filter through a fluted 18cm No. 1 filter paper into a 250ml graduated flask
8. Wash the precipitate and the paper thoroughly, with hot water collecting the washings in
the flask
9. Cool the flask and contents and make up to the mark (solution C)
10. Carry out titration against Fehling solution prepared and standardized
If the weight of sample is taken is Wg and the volume of sugar solution used in the titration is S
ml, then the percentage of original reducing sugar in the ice cream is:
25 × X
S× W
11. Pipette 10ml each of Fehling solution prepared and standardized as above into the flask
12. Add an accurately known volume of the standard dilute invert sugar solution and
complete the titration with the prepared sugar filtrate as described under standardization
of Fehling solution
13. Subtract the number of milligram of invert sugar found, from the amount contained in the
known volume of standard sugar solution added
Determination of reducing sugars after inversion
14. Transfer 50ml of the filtered solution (solution C) to a 200ml graduated flask, add 25ml of
water and 10ml of 6.34N hydrochloric acid
15. Invert as described in the preparation of standard dilute invert sugar solution
16. Determine the total invert sugar as described in the determination of reducing sugars
Expression of result
Calculation
Sucrose=[(reducing sugars after inversion) − (original reducing sugars)] × 0.95
[Source: Handbook of analysis BIS, SP: 18 (Part XI) – 1981]
141
142
Chapter 11
PANEER
143
144
11.1 Determination of Sensory Quality of Paneer
Aim: To determine sensory quality of paneer

Principle: Judging the quality of paneer by its taste and smell requires considerable skill, which
can pick out bad samples with a high degree of accuracy. The use of sensory technique is very
useful for preliminary assessment of milk product quality and handling practice. Paneer is
Paneer is the indigenous milk product prepared by the combined action of acid coagulation and
heat treatment of milk and subsequent drainage of whey.
Apparatus: A dish, cutting knife
Procedure:
1. Appearance: See the whole bulk paneer if there is any dirt or others
2. Flavor: smell the cut pieces of paneer for its odor and flavor
3. Texture: Observe the texture and surface of the cut piece
1. Appearance: Paneer shall be clear and free from dirt, surface discoloration, insects and
rodent contamination and from adulterants. It shall not have any free moisture
2. Flavor: Paneer shall have a pleasant odor and characteristic milk acidic flavor
3. Texture: Paneer shall have a closely-knit smooth texture, firm, cohesive and spongy body
- 143 -
11.2 Determination of Titrable Acidity of Paneer
Aim: To determine titrable acidity of paneer

Principle: Acidity in paneer is determined by back-titrating with standard HCl after adding
known excess of NaOH to the aqueous slurry of paneer.
Apparatus: Burette (with soda-lime guard tube), porcelain dishes (white, hemispherical of
approximately 60ml capacity), pipettes for 10ml, 1ml, pestle and mortar.
Chemicals:
Standard sodium hydroxide (0.1N): Prepare stock solution by dissolving NaOH pellet (or stick)
in water in the ratio 1:1 m/m. Tightly stopper the flask with a rubber bung and allow any
insoluble sodium carbonate to settle down for 3-4 days. Use the clear supernatant liquid for
preparing the standard 0.1N solution. About 8ml of stock solution is required per liter of distilled
water.
Phenolphthalein indicator: Dissolve 1g of phenolphthalein in 100ml of rectified spirit. Add 0.1N
sodium hydroxide solution until one drop gives faint pink coloration. Dilute with distilled water
to 200ml
Standard hydrochloric acid: 0.1N
Procedure:
1. Weigh accurately about 2g±0.1mg of paneer sample into a porcelain dish
2. Add 3ml of boiling distilled water and render the sample into a fine paste with a pestle
and mortar
3. Dilute with another 17ml of distilled water, washing off the adherents from the pestle
4. Cool to room temperature
5. Add 10ml of 0.1N sodium hydroxide
6. Add 1ml of 0.5% phenolphthalein indicator and titrate against 0.1N hydrochloric acid till
the pink color disappears
7. Stir vigorously throughout
Calculation:
10 − V
Titrable acidity (as lactic acid) percent by mass = × 0.9
M
Where, V = Volume of 0.1N hydrochloric acid required for titration
M = Mass in g of sample of paneer
[Source: Bureau of Indian Standards IS 10484-1983]
144
11.3 Determination of Fat Content in Paneer
Aim: To determine fat content in paneer by Van Gulik method.

Principle: The fat present in paneer is in the form of an emulsion. This emulsion is broken with
sulfuric acid and fat separation facilitated by amyl alcohol.
Apparatus: Gerber centrifuge, Van Gulik butyrometer, pipettes (10ml and 1ml), funnel, balance
(at least 2 decimals), water bath (65°C).
Chemicals: Van Gulik sulfuric acid (sp. gr. 1.53 to 1.60, prepared by adding 670ml of Gerber
sulfuric acid in 330ml of distilled water), amyl alcohol.
Procedure:
1. Weigh into a Van Gulik beaker placed on a rubber stopper 3g grated and ground paneer
and fix the glass firmly to the Van Gulik butyrometer
2. Add sulfuric acid (about 15ml) until the surface reaches close to the edge of the Van
Gulik glass
3. The butyrometer is placed in the water bath at 65°C and often shaken (do not invert
butyrometer). When all paneer has dissolved, shake vigorously
4. Add 1ml amyl alcohol in the Van Gulik glass
5. Add more sulfuric acid until the liquid surface reaches up to about the 35% mark on the
butyrometer scale. Close the butyrometer with the small rubber stopper and take care that
the big stopper is not pressed out. Then mix contents thoroughly by inverting the
butyrometer ten times
6. Centrifuge for 5 min in the Gerber centrifuge at normal speed of 1100-1200 rpm, then
place the butyrometer in the water bath at 65°C for 5 min, shake and repeat centrifugation
for 5 min
Place the butyrometer in the water bath at 65°C for 5 min and remove the small rubber stopper,
adjust the fat column so that the bottom of the fat column is on the zero mark on the butyrometer
and read the fat percentage
[Source: Modified from the method of determination of fat in cheese, Bureau of Indian Standard
IS: 1224 (Part II) – 1977]
145
11.4 Determination of Moisture in Paneer
Aim: To determine moisture content in paneer.

Principle: The moisture content of paneer is measured by drying off water at 100±1°C.
Apparatus: Flat bottom moisture dish with cover (of nickel, stainless steel, porcelain or other
suitable material not affected by boiling water, 7 to 8cm diameter and not more than 2.5cm deep,
provided with a short glass stirring rod having a widened flat end), desiccator, water bath, hot air
oven (100±1°C), balance (at least two decimals).
Procedure:
1. Weigh accurately about 2g±0.1mg of the material into a clean dish previously dried and
weighed along with glass rod
2. Mix the material uniformly with 4ml of hot, distilled water with the help of a small glass
rod (weighed earlier)
3. Wash off the particles of material adhering to the glass rod by pouring additional 1ml hot
distilled water
4. Heat the dish containing the material after uncovering in the electric oven maintained at
100±1°C for about 4 hrs
5. Cool the dish in the desiccator and weigh with the coven on
6. Repeat the process of drying, cooling and weighing at 30 min interval until the difference
between the two consecutive weights is less than 1mg
7. Record the lowest weight
Calculation
(M1 − M 2 )
Moisture % in paneer = ×100
(M1 − M)
Where,
M1 = Mass in g of the dish with sample before drying
M2 = Mass in g of the dish with the sample after drying
M = Mass in g of the empty dish
146
Chapter 12
CHHANA
147
148
12.1 Determination of Sensory Quality of Chhana
Aim: To determine sensory quality of chhana.

Principle: Chhana is the solid product obtained by the acid coagulation of milk at about its
boiling point followed by subsequent removal of whey. The coagulant used shall be sour (whey),
lactic acid or citric acid. Chhana shall not contain ingredient foreign to milk.
Apparatus: Cutting knife, glass dish
Milk sample: Only fresh, sweet, clean milk, free from colostrums and in every way fit for human
consumption shall be used. The milk shall be free from adulterants, preservatives and any matter
foreign to milk.
Procedure:
1. Appearance and color: The material shall be free from signs of fat or water seepage or
both, and moldiness. The color of the material shall be yellow to white
2. Odor and flavor: The material shall have a pleasant curdy flavor (slightly acidic flavor is
tolerated). It shall be free from objectionable flavors and odors
3. Texture and consistency: The material shall be of good texture and uniform consistency.
It shall be free from coarseness
4. The material shall be manufactured and packed in equipment and premixes maintained
under hygienic conditions. It shall also be stored and distributed under hygienic
conditions
- 148 -
12.2 Determination of Moisture of Chhana
Aim: To determine moisture of chhana.

Principle: Moisture content is determined by drying off the sample in oven.
Apparatus: Flat bottomed dishes (7-8cm in diameter and not more than 2.5cm deep provided
with a short glass stirring rod having a widened flat end), sand (prepared by digestion with
concentrated hydrochloric acid, followed by thorough washing with water, then drying and
ignition to dull red), well-ventilated oven (capable of operating at 102°C air temperature).
Procedure:
1. Heat the necessary number of metal dishes, each dish containing about 20g of prepared
sand and a stirring rod, in the oven for about one hour
2. Allow to cool in an efficient desiccator for 30 to 40 min
3. Weigh accurately about 3g of the prepared sample of cheese into a dish
4. Saturate the sand by the careful addition of a few drops of distilled water, and thoroughly
mix the wet sand with the cheese by stirring with the glass rod, smoothing out lumps and
spreading the mixture over the bottom of the dish
5. Place the dish on a boiling water bath for 20 to 30 min, then wipe the bottom of the dish
and transfer it with the glass rod, to the well-ventilated oven at 102±1°C.
6. The bulb of the oven control thermometer shall be immediately above the shelf carrying
the dish
7. Dishes shall not be place near the walls of the oven and should be insulated from the shelf
by suitable silica or glass supports
8. After 4 hrs, remove the dish to an efficient desiccator, allow to cool as before and weigh
9. Replace the dish in the oven for a further period of one hour at 102±1°C, remove to the
desiccator, and cool and weigh again
10. Repeat the process of heating, cooling and weighing for one hour till consecutive weights
agree to within 0.5mg
From the loss in weight observed, calculate the percentage by weight of moisture.
149
12.3 Determination of Fat Content in Chhana
Aim: To determine fat content in chhana.

Principle: The fat in chhana is in the form of emulsion. This emulsion is broken with sulfuric
acid and fat separation is facilitated by amyl alcohol.
Apparatus: Fat extraction apparatus (fat extraction tube or a Mojonnier fat extraction tube).
Electrically heated oven (set at 98 to 100°C)
Chemicals: Hydrochloric acid (sp. gr. 1.125), ethyl alcohol (95-96% by volume), light petroleum
ether (boiling range 40-60°C), diethyl ether (sp. gr. 0.720, peroxide-free). Diethyl ether may be
maintained free from peroxide by adding wet zinc foil (approximately 80cm2/liter, cut in strips
long enough to reach at least half way up to the container) that has been completely immersed in
dilute acidified copper sulfate for 1 min and subsequently washed with water.
Preparation of sample: Samples shall be prepared for chemical analysis by passing them
quickly through suitable grater by grinding them quickly in a mortar and returning them to the
sample container or by cutting them into small pieces with a sharp knife in the container.
Procedure:
1. Weigh accurately 1.0 to 1.5g of the prepared sample into the extraction tube
2. Add 10ml of the hydrochloric acid and boil gently with shaking, either over a flame or in
a boiling water bath, until all solid particles are dissolved
3. Cool the tube in running water
4. Add 10ml of alcohol and again mix
5. Complete extraction of the fat is dependent on satisfactory mixing at each stage
6. Proceed further in fat extraction apparatus as in determination of fat by rose Gottlieb
method and proceed with the blank determination using the same method
Deduct the value of weight of blank with value of weight of sample test. Note the actual fat
content.
[Source: Bureau of Indian Standards IS: 5162-1969, IS: 2785-1964, IS: 1479 (Part II) – 1961]
150
151
Chapter 13
YOGHURT
152
153
13.1 Determination of Sensory Quality of Fermented Milk Product,
Yoghurt
Aim: To determine sensory quality of fermented milk product, yoghurt.

Principle: Judging the quality of yoghurt by its taste and smell requires considerable skill, which
useful for preliminary assessment of milk quality and handling practice. Yoghurt is smelled and
In the case of large containers, a sample of at least 500g should be taken. Before evaluation it is
recommended that the samples be kept at the temperature of 4±2°C. For the evaluation of
appearance, the samples should, if possible, be presented in the original packages. Fo the
evaluation of consistency and flavor, individual portions of at least 50-100g should be available
for each assessor. During the evaluation the samples should have a temperature of 12±2°C.
Procedure:
The sensory evaluation of fermented milk products, yoghurt should be carried out in relation to
appearance, consistency and flavor.
1. The evaluation of appearance can be carried out simultaneously by the whole panel with
separate scoring; involving the filling and the surface of the product, color, visible purity,
presence of foreign matters, spots of mold, seepage of whey and phase separation. The
evaluation is made by examination in the opened package, if necessary by pouring out the
product from the package.
2. The evaluation of consistency involves thickness, stickiness and coarseness. Evaluation
can be made by blending the product with a (black) spoon before malaxating the sample
in the mouth. The defects mentioned in this method are applicable to fluid products as
well as fermented products with high viscosity.
3. The evaluation of flavor is make by smelling and tasting the product
- 152 -
Different categories of fermented milk products should be evaluated in accordance with the
International Standard.
International table of Fermented Milk Product Quality Terms
1. Appearance 3. flavor
Overfilled Watery
Underfilled Flat
Shrunken Bitter
Heterogeneous surface Cooked
Untypical color Burnt
Brown color Smoked
Non-uniform color Oily
Marbled Chemical flavor
Air bubbles Feed flavor
Foreign matter Light induced flavor
Separation of whey Defective aromatization
Mold Defective ingredients
Yeast Cheesy
Separation of phases Malty
Sedimentation Metallic
Lack of or poor distribution of ingredients Musty
Oxidized
2. Consistency Acid
Setting Sharp
Lumps or flakes Harsh
Dripping Sour
Uneven Tallowy
Gritty Yeasty
Sticky Rancid
Too thick Astringent
Too fluid Unclean
Ropy/Stringy Stale/Old
Dried Too sweet
Brittle Too salty
Gelatinous Soapy, alkaline
- 153 -
13.2 Determination of Fat Content in Yoghurt
Aim: To determine fat content in yoghurt.

Principle: When sulfuric acid is added to milk, the proteins will be digested thus destroying the
Addition of amyl alcohol has the function of separation fat from other components.
amyl alcohol, 10ml pipette, butyrometer stoppers, butyrometer stand.
colorless.
Procedure:
1. Measure 10ml of clear Gerber sulfuric acid into the butyrometer that should be clean and
absolutely free from fat residues
2. Pipette out 10ml of a well-mixed sample and transfer it carefully to the butyrometer
without allowing it to mix with the acid
3. Measure 2ml amyl alcohol into the butyrometer and proceed as in the case of milk except
that the butyrometer should be shaken and centrifuge immediately in order to avoid plug
formation
4. The butyrometer should be centrifuged twice with intermediate heating of the butyrometer
in a 65°C water bath for 5 min
The percentage of fat in the given milk sample of yoghurt is given by:
Fat content = Fat content reading ×1.075
[Source: Quality Handbook, Dairy Development Corporation]
154
13.3 Determination of Titrable Acidity in Yoghurt (Potentiometric
Method)
Aim: To determine the titrable acidity in yoghurt.

Principle: The titrable acidity of yoghurt is the number of milliliters of 0.1mol/liter sodium
hydroxide solution required to titrate a quantity of yoghurt to the pH of 8.3. It is conventionally
expressed as grams of lactic acid per 100g of product.
Suspension of a test portion in water is used for acidity test. It is a potentiometric titration with
0.1 mol/liter solution of sodium hydroxide to the pH of 8.30.
Apparatus: Analytical balance, pH meter equipped with a measuring and a reference electrode,
calibrated by means of two buffer solutions of pH 7 and 9 respectively, spoon, spatula.
Chemicals: Deionized water or distilled water, free from carbon dioxide by boiling for 10 min
before use, sodium hydroxide, standard volumetric solution, 0.1±0.0002 mol/liter, carbonate free.
Procedure:
1. Weigh approximately 10g of the prepared test sample to the nearest 10mg into a beaker of
capacity 50ml
2. Add approximately 10ml of water and mix
3. Insert electrodes of the pH meter into the suspension and ensure that they are properly
immersed
4. Titrate the contents of the beaker, whilst stirring, using the sodium hydroxide solution to a
pH of 8.30
5. Record the volume in milliliters of sodium hydroxide solution used to the nearest 0.05ml
Calculation
The titrable acidity, expressed as grams of lactic acid per 100g of product is equal to:
V × 0.9
M
Where,
V = Volume in ml of the sodium hydroxide solution used for the titration
M = Mass in g of the test portion
0.9 = Conversion factor for lactic acid
[Source: International Dairy Federation, IDF Standard – 150: 1991]
155
13.4 Determination of Percent Acidity I Yoghurt
Aim: To determine the acidity of yoghurt.

Principle: Same as in milk.
Apparatus: Glass burette (50ml capacity with 0.1ml graduation), porcelain dish (white-bottomed
and 100ml capacity), 1ml and 10ml pipettes, stirring rod.
Chemicals: 0.1N sodium hydroxide, 1% phenolphthalein indicator.
Procedure:
1. Stir the sample of yoghurt and measure with a pipette 10ml of yoghurt into the beaker.
With the same pipette add 20ml of distilled water
2. Add 1ml of 0.5% phenolphthalein and titrate with 0.1N sodium hydroxide solution
3. Note the volume of 0.1N sodium hydroxide solution used
ml of 0.1N NaOH × 0.009
% Acidity in yoghurt = ×100
10ml of sample
156
13.5 Determination of Total Solids in Dahi and Yoghurt
Aim: To determine total solids content in dahi and yoghurt by gravimetric method.
Principle: Drying of sour milk and dahi for the determination of solids by the usual method
results in loss of volatile constituents like acetic acid, acetaldehyde, etc. Some of these could be
fixed and the estimation could then be done with a fair accuracy by the following method.
Apparatus: Shallow bottom dish either made of stainless steel, nickel, porcelain or silica (7-8cm
diameter and 2.5cm deep and provided with easily removable lid), hot air oven at 98-100°C,
water bath with boiling water, analytical balance, desiccator, tongs.
Chemicals: 0.1N strontium hydroxide, 0.5% phenolphthalein.
Procedure:
1. Weigh accurately 10g of sample in a previously weighed dish
2. Add 1ml of phenolphthalein indicator and titrate with 0.1N strontium hydroxide to faint
pink color
3. Note the exact amount of hydroxide
4. Calculate the requirement of strontium hydroxide/g sample in order to neutralize the
sample
5. Weigh accurately about 5g of the sample in dish. Weight = W1
6. Add the calculated quantity, Vml, of strontium hydroxide to neutralize the acids present
in this weight of sample without adding phenolphthalein and proceed as in the method for
the determination of total solids in milk. Final weight of solids obtained = W2
W2 − (V × 0.00429) ×100
Percent total solids in dahi or yoghurt =
W1
Where, 0.00429 is a conversion factor from ml to g of 0.1N Sr(OH)2
157
13.5 Determination of Total Solids Content in Yoghurt
Aim: To determine total solids content in yoghurt.

Principle: Total solids content is the mass fraction of substances remaining after completion of
the heating process. This is achieved by evaporation of the water from a test portion in the
presence of zinc oxide at a temperature of 102±2°C in a drying oven.
Apparatus: Analytical balance, desiccator, drying oven, flat bottom dishes, boiling water bath,
stirring rods, homogenizer (for homogenizing fruit yoghurt) or blender, spatula.
Chemicals: Zinc oxide, distilled water.
Procedure:
1. Heat an open dish containing approximately 2g of zinc oxide together with its lid and a
stirring rod placed on top of the lid, in the oven for 1 hr
2. Place the lid (with the stirring rod on top) on the dish, immediately transfer to the
desiccator, allow to cool to room temperature and weigh the dish with the lid and rod
3. Move the zinc oxide to one side of the prepared dish by tilting, place on the clear space
approximately 1.0g of the prepared test sample, replace the lid on the dish with the
stirring rod on top and weigh the dish
4. Add 5ml of water to the dish. By mean of the stirring rod, thoroughly mix the diluted test
portion and the zinc oxide. Spread the mixture evenly over the bottom surface of the dish.
Leave the stirring rod with the flattened end used for mixing resting in the mixture and the
other end resting on the rim of the dish
5. Heat the dish on the boiling water bath or steam bath ensuring that the maximum possible
surface area of the bottom of the dish is exposed to the steam. Continue the heating
process for approximately 30 min with frequent mixing of the contents of the dish during
the early stages of drying, so as to obtain maximum evaporation of liquid
6. Remove the dish from the water bath or steam bath and place it in the drying oven for 3
hrs
7. Allow the dish to cool in the desiccator and weigh it
8. Again heat the dish and the contents together with its lid for a further 1 hr. Cool it again in
the desiccator and weigh it again
9. Repeat the process of reheating and weighing until the difference in mass between two
successive weighing does not exceed 1mg. Record the lowest mass
158
The total solids content, expressed as a percentage by mass is equal to:
m2 − m0
× 100 − 0.1a
m1 − m0
Where,
m0 = The mass in g of the dish (including zinc oxide, lid, and the stirring rod)
m1 = The mass in g of the dish (including zinc oxide, lid, and stirring rod) + the test
portion
m2 = The mass in g of the dish, lid, stirring rod and the dried test portion (including zinc
oxide)
a = The mass in g of the lactic acid as obtained in the titrable acidity test
[Source: International Dairy Federation IDF Standard – 151: 1991]
159
Chapter 14
CHEESE
160
161
14.1 Determination of Sensory Quality of Cheese
Aim: To determine sensory quality of cheese

Principle: Judging the quality of cheese by its taste and smell requires considerable skill, which
useful for preliminary assessment of milk quality and handling practice. Cheese is smelled and
Apparatus: Petri dish, cheese trier
In the case of large cheeses, an adequate sample should be taken by a cheese trier (figure 8) or by
cutting a sector. In the case of retail packaging, an adequate number of packages should be made
available. Before evaluation it is recommended that the sample be kept at the temperature
mentioned on the packages or laid down in national legislation. During the evaluation, the
samples should have a temperature of 14±2°C. For special cheeses other temperatures may be
chosen, within the limit of ±2°C.
Figure 8: Sampling of a loaf-shaped cheese from the side by means of a trier
- 161 -
Figure 9: Sampling a loaf-shaped cheese by cutting out 1 sector
Procedure:
1. The sensory evaluation should be carried in relation to:
Appearance – exterior
Appearance – interior
Consistency (body and texture)
2. The evaluation of the appearance – exterior (for example shape, rind/surface) is made by
visual examination of the whole cheese
3. The evaluation of the appearance – interior (for example openings, color) is made by
visual examination of the cut surface or a core sample of the cheese
4. The evaluation of the consistency is carried out using defined pieces of cheese obtained
by cutting or from a core sample, by bending followed by pressing and rubbing between
the forefinger and thumb as well as by chewing
5. The flavor is evaluated by smelling the cut cheese or core sample. Defined pieces of
cheese are chewed and salivated to evaluate the taste.
Different categories of cheese should be evaluated in accordance with the International Standard.
- 162 -
International Table of Cheese Quality Terms
1. Appearance – Exterior 2. Rind/Surface
Too flat Thick
Too high Thin
Deformed Rough
Vaulted (blown) Discolored
Concave Cracked
Oblique Dry
Soiled Wet
Dirty Rotten
Fatty
3. Appearance Smeary
Openings: Wrinkled
No holes Speckled
Too few Spots of mold
Too many Too much smear
Pin holed Too little smear
Too large Too little mold
Blown Irregular mold
Not typical Incorrect mold
Collapsed Mold under covering
Distorted Smear under covering
Uneven Holes
Glossy openings Corroded
Nesty openings
Cracks 4. Consistency/Body and Texture
Hoop side moldy Hard
Unevenly moldy Firm
Foreign mold Coarse
Spots of putrefaction Lumpy
Foreign material Curdy
Many holes near the surface Crumbly
Granular (grainy)
Color: Gritty
Discolored Mealy
Uneven color Chalky
Streaky Short
Marbled Brittle
Speckled Tough
Mottled Sticky
Pale/Dull Long
Bleached near the surface Springy
Red color near the surface Smooth
Soft
Hoop side soft
163
5. Flavor 4. Consistency/Body and Texture (continued)
Unclean Pasty
Foreign flavor Smeary
Uncharacteristic Thin (watery)
Rancid Dripping
Tallowy Spongy
Soapy Layered
Putrid Uneven
Ammoniacal
Flat
Sharp
Sweet
Acid
Bitter
Harsh
Salty
Metallic
Chemical
Sulfide
Stale
Burnt
Musty-flat
Musty
Fermented
Yeasty
Butyric acid
Feedy
Malty
Fruity
Weedy
Cooked
[Source: International Dairy Federation IDF Standard, 99C: 1997]
164
14.2 Determination of Salt Content in Cheese
Aim: To determine salt content in cheese

Principle: The salt, or sodium chloride, content of cheese is variable. In testing cheese for salt, a
direct titration approach cannot be used because of the interfering nature of proteins. A modified
Volhard test is employed in which cheese is digested by being boiled with a measured excess of
0.1N AgNO3 solution in the presence of nitric acid, nitrobenzene and potassium permanganate
prior to titration of the excess silver ions.
The principle in the salt test is that the amount of standard 0.1N silver nitrate (AgNO3) added in
excess reacts with the available sodium chloride as:
NaCl +AgNO3 → NaNO3 + AgCl
Then the AgNO3 not used up in the first reaction is titrated back in the presence of a ferric
ammonium sulfate indicator with 0.1N potassium thiocyanate (KSCN) as:
AgNO3 + KSCN → AgSCN + KNO3
When all of the remaining silver nitrate reacts with KSCN, the ferric indicator reaches a reddish
brown end point. Once the amount of excess AgNO3 is determined, it is a simple chore to find,
by subtraction and use of chemical equivalents, the amount which reacted with the salt (NaCl).
Nitrobenzene is added to coat AgCl precipitate (thus preventing it from providing Ag ions that
will react with KSCN) and to create a more distinct end point capable of more easily detectable
0.03% salt in cheese. It eliminates the tedious filtration step without affecting either the
sensitivity or accuracy of the method.
Apparatus: 300ml Erlenmeyer flask, burette, volumetric pipette
Chemicals: 0.1N AgNO3 solution, conc. HNO3 free from halogen, 5% potassium permanganate
solution (KMnO4), nitrobenzene, ferric ammonium sulfate (saturated solution), 0.1N potassium
thiocyanate solution (KSCN).
Procedure:
1. Weigh 3g of cheese into a 300ml Erlenmeyer flask
2. Then add 25ml of 0.1N AgNO3 from a burette or volumetric pipette. This is more than
adequate to combine with the available chloride
3. Introduce 10ml of halogen-free HNO3 and 50ml of distilled water into the flask
4. Heat the mixture in the flask to boiling under a hood
5. As it boils, add 15ml of fresh 5% potassium permanganate (KMnO4) solution in three 5ml
portions. Add each successive 5ml portion after the porpoise color changes to yellow
6. Cool the digested yellowish solution to room temperature and add 2ml of nitrobenzene
and 2ml of saturated ferric ammonium sulfate separately with pipettes
7. Titrate the contents directly with 0.1N KSCN to brick red end point, while swirling the
flask. Run a reagent blank with 3g sucrose to destroy the excess permanganate
165
Calculation:
N 58.443 
 KSCN × ( KSCN B − KSCN S) × 1000 
V V
% Salt (NaCl) =   × 100

 Wt. of cheese taken 
 
Where,
V
KSCN B = Volume (ml) of KSCN equivalent to 25ml AgNO3 in the blank titration
V
KSCN S = Volume (ml) of KSCN equivalent to the unreacted silver nitrate in the silver
nitrate in the sample titration
N
KSCN = Normality of KSCN solution
[Source: Laboratory Guide in Dairy Chemistry Practical, FAO]
166
14.3 Determination of Salt Content in Cheese
Aim: To determine salt content in cheese

Principle: The salt added to the cheese while manufacturing can be titrated with silver nitrate
using potassium chromate as indicator thus giving the actual salt content.
Apparatus: 250-ml conical flask, burette, water bath at 50°C.
Chemicals:
0.1N silver nitrate solution
1% potassium chromate indicator
4N nitric acid: Add 500ml distilled water in a 1000ml measuring flask, add 280ml concentrated
nitric acid and fill to the mark with distilled water
0.5N sodium citrate solution: 147g trisodium citrate (2H2O) is dissolved in a 1000ml measuring
flask, 500-600ml distilled water is added and 3-4 droplets of 1% phenolphthalein is added and a
10-20% NaOH solution is added until change from colorless to pink, water is then filled up to
1000ml
Procedure:
1. Weigh by difference accurately about 3g of cheese into the conical flask. Weight of
cheese = W
2. Add 20ml citrate solution and 80ml water at 55°C. An empty conical flask is filled with
the same chemical but without cheese, this giving a blank sample
3. Place the conical flasks in a water bath at 50°C for 1 hr. Shake once in a while. After one
hour the cheese will normally be dissolved
4. While stirring add first 10ml of the 4N nitric acid and then add 1ml of potassium
chromate indicator and titrate using the 0.1N silver nitrate solution until a chocolate red
color persists. Note down the volume V in milliliters of silver nitrate solution used. Also
titrate the blank sample; V0 is the volume in milliliters of 0.1N silver nitrate used for the
blank sample
(V − V0 ) × 0.1N
% Salt in cheese = × 5.84
W
5.84 is the conversion factor from mole of silver nitrate to gram of salt multiplied by 100.
167
14.4 Determination of Chloride Content in Cheese
Aim: To determine chloride content in cheese.

Principle: This international standard specifies a potentiometric titration method for the
determination of the chloride content of cheese and processed cheese products. The method is
applicable to all cheeses and processed cheese products containing more than 0.2% (m/m)
chloride ion. Chloride content of cheese and processed cheese products is defined by the mass
faction of substances determined by the procedure specified below as a percentage by mass of
chloride ion or sodium chloride or any other chloride. A suspension of test portion is prepared in
water. Acidification with nitric acid and subsequent potentiometric titration of chloride ion with
standard volumetric silver nitrate solution gives the amount of chlorides in cheese.
Apparatus: Device for grinding or grating cheese (capable of being easily cleaned), analytical
balance, blender, potentiometer (provided with measuring electrode suitable for the determination
of chloride, for example a silver electrode, and a reference electrode, for example a mercury (I)
sulfate), vessel suitable for blending and titrating, graduated cylinders (capacities 50 and 100ml),
burette graduated in 0.1ml (capacity 50ml) or an automatic plunger burette (preferably made of
brown glass), stirrer.
Chemicals: All reagents shall be of analytical grade. The water used shall be distilled water or
water of at least equivalent purity.
Silver nitrate (0.08 to 0.12 mol/liter): Dissolve 13.6 to 20.4g of silver nitrate in water which is
practically free from carbon dioxide and dilute to 1000ml. standardize the solution against
sodium chloride, which has previously been dried at 300°C, expressing the concentration of
silver nitrate solution to four decimal places. Store the solution away from direct sunlight.
Nitric acid: Approximately 4 mol/liter
Procedure:
1. Weigh to the nearest 0.001g, 2 to 5g of sample into the vessel
2. Add 30ml of water at about 55°C. Suspend the test portion using a blender. Rinse the
blender with approximately 10ml of water, collecting the reference electrode into the
suspension
3. Titrate the contents of the vessel with the standard volumetric silver nitrate solution from
the burette, stirring continuously, until the end point has nearly been reached
4. Titrate continuously until reaching the end point, which corresponds to the maximum
potential difference observed between two successive equal additions (of about 0.05ml) of
the silver nitrate solution
5. Carry out a blank test using the reagents but omitting the test portion
168
Calculation
Calculate the chloride content, as a percentage by mass, by means of the formula:
( V1 − V0 ) × C × f
m
Where,
V0 = volume in ml of the standard volumetric silver nitrate solution used in the blank test
V1 = volume of the standard volumetric silver nitrate solution used in the determination
C = the actual concentration (moles/liter) of the standard volumetric silver nitrate solution
m = mass in g of the test portion
f = factor expressing the results as a percentage of any chloride
The numerical values are, for example:
f = 3.55 for expression as % Cl
f = 5.84 for expression as % NaCl
f = 7.46 for expression as % KCl
Report the result to the second decimal place.
[Source: International Dairy Federation IDF 88A: 1988]
169
14.4 Determination of Total Solids Content in Cheese (Reference
Method)
Aim: To determine total solids content in cheese.

Principle: The total solids content is the mass remaining after completion of the heating process.
The total solids content is determined by evaporating the water from the sample in the presence
of sand at a temperature of 102°C in a drying oven.
Apparatus: Analytical balance, desiccator with efficient drying agent, drying oven, flat bottom
dishes, short glass stirring rods, quartz sand or sea sand.
Procedure:
1. Heat a dish containing approximately 25g of sand with its lid alongside and a stirring rod
on top of the lid, in the drying oven at 102°C for 1 hr
2. Place the lid (with the stirring rod on top) on the dish, immediately transfer the dish to a
desiccator, allow to cool for at least 45 min, and weigh the dish, with lid and rod. Weight
= Wg±0.1g
3. Tilt the sand to one side of the dish, place on a clear space about 3g of the prepared
sample, replace the lid with the stirring rod on top and weigh the dish. Weight = W1
4. Thoroughly mix together the rest portion and the sand and spread the mixture evenly over
the bottom of the dish. Leave the stirring end of the rod in the mixture with the other end
resting on the rim of the dish
Note 1: Mixing of sand and hard cheeses may be facilitated by adding sufficient water
(approximately 3ml) to saturate the sand.
Note 2: With cheeses which melt to a horn-like mass at a temperature of 102°C, it is
recommended that the dish containing the crushed cheese mass shall first be heated on
water or steam bath, exposing maximum surface of dish bottom to direct steam. The
contents of the dish shall be thoroughly mixed with glass rod from time to time to prevent
the formation of a hardened surface layer
5. Lay the stirring rod flat inside the dish, dry the bottom of the dish and heat the dish, with
its lid along side, in the drying oven at 102°C for 2 hrs
6. Place the lid on the dish and allow the dish to cool in the desiccator and then weigh it
7. Heat the dish and lid as described in 5 but for 1 hr, place the lid on the dish, and allow the
dish to cool in the desiccator and then weigh it. Weight = W2
8. Repeat the operations described in 7 until the difference in mass at 2 successive
weighings is not more than 0.5mg. Record the lowest mass as the mass of the dish at this
stage
170
Calculation
W2 − W
Total solids of the sample = ×100
W1 − W
[Source: International Dairy Federation IDF Standard 4A: 1982]
171
14.6 Determination of Fat Content in Cheese
Aim: To determine fat content in cheese by Van Gulik method.

Principle: The fat in cheese is in the form of an emulsion. This emulsion is broken with sulfuric
Apparatus: Gerber centrifuge, cheese butyrometer 40% scale, stoppers for butyrometer, pipettes
(10ml and 1ml), seamless funnel and stopper, weighing balance (at least two decimals), water
bath (65°C), small scoop of suitable material, glass rod, grater or pestle and mortar.
Chemicals: Gerber sulfuric acid (sp. gr. 1.53 to 1.60; prepare by adding 670ml of Gerber sulfuric
acid in 330ml of distilled water), amyl alcohol.
Procedure:
1. Grind samples of cheese
2. Weigh into stoppered funnel 3.00g of ground cheese
3. Transfer 10ml of sulfuric acid into butyrometer by means of a 10-ml pipette for sulfuric
acid or the automatic measure, taking care not to wet the neck of the butyrometer with the
sulfuric acid.
4. Add gently from the wash bottle sufficient warm water (30 to 40°C) to form a layer of
about 6mm deep on top of the acid, allowing the water to flow down the side of the bulb
5. Insert the neck of the funnel containing 3g of cheese into the neck of the butyrometer.
Withdraw the stopper from the neck of the funnel and transfer all the cheese to the
butyrometer with the aid of the glass rod or spatula
6. Measure 1ml of amyl alcohol into the butyrometer by means of the 1-ml pipette for amyl
alcohol or the automatic measure. Do not wet the neck of the butyrometer with alcohol
7. Add warm water (30 to 40°C) from wash bottle until the butyrometer is filled to about
5mm below the shoulder
8. Close the neck of the butyrometer firmly with the stopper without disturbing the contents.
When a double-ended stopper is used, screw it in until the widest par is at least level with
the top of the neck. When a lock stopper is used, insert it until the rim is in contact with
the neck of the butyrometer
9. Shake the butyrometer carefully, without inverting it, until the contents are thoroughly
mixed, the sample is dissolved and no any particles are seen in the liquid. Then invert the
butyrometer for a few times to mix the contents thoroughly
10. Transfer the butyrometer quickly, with the bulb uppermost, into a water bath having a
temperature of 65°C and leave it there for not less than 3 min and not more than 10 min
11. Transfer it to the centrifuge; centrifuge it at the maximum speed of 1100-1200 rpm for 5
min
172
Transfer it into a water bath having a temperature of 65°C and allow it to stand in the water bath
for at least 3 min. before taking a reading, adjust the position of the fat column to bring the lower
end of the column on to a main graduation mark zero and read the fat percentage.
[Source: Bureau of Indian Standard IS: 1224 (Part II) – 1977]
173
14.7 Determination of pH of Cheese
Aim: To determine pH of cheese.

Principle: pH may be defined as the negative logarithm of the hydrogen ion concentration. The
measurement of pH is finding increasing use in the dairy industry since it provides in many cases
a more meaningful measurement than titrable acidity. pH values below 7 are acid while above 7
are alkaline, pH 7 being considered neutral. pH is a measure of active acidity.
Apparatus: pH meter (pH meter is a device capable of measuring small differences in voltage
developed between two electrodes immersed in the sample. The electrical changes are converter
into direct pH readings. The pH meter is standardized by the use of standard buffer solution
which should be carefully handled to prevent contamination. For fresh cheese, special cheese
electrodes are available), beaker, grater.
Chemicals: Standard buffer solutions for standardizing the pH meter (for example pH 4 and 9)
Procedure:
1. Grate the cheese sample and pour into a clean, dry beaker
2. To grated cheese sample add a few drops of distilled water
3. Carefully press electrodes into the cheese-containing beaker and determine the pH of
cheese directly into pH meter
The pH of the cheese sample comes on the digital screen of the pH meter
[Source: Laboratory Guide in Dairy Chemistry Practicals, FAO]
174
14.8 Determination of Nitrogen Content in Cheese
Aim: To determine nitrogen content in cheese by Kjeldahl method

Principle: Nitrogen content in cheese is the mass fraction of substances determined according to
the procedure specified in this method. It is expressed as a percentage by mass.
The test portion is digested with a mixture of concentrated sulfuric acid and potassium sulfate,
using copper (II) sulfate as a catalyst to thereby convert organic nitrogen present to ammonium
sulfate. Addition of excess sodium hydroxide to the cooled digest liberates ammonia. Distillation
of this ammonia into an excess of boric acid solution and titration with hydrochloric acid solution
gives the ammonia content resulting from the digestion. The amount of ammonia is now used to
calculate the amount of nitrogen in the sample.
Apparatus: Water bath (maintained at 30±1°C), Kjeldahl flasks (500ml or 800ml capacity),
analytical balance (sensitivity 0.001g), boiling aids (glass beads of diameter approximately
5mm), burette or automatic pipette for delivering 1.0ml portions of the copper sulfate solution,
graduated measuring cylinders (50ml, 100ml and 500ml), distillation apparatus (made of
borosilicate glass or other suitable material to which a Kjeldahl flask can be fitted consisting of
an efficient splash head connected to an efficient condenser with straight inner tube and an outlet
tube attached to its end; the connecting tubing and stopper shall be close filling and preferably
made of neoprene), anti-bumping granules (for example, of fused alumina), conical flasks
(500ml, graduated at 200ml), burette (50ml).
Chemicals: All reagents shall be of recognized analytical grade. The water used shall be glass-
distilled or water of at least equivalent purity.
Potassium sulfate (K2SO4)
Copper solution: Dissolve 5.0g copper (II) sulfate pentahydrate (CuSO4.5H2O) in water and
dilute to the mark in a 100ml one-mark volumetric flask
Sulfuric acid: At least 98% m/m; density = 1.84g.ml at 20°C
Antifoaming agent
Sodium hydroxide solution: Low in nitrogen, containing 50g sodium hydroxide per 100g
solution)
Indicator solution: Dissolve 0.1g of methyl red and dilute to 50ml in 95% v/v ethanol. Dissolve
0.5g of bromocresol green and dilute to 250ml in 95% v/v ethanol. Mix 1 part of methyl red and
5 parts of bromocresol green solution (or combine both the solution) just before use (use only
fresh mixture)
Boric acid solution: Dissolve 40g of boric acid (H3BO3) in 1 liter of hot water, allow to cool, add
3ml of the indicator, mix and store in a borosilicate glass bottle
Hydrochloric acid standard: Strength = 0.1±0.0005 Mol/liter
Ammonium sulfate: Minimum assay 99.9% on dried material (immediately before use, dry the
ammonium sulfate at 102±2°C for not less than 2 hr and cool to room temperature in a
desiccator)
175
Tryptophan or lysine hydrochloride: Minimum assay 99%
Sucrose: Nitrogen content not more than 0.002% m/m
Preparation of sample: Warm the sample to 38±1°C by means of water bath. Gently mix the
sample immediately prior to weighing the test portion.
Procedure:
A: Test portion and pretreatment
1. To the Kjeldahl flask add some boiling aids for example three glass beads, 15g of the
potassium sulfate, 1.0ml of copper sulfate solution, approximately 5g of the prepared test
sample, weighed to the nearest 0.1mg, and 25ml of sulfuric acid, using the acid to wash
down any copper sulfate solution, potassium sulfate or test portion left on the neck of the
flask
2. Gently mix the contents of the flask
B: Determination
Digestion
1. Carry out digestion using a digestion apparatus the heater source of which can be adjusted
to bring 250ml of water (including boiling aids) with an initial temperature of 25°C to a
rolling boil in approximately 5-6 min
2. To determine the maximum heater setting to be used during digestion, preheat the source
at the heater setting being evaluated
3. In the case of gas heater the preheat period shall be 10 min and for an electric heater the
preheat period shall be 30 min
4. Determine the heater setting that brings water from 25°C to a rolling boil in 5-6 min for
rack of the heaters. This is the maximum heater setting to be used during digestion
5. Heat the Kjeldahl flask on the digestion apparatus using a heater setting low enough such
that charred digest does not foam up the neck of the Kjeldahl flask
6. Digest at this heat setting for at least 20 min or until white fumes appear in the flask
7. Increase the heater setting to half the maximum setting determined above and continue
heating for 15 min
8. At the end of 15 min increase the heat to maximum setting determined above
9. After the digest clears (clear with light blue-green color) continue boiling for 1-1.5 hrs at
maximum setting. The total digestion time will be between 1.8-2.25 hrs
10. To determine the specific boiling time required for analysis conditions in a particular
laboratory using a particular set of apparatus, select a high-protein, high fat milk sample
and determine its protein content using different boil time (1-1.5 hrs) after clearing
11. The mean protein result increases with increasing boil time, becomes consistent and then
decreases when boil time is too long. Select the boil time that yields the maximum protein
result
12. At the end of the digestion, the digest shall be clear and free of undigested material
176
13. Allow the acid digest to cool to room temperature over a period of approximately 25 min
14. The cooled digest should be liquid or liquid with a few crystals at the bottom of the flask
15. After the digest has cooled to room temperature, add 300ml of water (for 800ml flasks acc
400ml of water) plus three or four drops of the antifoaming agent using the water to wash
down the neck of the flask
16. Mix the contents thoroughly and ensure that any crystals which separate out are dissolved
17. Add some anti-bumping granules
18. Allow the mixture to cool to room temperature prior to distillation
C: Distillation
1. Add 75ml of sodium hydroxide solution to the cooled, diluted digest by carefully pouring
the solution down the inclined neck of the flask so as to form a layer at the bottom of the
bulb of the flask
2. Immediately after the addition of sodium hydroxide solution to the Kjeldahl flask,
connect it to a distillation apparatus, the tip of whose condenser outlet tube is immersed in
50ml of the boric acid solution contained in a conical flask
3. Vigorously swirl the Kjeldahl flask to mix contents thoroughly and boil gently at first to
prevent excessive frothing
4. When 100ml to 125ml of distillate have been collected, lower the conical flask until the
tip of the condenser outlet tube is approximately 40mm above the 200ml flask
5. Continue distillation until irregular boiling (bumping) starts and then immediately stop
the heating
6. Disconnect the Kjeldahl flask and rinse the tip of the condenser outlet tube with a little
water, collecting the rinsings in the conical flask
7. The distillation rate shall be such that approximately 150ml of distillate are collected
when irregular boiling (bumping) starts and the volume of the contents of the conical
flask will be approximately 200ml
8. The efficiency of the condenser shall be such that the temperature of the contents of the
conical flask does not exceed 25°C during the distillation
D: Titration
1. Titrate the boric acid receiving solution with the standard volumetric hydrochloric acid
solution to the first trace of pink. Estimate the burette reading to 0.01ml
E: Blank test
1. Carry out a blank test following the procedure described above taking 5ml of water with
about 0.85g sucrose instead of the test portion
2. The blanks for sample materials other than milk should contain a mass of sucrose that will
consume approximately the same amount of acid during digestion as an average sample of
that type
177
Calculation of nitrogen content
The nitrogen content, expressed as a percentage by mass is equal to:
1.4007(Vs − Vb )M
W
Where,
Vs = the volume in ml of the standard volumetric solution of acid used in the
determination
Vb = the volume in ml of the standard volumetric solution of acid used in the blank test
M = the exact molarity to four decimal place of the standard volumetric solution of acid
W = the mass in g of the test portion
Round the result to the nearest 0.001%
Calculation of crude protein content
The crude protein expressed as a percentage by mass is obtained by multiplying the nitrogen
content by 6.38
Note: What is the use of lysine hydrochloride and tryptophan??? ---Basanta
[Source: International Dairy Federation IDF 20B: 1993]
178
14.9 Determination of Protein in Cheese
Aim: To determine protein content in cheese by Kjeldahl method.

Principle: The protein content in cheese is determined by acid-digestion (with conc H2SO4) to
give ammonium sulfate, degradation of the latter with excess NaOH to give NH3, entrapment of
NH3 in an excess of standard acid, and calculation of NH3 trapped by back titrating the acid with
standard NaOH. The NH3 content is be used to calculate the amount of nitrogen present in the
sample. The relation between nitrogen content and protein content in cheese is in turn used to
calculate the protein content.
Apparatus: Round bottom flask A of 100ml capacity fitted with a rubber stopper through which
passes one end of the connecting bulb tube B. The other end of the bulb tube B is connected to
the condenser C which is attached by means of a rubber tube to a dip tube D which dips into the
liquid contained in a beaker E of 250ml capacity.
Chemicals: Concentrated sulfuric acid (sp. gr. 1.84), copper sulfate, potassium sulfate or
anhydrous sodium sulfate (nitrogen-free), sodium hydroxide solution (dissolve 225g of sodium
hydroxide in 500ml of water), standard sodium hydroxide solution (0.1N), standard sulfuric acid
(0.1N), methyl red indicator (dissolve 1g of methyl red in 200ml of rectified spirit, 95% v/v).
Procedure:
1. Transfer carefully about 0.5g of accurately weighed sample to the Kjeldahl flask, taking
precaution to see that particles of samples do not stick in the neck of the flask
2. Add 25ml of concentrated sulfuric acid through the neck of the flask so that it washes the
material, if any sticking to the sides of the flask
3. Add about 0.2g copper sulfate into the flask. Place the flask in an inclined position. Heat
below the boiling point of the acid until frothing ceases
4. Add 10g potassium sulfate or anhydrous sodium sulfate. Increase heat until acid boils
vigorously and digest for 30 min after the mixture becomes clear and pale green or
colorless
5. Wash down with the minimum quantity of concentrated sulfuric acid, particles if any
sticking to the sides and continue digestion for 60-90 min
6. Cool the contents of the flask and transfer quantitatively to a 1000ml round bottomed
flask A and dilute to 250ml
7. Add with shaking a few pieces of pumice stones to prevent bumping
8. Add about 50ml of sodium hydroxide solution or more, carefully through the side of the
flask so that it does not mix at once with the acid solution but forms a layer below it
9. Assemble the apparatus taking care that the tip of the condenser extends below the surface
of a known quantity of standard sulfuric acid contained in the beaker E
10. Wash the dip tube D carefully so that all traces of condensate are transferred to the beaker
E; add two or three drops of methyl red indicator and titrate with standard sodium
hydroxide solution
179
Note: Where is the distillation done??? -----Basanta
11. Carry out a blank using all reagents in the same quantities and 0.5g sucrose in place of the
sample
Calculation
Protein is calculated by multiplying nitrogen content by the factor 6.38
8.93(B − A)N
Protein percent by weight =
W
Where,
B = volume in ml of standard sodium hydroxide solution used to neutralize the acid in the
blank determination
A = volume in ml of standard sodium hydroxide solution used to neutralize the excess of
acid in the test with the material
N = normality of standard sodium hydroxide solution
W = weight in g of the material taken for the test
180
Chapter 15
KHOA
181
182
15.1 Determination of Sensory Quality of Khoa
Aim: To determine sensory quality of khoa

Principle: Khoa is a heat coagulated milk product obtained by partial dehydration of milk of
buffalo, cow, sheep and goat or their admixture. Milk solids stably processed may also be used. It
shall not contain any ingredient foreign to milk except the addition of citric acid. The
determination of sensory quality involves testing by trained panelists for appearance and color,
odor and flavor, and texture and consistency.
Apparatus: Cutting knife, glass dish
Procedure:
Only fresh, sweet, clean milk, free from colostrums and in every way fit for human consumption
shall be used. The milk shall be free from adulterants, preservatives and any matter foreign to
milk.
1. Appearance and color: Khoa shall be free from signs of free fat, water seepage and
moldiness. The color of khoa shall be white to pale yellow, may be with a tinge of brown.
2. Odor and flavor: The khoa shall have its characteristic flavor. It shall be free from
objectionable flavors and odors
3. Texture and consistency: The khoa shall confirm to its type and designation. It shall
have uniform texture and consistency. The surface of khoa should not appear dry and nor
should it be sandy
4. The product shall be manufactured and packed in equipment and premises maintained
under hygienic conditions. It shall also be stored and distributed under hygienic
conditions
- 182 -
15.2 Determination of Titrable Acidity of Khoa
Aim: To determine titrable acidity of khoa

Principle: The titrable acidity of khoa is determined by titrating a warm emulsion (prepared by
grinding in water) against standard NaOH and expressing the result in terms of percentage lactic
acid (m/m)
Apparatus: Burette, porcelain dish, pipette
Chemicals: 0.1N standard NaOH solution, 0.1N standard HCl, phenolphthalein indicator
solution (0.5% in 50% ethyl acohol)
Procedure:
A: Direct Method:
1. Weigh accurately about 2g of khoa in a porcelain dish
2. Add 3g of hot water and render into a fine paste using a pestle and mortar
3. Dilute further adding 17ml hot water, washing off any adherents from the pestle
4. Add 1ml of phenolphthalein indicator solution and titrate against standard hydroxide
solution
5. Stir vigorously throughout; persistence of a slight pinkish tinge for 30 sec indicates the
end point
Calculation
9 × AN
Titrable acidity (as lactic acid), percent by mass =
M
Where,
A = volume in ml of standard sodium hydroxide required for titration
M = mass in g of material taken for the test
B: Indirect Method:
1. Weigh accurately about 2g of khoa in a porcelain dish
2. Add 3ml of hot water and render into paste using a pestle and mortar
3. Dilute by further adding 17ml hot water, washing off any adherents from the pestle
4. Add 10ml of standard sodium hydroxide solution to the diluted product and after adding
1ml of phenolphthalein indicator solution, titrate against standard hydrochloric acid. Stir
vigorously throughout
5. The end point is indicated when the pink color disappears completely
183
Calculation
(10 − V)
Titrable acidity (as lactic acid), % by mass = × 0.9
M
Where,
V = normality of standard hydrochloric acid
M = mass in g of the material taken for the test
[Source: Bureau of Indian Standard IS 4883-1968]
184
15.3 Determination of Moisture of Khoa
Aim: To determine moisture of khoa

Principle: Moisture is determined by drying off the sample in oven.
Apparatus: Flat bottomed dishes (7-8cm in diameter and not more than 2.5cm deep provided
with a short glass stirring rod having a widened flat end), sand (prepared by digestion with
concentrated hydrochloric acid, followed by thorough washing with water, then drying and
ignition to dull red), well-ventilated oven (capable of operating at 102°C air temperature).
Procedure:
1. Heat the necessary numbers of metal dishes, each dish containing about 20g of prepared
sand and a stirring rod, in the oven for about 1 hr
2. Allow to cool in an efficient desiccator for 30-40 min
3. Weigh accurately about 3g of the prepared sample of cheese into a dish
4. Saturate the sand by careful addition of a few drops of distilled water, and thoroughly mix
the wet sand with the cheese by stirring with the glass rod, smoothing out lumps and
spreading the mixture over the bottom of the dish
5. Place the dish on a boiling water bath for 20-30 min, then wipe the bottom of the dish and
transfer it with the glass rod, to the well-ventilated oven at 102±1°C
6. The bulb of the oven control thermometer shall be immediately above the shelf carrying
the dish
7. Dishes shall not be placed near the walls of the oven and should be insulated from the
shelf by suitable silica or glass supports
8. After four hours, remove the dish to an efficient desiccator, allow to cool as before and
weigh
9. Replace the dish in the oven for a further period of one hour at 102±1°C, remove to the
desiccator, cool, and weigh
10. Repeat the process of heating, cooling and weighing for one hour till consecutive
weighings agree within 0.5mg
From the loss in weight observed, calculate the percentage by weight of moisture.
[Source:
Bureau of Indian Standards IS: 2785-1964]
185
15.4 Determination of Fat Content in Khoa
Aim: To determine fat content in khoa.

Principle: The fat in khoa is in the form of an emulsion. This emulsion is broken with sulfuric
Apparatus: Fat extraction apparatus (fat extraction tube or a Mojonnier fat extraction tube),
electrically heated oven (set at 98-100°C).
Chemicals: Hydrochloric acid (sp. gr. 1.125), ethyl alcohol (95-96% by volume), light petroleum
ether (boiling range 40-60°C), diethyl ether (sp. gr. 0.720, peroxide-free). Diethyl ether may be
maintained free from peroxide by adding wet zinc foil (approximately 80cm2/liter, cut in strips
long enough to reach at least half way up to the container) that has been completely immersed in
dilute acidified copper sulfate for 1 min and subsequently washed with water.
Preparation of sample: Samples shall be prepared for chemical analysis by passing them
quickly through suitable grater by grinding them quickly in a mortar and returning them to the
sample container or by cutting them into small pieces with a sharp knife in the container.
Procedure:
1. Weigh accurately 1.0 to 1.5g of the prepared sample into the extraction tube
2. Add 10ml of the hydrochloric acid and boil gently with shaking, either over a flame or in
a boiling water bath, until all solid particles are dissolved
3. Cool the tube in running water
4. Add 10ml of alcohol and again mix
5. Complete extraction of the fat is dependent on satisfactory mixing at each stage
6. Proceed further in fat extraction apparatus as in determination of fat by rose Gottlieb
method and proceed with the blank determination using the same method
Deduct the value of weight of blank with value of weight of sample test. Note the actual fat
content.
[Source: Bureau of Indian Standards IS: 4883-1968, IS: 2785-1964, IS: 1479 (Part II) – 1961]
186
187
Chapter 16
SKIM & WHOLE MILK POWDER
188
189
6.1 Determination of Sensory Quality of Powder Milk
Aim: To determine sensory quality of powder milk

Principle: Judging the quality of skim milk by its taste and smell requires considerable skill,
which could only be acquired by practice. Sensory tests are used in all dairies and an experienced
person can pick out bad samples with a high degree of accuracy. The use of sensory technique is
very useful for preliminary assessment of milk product quality and handling practice. Skim milk
is smelled and observed visually to see if there are any sensory defects.
Apparatus: 250ml beaker, spatula, glass rod.
In the case of bulk powder, a sample of at least 250g should be made available for sensory
evaluation, and in the case of retail packages, an adequate number. The samples available should
be adequate for the preparation of reconstituted milk for evaluation and possible re-evaluation by
the panel and for an appropriate quantity of undissolved powder to follow the reconstituted
product for evaluation.
The required quantity of samples is reconstituted by dissolving milk powder in water according
to the following formula:
1000
g of milk powder in 90g of water
100 − F
Where, F is the fat content of the milk powder. The reconstitution should be made in
microbiologically pure, colorless and tasteless water at 25°C, except in the case of whole milk
powder not clamed to be soluble in cold water, where the corresponding temperature should
preferably be 40°C.
The solution should be prepared approximately 1 hr before evaluation and dispensed in clear,
tasteless and odorless glasses. The glasses containing the reconstituted milk as well as the
remaining powder sample should be adequately covered until the evaluation takes place.
Reconstituted milk should be kept under conditions, which avoid the influence of light.
During the evaluation, the reconstituted milk should be maintained at a temperature of 20°C.
Deviations of more than ±2°C from this temperature should be avoided.
Procedure:
The sensory evaluation of reconstituted milk as well as powder should be carried out in relation
to appearance and flavor.
1. Appearance involves the following main features: color, visible purity, absence of lumps,
flakes, or hard granules.
2. Flavor involves the following features: taste and odor.
- 188 -
Different categories of milk powder should be evaluated in accordance with the international
standard.
International Table of Milk Powder Quality Terms
Lumps Cooked
Hard granules Chalky
Flakes Feed
Brown Flat
Uneven color Burnt
Scorched particles Bitter
Free fat Oxidized
Free protein Tallowy
Fishy
Cardboardy
Metallic
Oily
Stale
Rancid
Salty
Acid
Foreign matter
Chemical flavor
Putrid
[Source: International Dairy Federation IDF Standard: 99C: 1997]
- 189 -
6.2 Determination of Fat Content of Dried Milk
(Rose Gottlieb Reference Method)
Aim: To determine fat content of dried milk by Rose Gottlieb method.

Principle: The fat content is determined by gravimetric method. This method specifies the
reference method for the determination of the fat content of dried high fat milk (by dried high-fat
milk is understood dried milk with a fat content of 40% m/m or more), dried whole, dried
partially skimmed and dried skimmed milk, dried whey, dried butter milk and dried butter serum.
It is expressed as a percentage by mass.
Rose Gottlieb method entails: extraction of an ammoniacal ethanolic solution of a test portion
with diethyl ether and light petroleum, removal of the solvents by distillation or evaporation, and
determination of the mass of the substances extracted which are soluble in light petroleum.
Apparatus: Analytical balance, centrifuge (rotational frequency of 500 to 600/min to produce a
gravitational field of 80-90g), distillation or evaporation apparatus (to enable the solvents and
ethanol to be distilled form the fat-collecting flask or to be evaporated from beakers and dishes at
a temperature not exceeding 100°C), drying oven (electrically heated at a temperature of
102±2°C), water bath (capable of being controlled at 65±5°C), Mojonnier-type fat-extraction
flasks, rack (to hold the fat extraction flasks or tubes), wash bottle, fat-collecting vessels (boiling
flasks, flat-bottomed of capacity 125ml, conical flasks of capacity 250ml), boiling aids (fat-free,
of non-porous porcelain), measuring cylinders, (capacities 5 and 25ml), pipettes (graduated of
capacity 10ml), tongs (made of metal, suitable for holding flasks, beakers or dishes).
Chemicals: Ammonia solution (containing approximately 25% m/m of NH3, density 910g/liter at
20°C), ethanol or ethanol denatured (at 94% v/v), congo red solution (dissolve 1g congo red in
water and dilute to 100ml), diethyl ether (free from peroxides, containing no or not more than
2mg/kg of antioxidants and complying with requirements for the blank test), light petroleum
(having any boiling range between 30 and 60°C), mixed solvent (prepared shortly before use by
mixing equal volumes of diethyl ether and light petroleum).
Procedure:
1. Thoroughly mix the laboratory sample by repeatedly rotating and inverting the container
(if necessary, after having transferred all of the laboratory sample to an air-tight container
of sufficient capacity to allow this operation to be carried out)
2. Mix the test sample by gently stirring or rotating and inverting the container and
immediately weigh to the nearest 1mg, directly or by difference, into fat-extraction flask
one of the following test portions:
a) About 1g of dried high-fat milk, of dried whole milk or of dried butter serum
b) About 1.5g of dried partially skimmed milk
c) About 1.5g of dried skimmed milk
d) About 1.5g of dried whey
e) About 1.5g of dried buttermilk
190
The test portion shall be delivered as completely as possible into the lower (small) bulb of
the extraction flask. Weight of test portion = m0
3. Carry out a blank test simultaneously with the determination, using the same procedure
and same reagents, but replacing the dispersed test portion as in 5 by 10ml water
4. Dry a vessel with a few boiling aids in the oven for 1 hr. Allow the vessel to cool
(protected from dust) to the temperature of the weighing room (glass vessel for at least 1
hr, metal dish for at least 0.5 hr). Using tongs (to avoid, in particular, temperature
variations), place the vessel on the balance and weigh to the nearest 0.1mg
5. Add 10ml of water at 65±5°C so as to wash the test portion into the small bulb of the
flask, and mix thoroughly until the product is completely dispersed. Cool in running water
6. Add 2ml of the ammonia solution, or an equivalent volume of a more concentrated
ammonia solution, and mix thoroughly with the dispersed test portion in the small bulb of
the flask. After the addition of the ammonia, continue the determination without delay
7. Heat the flask at 65±5°C in the water bath for 15 to 20 min with occasional shaking and
then cool to laboratory temperature
8. Add 10ml of ethanol and mix gently but thoroughly by allowing the contents of the flask
to flow backward and forward between the two bulbs; avoid bringing the liquid too near
to the neck of the flask. If desired, add two drops of the congo red solution
9. Add 25ml of the diethyl ether, close the flask with a cork saturated with water or with a
stopper wetted with water, and shake the flask vigorously, but not excessively (in order to
avoid the formation of persistent emulsions), for 1 min with the flask in a horizontal
position and the small bulb extending upward, periodically allowing the liquid in the large
bulb to run in the small bulb
If necessary, cool the flask in running water, then carefully remove the cork or stopper
and rinse it and the neck of the flask with a little of the mixed solvent using the wash
bottle so that the rinisings run into the flask or the prepared fat-collecting vessel.
10. Add 25ml of light petroleum, close the flask with the rewetted cork or rewetted stopper
(by dipping in water), and shake the flask gently for 30 sec
11. Centrifuge the closed flask 1 to 5 min at a rotational frequency of 500 to 600/min. If a
centrifuge is not available, allow the closed flask to stand in the rack for at least 30 min
until the supernatant layer is clear and distinctly separated from the aqueous layer. If
necessary, cool the flask in running water.
12. Carefully remove the cork or stopper and rinse it and the inside of the neck of the flask
with a little of the mixed solvent so that the rinsings run into the flask or the fat-collecting
vessel.
If the interface is below the bottom of the stem of the flask, raise it slightly above this
level by gently adding water down the side of the flask to facilitate the decapitation of
solvent.
13. Holding the extraction flask by the small bulb, carefully decant as much as possible of the
supernatant layer into the prepared fat-collecting vessel containing a few boiling aids in
the case of flasks (optional with metal dishes), avoiding decapitation of the aqueous layer.
191
14. Rinse the outside of the neck of the extraction flask with a little of the mixed solvent,
collecting the rinsings in the fat-collecting vessel and taking care that the mixed solvent
does not spread over the outside of the extraction flask.
If desired, the solvent or part of the solvent may be removed from the vessel by
distillation or evaporation as described elsewhere.
15. Add 5ml of ethanol to the contents of the extraction flask, using ethanol to rinse the inside
of the neck of the flask and mix it.
16. Carry out a second extraction by repeating the operations described in 9 to 14 inclusive,
but using only 15ml of the diethyl ether and 15ml of the light petroleum; use the ether to
rinse the inside of the neck of the extraction flask. If necessary, raise the interface to
slightly above the middle of the stem of the flask to enable the final decapitation of
solvent to be as complete as possible.
17. Carry out a third extraction without addition of ethanol by again repeating the operations
described in 9 to 13 inclusive, but using only 15ml of the diethyl ether and 15ml of light
petroleum. Use ether to rinse the inside of the neck of the extraction flask.
If necessary, raise the interface to slightly above the middle of the stem of the flask to
enable the final decapitation of solvent to be as complete as possible.
18. Remove the solvents (including ethanol) as completely as possible from the flask by
distillation, or from the beaker or dish by evaporation, rinsing the inside of the neck of the
flask with a little of the mixed solvent before commencing the distillation.
19. Heat the fat-collecting vessel (flask placed on its side to allow solvent vapor to escape) for
1 hr in the drying oven, controlled at 102±2°C. Remove the fat-collecting vessel from the
oven, allow to cool (not in a desiccator, but protected from dust) to the temperature of the
weighing room (glass vessel for at least 1 hr, metal dish for at least 0.5 hr) and weigh to
the nearest 0.1mg. Do not wipe the vessel immediately before weighing. Place the vessel
on the balance using tongs (to avoid, in particular, temperature variations).
20. Repeat the operations described in 19 until the mass of the fat-collecting vessel decrease
by 0.5mg or less, or increases, between two successive weighings. Record the minimum
mass as the mass of the fact-collecting vessel and extracted matter.
21. Add 25ml of the light petroleum to the fat-collecting vessel in order to verify whter or not
the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the fat
is dissolved.
22. If the extracted matter is wholly soluble in the light petroleum, or in case of doubt extract
the fat completely for the vessel by repeatedly washing with warm light petroleum.
23. Allow any trace of insoluble material to settle and carefully decant the light petroleum
without removing any insoluble material. Repeat this operation three more times, using
light petroleum to rinse the inside of the vessel.
24. Finally, rinse the outside of the top of the vessel with mixed solvent so that the solvent
does not spread over the outside of the vessel. Remove light petroleum vapor from the
vessel by heating the vessel for 1 hr in the drying oven, controlled at 102±2°C, allow to
cool and weigh.
192
25. Take the mass of fat as the difference between the mass determined in 20 and this final
mass
Calculation
The fat content, expressed as a percentage by mass, is equal to:
(m1 − m 2 ) − (m3 − m 4 )
×100
m4
Where,
m0 = the mass in g of the test portion
m1 = the mass in g of the fat-collecting vessel and extracted matter
m2 = the mass in g of the prepared fat-collecting vessel or, in the case of undissolved
material, of the fat-collecting vessel and insoluble residue
m3 = the mass in g of the fat-collecting vessel used in the blank test and any extracted
matter
m4 = the mass in g of the prepared fat-collecting vessel used in the blank test or, in the
case of undissolved material, of the fat-collecting vessel and insoluble residue.
Report the result to the nearest 0.01% (m/m)
193
16.3 Determination of Water Content in Dried Milk
Aim: To determine water content in dried milk

Principle: Water content is the loss of mass after drying at 102±2°C to a constant weight. It is
expressed in g/100g.
Apparatus: Analytical balance, drying oven (at 102±2°C), desiccator, flat-bottomed dishes
(depth approximately 25mm, diameter approximately 50mm, glass, stainless steel, nickel or
aluminum, provided with well-fitting readily removable lids), bottles.
Sample preparation: Transfer the whole of the sample of dried milk to a dry, stoppered bottle of
capacity approximately twice the volume of sample. Mix intimately by rooting and shaking the
bottle. In case complete homogeneity is not obtained by this procedure, take the test portions (for
two single determinations) from the prepared test sample at two points as far apart as possible.
Procedure:
1. Heat an uncovered dish and its lid in the oven, controlled at 102±2°C, for 1 hr
2. Place the lid on the dish, transfer the covered dish to the desiccator, allow it to cool to the
temperature of the balance room and weigh to the nearest 0.1mg
3. Put approximately 1-3g of the prepared test sample into the dish, cover with the lid and
weigh to the nearest 0.1mg
4. Uncover the dish and place it with its lid in the oven controlled at 102±2°C, for 2 hr
5. Replace the lid, transfer the covered dish to the desiccator, allow it to cool to the
temperature of the balance room and weigh to the nearest 0.1mg
6. Uncover the dish and heat it again, with its lid, in the oven for 1 hr. Then repeat operation
5.
7. Repeat this process until the difference in mass between two successive weighings does
not exceed 0.5mg. Record the lowest mass
Note: Drying will usually be complete after the first 2 hrs
Calculation
 M − M0 
The water content, expressed in g/100g =  2  × 100
 M1 − M 0 
Where,
M0 = mass in g of the dish and the lid
M1 = mass in g of the dish, the lid and the test portion before drying
M2 = mass in g of the dish, the lid and the test portion after drying
Report the result to the nearest 0.01g/100g
[Source: International Dairy Federation EDF Standard 26A: 1993]
194
16.4 Determination of Moisture and Total Solids
Aim: To determine moisture and total solids of the skim and whole milk powder
Principle: The moisture and total solids are determined by drying off extra water by heating at
102±1°C in an oven to a constant weight.
Apparatus: Flat bottom dishes (with cover, of nickel or other suitable metal, not affected by
boiling water, 70-80mm in diameter and not more than 25mm deep, provided with short glass
stirring rods having a widening flat end), well ventilated oven (temperature maintained at
102±1°C).
Procedure:
1. Weigh accurately about 5g±0.1mg of the sample into a flat bottom glass or aluminum dish
(with cover) previously dried and weighed
2. Heat the dish containing the material after uncovering in an oven maintained at 102±1°C
for about 5 hrs
3. Cool in a desiccator and weigh with the cover on
4. Repeat the process of drying, cooling and weighing at 30 min intervals until the
difference between two consecutive weighings is less than 1mg
5. Record the mass, preserve the dish containing this dried material in a desiccator for the
determination of total solids.
Interpretation of Result:
Calculation
Percent moisture: From the loss in mass, calculate the moisture, percent by mass in the sample
taken for the test.
Percentage of total solids: From the mass of dry residue, calculate the percentage of total solids
in the sample.
[Source: Handbook of Food analysis BIS, SP: 18 (Part XI) 1981]
195
16.5 Determination of Titrable Acidity in Skim and Whole Milk Powder
Aim: To determine titrable acidity in skim and whole milk powder.

Principle: Developed acidity in milk sample will commonly have connection with the
development of lactic acid by microbiological fermentation of the lactose. The increase in acidity
(fall in pH) is a measure of the degree of sourness.
Apparatus: Burette with soda lime guard tube, porcelain dishes of 60ml capacity, stirring rods
(flattened at one end), pipettes (10ml and 1ml).
Chemicals: Standard sodium hydroxide solution (0.1N), 1% phenolphthalein indicator solution
(1g of phenolphthalein in 100ml of alcohol and add 0.1N NaOH solution until one drop gives a
faint pink color; dilute with distilled water to 200ml), rosaniline acetate stock solution (dissolve
0.12g rosaniline acetate in 50ml rectified spirit containing 0.5g glacial acetic acid and make up to
100ml with rectified spirit), rosaniline acetate bench solution (dilute 1ml of the stock solution to
500ml with a mixture of rectified spirit and distilled water in equal proportion by volume).
Procedure:
1. Weigh accurately about 1g±0.1mg of the sample into each of the two porcelain dishes.
2. Add 10ml of boiling water to each dish and stir with the flat end of a glass rod until a
perfectly smooth liquid is obtained.
3. Cool to room temperature; use the contents of one dish as a blank by stirring in 2ml of
bench solution of rosaniline acetate.
4. Add 1ml of phenolphthalein indicator solution to the other dish followed by standard
sodium hydroxide solution introduced drop by drop from the burette until by comparison
the color matches the pink tint of the blank.
5. Stir vigorously throughout. The time taken for the complete titration shall not 20 seconds.
Interpretation of Result:
Calculation
9AN
Titrable acidity (as lactic acid), percent by mass =
M
Where,
A = volume in ml of the standard sodium hydroxide required for titration
N = normality of the standard sodium hydroxide required for titration
M = mass in g of milk powder taken for the test
[Source: Handbook of Food analysis BIS, SP: 18 (Part XI) – 1981]
196
16.6 Determination of Titrable Acidity of Dried Milk (Reference Method)
Aim: To determine titrable acidity in dried milk.

Principle: Titrable acidity of dried milk is the number of milliliters of 0.1N NaOH solution
required to titrate a quantity of the reconstituted milk corresponding to 10g of solids not fat to a
pH of 8.4. Preparation of reconstituted milk is done by addition of water to a test portion of dried
milk corresponding accurately to 5g of solids not fat.
The amount of sodium hydroxide solution required is a function of the amount of natural
buffering substances present in the product and of the development or addition of acid or alkaline
substances.
Apparatus: Analytical balance, pH meter (with slope control, capable of being read to 0.01 pH
unit, with glass electrode and a suitable reference electrode, calibrated using two buffer solutions
with pH approximately 7 and 9 respectively known to be within ±0.01 pH unit), magnetic stirrer,
burette (graduated in 0.1ml with an accuracy of 0.05ml), measuring cylinder (50ml capacity),
conical flask (100ml or 150ml capacity, with ground glass neck and ground glass stopper; the
neck shall be large enough to accommodate the two electrodes, the burette tip and the nitrogen
line).
Chemicals: Sodium hydroxide, standard volumetric solution 0.1±0.0002N (protect this solution
against penetration of CO2), nitrogen.
Procedure:
Preparation of the test sample
1. Transfer the sample to a clean, dry container 9provided with an airtight lid) of capacity
about twice the volume of the sample.
2. Close the container immediately and thoroughly mix the contents by repeatedly shaking
and inverting the container. During these operations, exposures of the sample to the
atmosphere should be avoided as far as possible, to minimize absorption of water.
Test portion
1. Weigh 500/a±0.01g of test sample into conical flask, a being the solids-not-fat content of
the sample, expressed as percentage.
Note: The solids-not-fat content of the sample may be calculated by subtracting the fat content
and the moisture from 100.
Determination
2. Reconstitute the test portion with 50ml water, from the measuring cylinder, at about 20°C,
agitating vigorously, and allow it to stand for about 20 min
3. Titrate the contents of the conical flask by adding the sodium hydroxide from the burette
until the pH, measured with the pH meter, persists for about 5 sec at 8.40; during the
titration, the solution should be stirred using the magnetic stirrer, and the absorption of
carbon dioxide from the air should be avoided by flushing the conical flask with nitrogen.
The titration should be completed within 1 min.
197
4. Record the volume, in milliliters, of sodium hydroxide solution used, to the nearest
0.05ml.
Calculation
The titrable acidity of the sample is equal to:
2×V
Where, V is the volume, in ml, of 0.1N sodium hydroxide used for the titration.
Express the result to one decimal place.
[Source: International Dairy Federation IDF, 86: 1981]
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16.7 Determination of Solubility Index in Skim and Whole Milk Powder
Aim: To determine solubility index in skim and whole milk powder.

Principle: Solubility index is the amount of sediment left after mixing the milk powder in water.
This is determined by centrifuging the reconstituted milk and measuring the ml of sediment
found after complete centrifugation.
Apparatus: Balance, centrifuge, centrifuge tubes, mixing jars, mixer, siphon tube
Procedure:
Reconstitution
1. Add 14g of sample to 100ml distilled water at a temperature of 24°C in the mixing jar
2. Place the jar in a mixer and stir for exactly 90 sec
3. Allow the sample to stand until foam has separated sufficiently to permit its complete
removal by a spoon. The period of standing after mixing should not exceed 15 min
4. After removal of foam, mix the sample thoroughly with a spoon for 5 sec
Removal of cream and soluble fraction
5. Fill up the centrifuge tube immediately with the reconstituted milk to the 50-ml mark
6. Centrifuge the tube for 5 min at the required speed
7. Immediately siphon off the transparent liquid to within 5ml of the surface of the sediment
level, taking care not to disturb the sediment layer
8. Add about 25ml distilled water at a temperature of 24°C and shake the tube to disperse
the sediment, dislodging it, if necessary, with a wire
9. Fill the tube to the 50ml mark with distilled water at a temperature of 24°C. Invert and
shake to mix the contents thoroughly.
10. Again centrifuge at the required speed for 5 min
Determination of solubility index
11. Hold the tube in a vertical position with the upper level of the sediment on a level with the
eye and read the milliliters of sediment in the tube to the nearest graduated scale division.
12. The sediment is easily distinguished when the tube is held between the eye and a strong
source of light.
199
Calculation
Solubility Index – report the solubility index as the milliliters of sediment in the tube.
200
16.8 Determination of the Dispersibility and Wettability of Instant Dried
Milk
Aim: To determine the dispersibility and wettability of instant dried milk.

Principle: The degree to which a dried milk is instant depends on various properties, which can
be classified as wettability, sinkability, solubility and dispersibility. The first three all affect the
last to some extent and hence dispersibility is defined and determined in this method.
Dispersibility is defined by mass of dry matter of the sample that can be dispersed in water,
determined by the procedure specified. At test portion of the sample, of known water content is
evenly spread on the surface of water adjusted to 25°C, the mixture stirred manually for a short
time and part of the mixture filtered through a sieve and the total solids content of the collected
liquid determined. Dispersibility is calculated from the mass of the test portion and the values for
water content and total solids content.
Apparatus: Container (capacity about twice the volume of the laboratory sample with air-tight
lid), balance (top pan, capable of weighing 0.1g), scoop (suitable for weighing out the test
portion), thermometer (suitable for indicating a temperature of 25±1°C), glass beaker (with spout,
capacity 600ml, external diameter 90±2mm, overall height 126±3mm, graduated at 150 and
250ml with the rim lying in a horizontal plane parallel to the base), glass plate (120×120mm,
outside diameter 80±0.3mm, with the ends ground, parallel and at right angles to the longitudinal
axis), stand and clamp for holding the glass tubing, brush suitable for removing the test portion
from the scoop, spatula (stainless steel), stop watch, test sieve (diameter 200mm, woven metal
wire cloth), conical flask with stopper (capacity 250ml), glass funnel (suitable for transferring the
contents of the sieve receiver).
Chemicals: During the analysis use only distilled water or water of equivalent purity.
Procedure:
Pretreatment of test sample
Before proceeding with the operations, ensure that the test sample has been at laboratory
temperature (20-25°C) for at least 48 hrs. This treatment is necessary with all test samples so that
any influence on dispersibility attributable to the physical state of the fat is constant from sample
to sample.
Water content of pretreated test sample
Carry out, in duplicate, the procedure to obtain two single values (to the nearest 0.01% m/m) for
the water content of the pretreated test sample.
Record the mean of these values, to the nearest 0.1% (m/m) as the water content.
Test method
1. Carry out the test method in duplicate to obtain single values for dispersibility.
2. Mix the pretreated test sample by very gently rotating the container a few times and weigh
out in the scoop, 26±0.1g of instant dried whole milk.
201
3. Weigh out 250±0.1g of water, adjusted to 25±1°C, in a dry glass beaker. The portion
above the final water level should remain dry.
4. Place the beaker on the base of the stand, place the glass plate centrally on top of the
beaker and place the glass tubing on the glass plate, clamping the tubing so that it is
centrally located above the beaker and the glass plate is free enough to be withdrawn.
5. Transfer the entire test portion to within the glass tubing, using the brush if necessary and
spread the test portion evenly over the glass plate with the spatula.
6. Start the stop watch and when after 1 min its main hand again indicates 0/60sec, withdraw
the glass plate with one hand (holding the beaker with the other hand) so that the test
portion progressively falls onto the surface of the water. The withdrawal of the plate shall
be performed with a gently continuous movement and shall be accomplished in
approximately 2.5 sec.
7. Immediately remove the beaker from below the glass tubing and when the main hand of
the stop watch indicates 5 sec insert the spatula down the side of the beaker until it
touches the bottom.
8. During the next 5 sec stir the contents of the beaker with spatula making 1 complete
stirring movement per sec, i.e., a smooth continuous movement of the spatula across the
beaker for one side to the opposite side and back and occupying 1 sec, with the end of the
spatula blade in continuous contact with the bottom of the beaker and slightly tilting the
spatula away from the side of the beaker at the end of each half stirring movement so as to
minimize accumulation of unwetted dried milk on the sides of the beaker.
9. Without interruption, continue the stirring for 15 sec in the same manner except that the
spatula is maintained in a vertical position throughout.
10. While making 20 complete stirring movements in 20 sec, continuously rotate the beaker
on its base so that approximately one complete turn (360°) is achieved during the stirring.
11. After completion of the stirring allow the contents of the beaker to stand for 30 sec, i.e.,
until the main hand of the stop watch indicates 55 sec and then without disturbing any
sediment, quickly pour off the liquid down to approximately the 150ml graduation mark
distributing the decanted liquid as evenly as possible over the test sieve below which is
fitted the receiver.
12. Do not tilt or move the sieve during the sieving. To facilitate the passage of the liquid
through the sieve, the sieve is wetted before any use by rinsing it with water. All excess
water is removed from the sieve by wiping it with a towel; the top and bottom surfaces of
the wire cloth are only superficially wiped.
13. 30 sec after the beginning of the sieving operation, i.e., when the main hand of the stop
watch has returned to the 25 sec position, transfer as completely as possible the contents
of the receiver to the conical flask by means of the glass funnel and stopper the flask.
14. Thoroughly mix the liquid in the flask by repeatedly inverting the flask. Carry out, in
duplicate, the procedure described to obtain two single values (to the nearest 0.01% m/m)
for the total solids content of the liquid. Record the mean of these values, to the nearest
0.1% (m/m) as the total solids content.
202
Calculation
Calculate each duplicate single value for dispersibility as a percentage, using the following
formula:
a) Instant dried skim milk:
T × 962
D=
100 − ( W + T )
b) Instant dried whole milk:
T × 735
D=
100 − ( W + T )
Where,
D = dispersibility in %
T = total solids content in % (m/m) of the liquid
W = the water content in % (m/m) of the pretreated test sample
Provided these values comply with the requirements, report the mean value expressed to the
nearest 1% as the dispersibility of the laboratory sample.
[Source: International Dairy Federation IDF 87: 1979]
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Chapter 17
SWEETENED CONDENSED MILK
204
205
17.1 Determination of Sensory Quality of Sweetened Condensed Milk
Aim: To determine sensory quality of sweetened condensed milk.

Principle: Sweetened condensed milk commonly sold in tin cans is judged on the basis of its
sensory characteristics, such as the appearance of the surface layer of the product, color, body
and texture and flavor. This standard specifies an acceptable procedure for sensory evaluation of
sweetened condensed milk. It is expected that this will prove helpful to manufacturers and
consumers for evaluating the product for its sensory properties on rational basis.
Sensory terms:
Strong: It is a flavor defect, which is suggestive of caramelized sugar and is usually
accompanied by browner tint to the natural color.
Buttons-lumpy: It is a body defect, which is characterized by the presence of round and firm
lumps with stale odor at the surface of the product
Gassy: Results from gas formation by yeast in the product, this defect is recognized by bloated or
huffed can and a yeasty odor. Such products shall not be tasted.
Sandy, coarse, mealy: A textural defect in which the product lacks smoothness and is felt in the
mouth. This is caused by the increase in crystallization of lactose.
Settled: It is a defect when solids deposit in the form of a layer in the bottom of the can.
Thickened: This is a body defect, which is manifested by a gel formation.
Fat separation: This defect occurs in the form of a fatty film on the bottom surface, which is in
contact with the product.
Fruitiness: Flavor defect of microbial origin
Apparatus: Spatula, glass dish
Procedure:
The sensory evaluation of the sample is done as follows:
1. Package appearance
2. Color and appearance
3. Body and texture
4. Flavor
(i) Package appearance: Improper seal, rust spot/soiled, dull surface
(ii) Color and appearance: Browning, mold buttons, fat separation
(iii) Body and texture: Thickened, sandy/coarse, mealy, heavy, settled
(iv) Flavor: Caramelized, rancid/tallowy, metallic fruitiness
- 205 -
17.2 Determination of Fat content (Rose-Gottlieb Reference Method)
Aim: To determine fat content in sweetened condensed milk.

Principle: This method is applied for the determination of the fat content of all types of
evaporated milk and sweetened condensed milk (liquid sweetened and unsweetened concentrated
milk). It is expressed as percentage by mass.
Rose Gottlieb method entails: extraction of an ammoniacal ethanolic solution of a test portion
with diethyl ether and light petroleum, removal of the solvents by distillation or evaporation, and
determination of the mass of the substances extracted which are soluble in light petroleum.
Apparatus: Analytical balance, centrifuge (rotational frequency of 500 to 600/min), distillation
or evaporation apparatus (at a temperature not exceeding 100°C), drying oven (electrically heated
at a temperature of 102±2°C), water bath (capable of being controlled at 30-60°C), Mojonnier-
type fat-extraction flasks, wash bottle, fat-collecting vessels (boiling flasks, flat-bottomed of
capacity 125ml-250ml, or metal dishes; if metal dish is used, it shall preferably be of stainless
steel, shall be flat bottomed, preferably with a spout, and shall have a diameter of 80-100mm and
height of approximately 50mm), boiling aids (fat-free, of non-porous porcelain or silicon
carbide), measuring cylinders, (capacities 5 and 25ml), pipettes (graduated of capacity 10ml),
tongs (made of metal, suitable for holding flasks, beakers or dishes).
Chemicals: Ammonia solution (containing approximately 25% m/m of NH3, density 910g/liter at
20°C), ethanol or ethanol denatured (at 94% v/v), Congo red solution.
The preparation of Congo red solution is as follows: Dissolve 1g Congo red in water and dilute to
100ml), diethyl ether (free from peroxides, containing no or not more than 2mg/kg of
antioxidants and complying with requirements for the blank test), light petroleum (having any
boiling range between 30 and 60°C), mixed solvent (prepared shortly before use by mixing equal
volumes of diethyl ether and light petroleum).
Procedure:
Preparation of the test sample
1. Evaporated milk
Shake and invert the container. Open the container, pour the product slowly into a second
container (provided with an air-tight lid) and mix by repeated transfer, taking care to incorporate
in the sample any fat other constituents adhering to the wall and ends of the first container.
Finally, transfer the product as completely as possible to the second container.
If necessary, in the case of samples in sealed cans, condition the unopened container in the water
bath at 40-60°C. Remove and shake the can vigorously every 15 min. After 2 hrs, remove the can
and allow it to cool to room temperature. Remove the lid entirely and thoroughly mix the
contents by stirring with a spoon or spatula. If fat separates, do not test the sample.
206
2. Sweetened condensed milk
Open the container and mix thoroughly with a spoon or spatula. Use an up and down rotary
movement in such a way that the top layers and the contents of the lower corners of the container
are moved and mixed. Take care to incorporate in the sample any milk adhering to the wall and
ends of the container. Transfer the product as completely as possible to a second container
(provided with an air-tight lid). Close the container.
If necessary, in the case of samples in sealed cans, condition the unopened container in the water
bath at 40-60°C. Open and scrape out all milk adhering to the interior of the can, transfer to a
dish large enough to permit stirring thoroughly and mix until the whole mass in homogeneous.
In the case of a sample in a collapsible tube, open the tube and transfer the contents to a jar. Then
cut open the tube and scrape out all materials adhering to the interior and add to the contents of
the jar.
3. Test portion
Mix the test sample by stirring (in the case of sweetened condensed milk) or by gently inverting
the bottle three or four times (in the case of evaporated milk) and immediately weigh to the
nearest 1mg, directly or by difference, into a fat-extraction flask 4 or 5g of the test sample of
evaporated milk or, 2.0 to 2.5g of the test sample of sweetened condensed milk.
The test portion shall be delivered as completely as possible into the lower (small) bulb of the
extraction flask.
4. Blank test
Carry out blank test simultaneously with the determination, using the same procedure and same
reagents, but replacing the dissolved test portion with 10ml of water.
5. Preparation of fat-collecting vessel
Dry a vessel with a few boiling aids in the oven for 1 hr. Allow the vessel to cool (protected from
dust) to the temperature of the weighing room (glass vessel for at least 1 hr, metal dish for at least
0.5 hr). Using tongs (to avoid, in particular, temperature variations), place the vessel on the
balance and weigh to the nearest 0.1mg.
6. Determination
1. Add water at 30°C to the test portion to obtain a total volume of 10 to 11ml and shake
gently with slight warming (40-50°C) until the product is completely dispersed. Cool in
running water.
2. Add 2ml of ammonia solution, or an equivalent volume of a more concentrated ammonia
solution, and mix thoroughly with the diluted test portion in the small bulb of the flask.
After the addition of the ammonia, continue the determination without delay.
3. Add 10ml of the ethanol and mix gently but thoroughly by allowing the contents of the
flask to flow backward and forward between the tow bulbs; avoid bringing the liquid too
near to the neck of the flask. If desired, add two drops of the Congo red solution.
4. Add 25ml of the diethyl ether, close the flask with a cork saturated with water or with a
stopper wetted with water and shake the flask vigorously, but not excessively) in order to
avoid the formation of persistent emulsions), for 1 min with the flask in a horizontal
207
position and the small bulb extending upwards, periodically allowing the liquid in the
large bulb to run into the small bulb. If necessary, cool the flask in running water, then
carefully remove the cork or stopper and rinse it and the neck of the flask with a little of
the mixed solvent using the wash bottle so that the rinsings run into the flask or the
prepared fat-collecting vessel.
5. Add 25ml of the light petroleum; close the flask with the rewetted cork or rewetted
stopper (by dipping in water) and shake the flask gently for 30 sec.
6. Centrifuge the closed flask for 1 to 5 min at a rotational frequency of 500-600/min. If a
centrifuge is not available, allow the closed flask to stand in the rack for at least 30 min
until the supernatant layer is clear and distinctly separated from the aqueous layer. If
necessary, cool the flask in running water.
7. Carefully remove the cork or stopper and rinse it and the inside of the neck of the flask
with a little of the mixed solvent so that the rinsings run into the flask or into the fat-
collecting vessel.
If the interface is below the bottom of the stem of the flask, raise it slightly above this
level by gently adding water down the side of the flask to facilitate the decapitation of
solvent.
8. Holding the extraction flask by the small bulb, carefully decant as much as possible of the
supernatant layer into the prepared fat-collecting vessel containing a few boiling aids in
the case of flasks (optional with metal dishes), avoiding decantation of any of the aqueous
layer.
9. Rinse the outside of the neck of the extraction flask with a little of the mixed solvent,
does not spread over the outside of the extraction flask.
If desired, the solvent or part of the solvent may be removed from the vessel by
distillation or evaporation.
10. Add 5ml of the ethanol to the contents of the extraction flask, using the ethanol to rinse
the inside of the neck of the flask and mix.
11. Carry out a second extraction by repeating the operations described in 4 to 9 inclusive, but
using only 15ml of the diethyl ether and 15ml of the light petroleum; use the ether to rinse
the inside of the necks of the extraction flask.
12. Carry out a third extraction without addition of ethanol by again repeating the operations
described in 4 to 8 inclusive, but using only 15ml of diethyl ether and 15ml of the light
petroleum; use the ether to rinse the inside of the neck of the extraction flask.
13. Remove the solvents (including ethanol) as completely as possible from the flask by
distillation, or from the beaker of dish by evaporation, rinsing the inside of the neck of the
flask with a little of the mixed solvent before commencing the distillation.
208
1 hr in the drying oven controlled at 102°C. Remove the fat-collecting vessel from the
oven, allow it to cool (not in a desiccator, but protected from dust) to the temperature of
the weighing room (glass vessel for at least 1 hr, metal dish for at least 0.5 hr) and weigh
to the nearest 0.1mg.
15. Repeat the operations described in 14 until the mass of the fat-collecting vessel decreases
by 0.5mg or less, or increases between two consecutive weighings. Record the minimum
mass as the mass of the fat-collecting vessel and extracted matter.
16. Add 25ml of the light petroleum to the fat-collecting vessel in order to verify whether or
not the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the
fat is dissolved.
If the extracted fat is wholly soluble in the light petroleum, take the mass of fat as the
difference between the final mass of the vessel containing the extracted matter and its
initial mass.
17. If the extracted matter is not wholly soluble in the light petroleum, or in case of doubt
extract the fat completely from the vessel by repeatedly washing with warm light
petroleum.
Allow any trace of insoluble material to settle and carefully decant the light petroleum
without removing any insoluble material. Repeat this operation three more times, using
the light petroleum to rinse the inside of the neck of the vessel. Finally, rinse the outside
of the top of the vessel with mixed solvent so that the solvent does not spread over the
outside of the vessel. Remove light petroleum vapor from the vessel by heating the vessel
for 1 hr in the drying oven, controlled at 102°C, allow it to cool and weigh, as described
in 14 and 15.
18. Take the mass of fat as the difference between the mass determined in 15 and this finall
mass.
Calculation
(m1 − m 2 ) − (m3 − m 4 )
×100
m0
Where,
m0 = mass in g of the test portion (2)
m1 = mass in g of the fat-collecting vessel and extracted matter determined in 15
m2 = mass in g of the prepared fat-collecting vessel or, in the case of undissolved
material, of the fat-collecting vessel and insoluble residue determined in 17
m3 = mass in g of the prepared fat-collecting vessel used in the blank test and any
extracted matter determined in 15
209
m4 = mass in g of the prepared fat-collecting vessel used in the blank test, or, in the case
of undissolved material, of the fat-collecting vessel and insoluble residue determined in
17.
210
17.3 Determination of Total Milk solids
Aim: To determine total milk solids.

Principle: Total milk solids can be determined by drying off excess water from the sample. The
presence of sucrose in the sample is taken into account by subtracting its amount from the total
solids obtained after drying the sample.
Apparatus: Flat-bottom dish (nickel or other suitable material not affected by boiling water, 7-
8cm in diameter and not more than 2.5cm deep, provide with a short glass stirring rod having a
widening flat end), sand (which passes through 500 micron IS sieve and is retained on 180
micron IS sieve; it shall be prepared by digestion with concentrated hydrochloric acid, followed
by thorough washing with water till free from chlorides. It is shall then be dried and ignited to
dull red), well-ventilated oven (capable of operating at 98-100°C air temperature).
Procedure:
1. Heat a metal dish containing about 25g of the prepared sand with lid open and a stirring
rod, in the oven at 98-100°C for about 2 hrs.
2. Allow the dish (with lid on) to cool in an efficient desiccator for 30-40 min.
3. Tilt the sand to one side of the dish and weigh accurately about 1.5g of the material.
4. Add about 5ml of distilled water in the dish and thoroughly mix the sand with the sample
by stirring with the glass rod, smoothing out lumps and spreading the mixture over the
bottom of the dish.
5. Leave the rod in the mixture.
6. Place the dish on a boiling water bath for 20 min, carefully stirring the mixture from time
to time, then wipe the bottom of the dish and transfer it along with the glass rod to the
well-ventilated oven at 98-100°C.
7. The lid shall be place beside the dish. The bulb of the oven control thermometer shall be
immediately above the shelf carrying the dish. The dish shall not be placed near the walls
of the oven and should be insulated from the shelf by suitable silica or glass supports.
8. After 1.5 hrs, remove the dish, cover it with the lid and place in an efficient desiccator;
allow it to cool, and weigh.
9. Replace the dish in the oven for a further period of one hour at 98-100°C, remove to
desiccator, cool, and weigh again.
10. Repeat the process of heating, cooling and weighing after every one hour till consecutive
weighings agree to within 0.5mg.
11. For blank determination, run the blank using the same procedure as above except that in
place of the condensed milk sample, 2ml of distilled water is added. There should not be
any difference in mass.
211
Calculation
100(M 2 − M1 )
Total milk solids, percent by mass = −S
M
Where,
M2 = mass in g of the contents of dish after drying
M1 = mass in g of the sand taken for the test
S = percentage by mass of sucrose in the material
Accuracy of the method:
The minimum deviation between duplicate determinations should not exceed by more than 0.1%
of total milk solids.
212
17.4 Determination of Total Solids Content
(Reference Method)
Aim: To determine total solids content in sweetened condensed milk

Principle: Total solids content is the mass remaining after completion of the heating process of
the sample. It is expressed as a percentage by mass. Total solid is obtained after pre-drying on a
steam bath followed by evaporation of the water from a test portion at a temperature of 102°C in
a drying oven until constant weight.
Apparatus: Analytical balance, desiccator (silica gel with hygrometric indicator), boiling water
bath, drying oven (102±2°C), flat bottom dish (height 20-25mm, diameter 50-75mm, stainless
steel, nickel or aluminum provided with well-fitting, readily removable lid), water bath (at 30-
60°C), homogenizer.
Chemicals: The water shall be distilled water or water of at least equivalent purity.
Preparation of test sample: Shake the container with frequent inversion. Open this container
and pour milk slowly into another container made of glass, with an air-tight lid, taking care to
incorporate in the sample any fat or other constituents adhering to the walls of the original
container. Stir vigorously and close the container.
Heat the closed container in a water bath at 40-60°C. Remove and shake the can vigorously every
15 min. after 2 hrs, remove the container and cool to 20-25°C. Remove the lid and mix
thoroughly by stirring the milk with a spoon or spatula.
Procedure:
1. Heat a dish with its lid in the oven for at least 1 hr.
2. Place the lid on the dish, immediately transfer to desiccator, allow it to cool to room
temperature (at least 30 min) and weigh to the nearest 0.1mg.
3. Quickly weigh, to the nearest 0.1mg, 1 to 5g of the prepared sample (depending on
expected solids content) into the prepared dish.
4. In the case of milk or cream, tilt the dish to spread the portion evenly over the bottom of
the dish.
5. In the case of evaporated milk, add 3 to 5ml of water, tilt the dish to mix and spread the
test portion evenly over the bottom dish.
6. Pre-dry the dish for 30 min by heating it on the boiling water bath.
7. Heat the dish, with its lid alongside, in the oven for 2 hrs. Place the lid on the dish and
immediately transfer to a desiccator.
8. Allow the dish to cool to room temperature (at least 30 min) and weigh to the nearest
0.1mg.
9. Again heat the dish with its lid alongside in the oven but for only 1 hr. Place the lid on the
dish and immediately transfer the desiccator. Allow it to cool as in 8 and weigh to the
nearest 0.1mg.
213
10. Repeat the operation described in 9 until difference in mass between two consecutive
weighings does not exceed 1mg. Record the lowest mass.
Calculation
The total solids content, expressed as a percentage by mass, is equal to:
m 2 − m0
×100
m1 − m 0
Where,
m0 = mass in g of the dish with the lid
m1 = mass in g of the dish, lid and the test portion
m2 = mass in g of the dish, lid and dried test portion
Round the value obtained to the nearest 0.01g/100g
[Source: International Dairy Federation IDF, 21B: 1987]
214
17.5 Determination of Total Solids Content
(Reference Method)
Aim: To determine total solids content in sweetened condensed milk

Principle: Total solids content is the mass remaining after completion of the heating process of
the sample. It is expressed as a percentage by mass. Moisture content by this method entails
evaporation of the water from a test portion in the presence of sand at a temperature of 102±2°C
in a drying oven until constant weight. The difference in weight is moisture content.
Apparatus: Analytical balance, desiccator (silica gel with hygrometric indicator), drying oven
(at 102±2°C), flat bottom dish (height 20-25mm, diameter 50-75mm, stainless steel, nickel or
aluminum provided with well-fitting, readily removable lid), boiling water bath or steam bath,
water bath at (30-40°C), quartz or sea sand.
Quartz or sea sand should be such that it can pass through a woven wire sieve with nominal
aperture of 500µm but is retained by a sieve with a nominal size of 180µm, and which complies
with the following suitability test:
Place approximately 20g of sand in a dish with a stirring rod. Heat the open dish and sand,
stirring rod and lid in the oven for at least 2 hrs. Fit the lid, allow the closed dish to cool in the
desiccator to the temperature of the balance room and weigh to the nearest 0.1mg.
Moisten the sand with approximately 5ml of water, mix the sand and water with the stirring rod
and heat the dish and sand, lid and rod for at least 4 hrs in the oven. Fit the lid, allow the dish to
cool in the desiccator to the temperature of the balance room and weigh again to the nearest
0.1mg. The difference between two weighings shall not exceed 0.5mg.
Preparation of sample: Open the container and thoroughly mix the milk with a spoon or spatula.
Use an up and down rotary movement in such a way that the top layers and the contents of the
lower corners are moved and mixed. Take care to incorporate in the sample any milk adhering to
the wall and ends of the container. Transfer the milk as completely as possible to a second
container made of glass, provided with an air-tight lid. Close this container.
Heat the closed container in the water bath controlled at 30-40°C. Cool to 20-25°C. Stir the milk
in the container thoroughly. Mix until the whole mass is homogeneous. Close the container. In
case of a collapsible tube, open and transfer the contents to a jar. Cut open the tube and scrape out
all material adhering to the interior and transfer to the jar.
Procedure:
1. Heat a dish containing approximately 25g of sand, with its lid alongside and a stirring rod
on top of the lid, in the drying oven for at least 1 hr.
2. Place the lid (with the stirring rod on top) on the dish, immediately transfer the dish to a
desiccator, and allow to cool (at least 45 min), and weigh the dish, with lid and rod, to the
nearest 0.1mg.
3. Tilt the sand to one side of the prepared dish, place on a clear space about 2.0g of the
prepared sample, replace the lid with the stirring rod on top and weigh the dish to the
nearest 0.1mg.
215
4. Add 5ml of water to the test portion in the dish and mix with the stirring rod.
5. Thoroughly mix together the diluted test portion and the sand, and spread the mixture
evenly over the bottom of the dish.
6. Leave the stirring end of the rod in the mixture with the other end resting on the rim of the
dish.
7. Heat the dish on the boiling water bath, with as much as possible of the bottom of the dish
exposed to the steam, for approximately 30 min, stirring the mixture frequently in the
early stages of drying so that the mixture is well aerated and becomes crumbly.
8. Lay the stirring rod flat inside the dish, dry the bottom of the dish and heat the dish, with
its lid alongside, in the oven for 2 hrs.
9. Place the lid on the dish, and allow the dish to cool in the desiccator.
10. Allow the dish to cool (at least 45 min) and weigh to the nearest 0.1mg.
11. Again heat the dish with its lid alongside in the oven, but only for 1 hr.
12. Place the lid on the dish, and immediately transfer the dish to the desiccator, allow it to
cool and weigh to the nearest 0.1mg.
13. Repeat the operations described in 11 until the difference in mass between too successive
weighings is not more than 1mg. record the lowest mass.
Calculation
The total solids content expressed in grams per 100g is equal to:
m2 − m0
×100
m1 − m 0
Where,
m0 = mass in g of the dish (including sand), lid and stirring rod
m1 = mass in g of the dish, lid, stirring rod and test portion (before drying)
m2 = mass in g of the dish, lid, stirring rod and dried test portion
Round the value obtained to the nearest 0.01g/100g.
[Source: International Dairy Federation IDF Standard, 15B: 1991]
216
17.6 Determination of Sucrose Content
(Polarimetric Method, IDF)
Aim: To determine sucrose content in sweetened condensed milk.

Principle: The method, which employs the rotation of the plane of polarized light to estimate
sucrose content, is applicable to sweetened condensed milk of normal composition prepared from
whole, partially skimmed or skimmed milk that contains intact sucrose (no altered sucrose, that
is). Thus, sucrose content of sweetened condensed milk is the mass fraction of unaltered sucrose
determined by the procedure described in this method.
Apparatus: Drying oven (thermostatically controlled, maintaining a temperature of 102±1°C),
analytical balance, one-mark volumetric flasks (capacities 50ml, 100ml and 200ml), water bath
(40°C), water bath (fitted with circulator and circulating pump, maintaining a temperature of
60±1°C), polarimeter (a semi-automatic or manual instrument, accurate to 0.01 angular degrees,
or a fully automated electronic instrument with digital read-out, accurate to not less than 0.01
angular degrees; wavelength of the polarized light beam 589.44 nm of sodium D-line; the
instrument shall have a compartment to hold the jacketed polarimeter tube), polarimeter tube
(jacketed, of 200mm optical path length), quartz control plate (fitted with water jacket, serving as
Polarimetric reference standard; rotation 8.5 angular degrees), beakers (capacity 100ml), conical
flasks (stoppered, of capacity 250ml), pipettes (capacity 40ml), filter funnel (diameter
approximately 7cm), filter paper (medium grade, of diameter approximately 15cm).
Chemicals:
Zinc acetate solution: Dissolve in water 21.9g of zinc acetate dehydrate (CH3COO)2Zn.2H2O)
and 3ml of glacial acetic acid, dilute to 100ml and mix.
Potassium hexacyanoferrate (II) solution: Dissolve in water 10.6g of potassium hexacyanoferrate
(II) trihydrate (K4[Fe(CN)6]3H2O), dilute to 100ml and mix.
Hydrochloric acid solution: Add 65ml of conc hydrochloric acid (ρ20°C = 1.18g/lit) to water,
dilute to 100ml and mix.
Ammonia solution: Add 10ml of concentrated ammonia solution (ρ20°C = 0.88g/lit) to water,
dilute to 100ml and mix.
Acetic acid: Add 12ml of glacial acetic acid (CH3COOH) to water, dilute to 100ml and mix.
Sucrose standard solution: Transfer a quantity of finely ground sucrose (C12H22O11) to a dish
(maximum depth of sucrose layer 20mm) and heat in the oven at 102°C for 1 hr. Allow it to cool
to room temperature in a desiccator and weigh 2.6g, to the nearest 1mg, in a glass weighing boat.
Transfer, using water, to a 100-ml volumetric flask. Add water to within few milliliters of the
mark, stopper and swirl to mix and complete the dissolution. Place the flask in the water bath at
20°C and, when the solution has attained the bath temperature, fill to the mark with water and
mix. Note that this solution is stable for 1 day only.
217
Preparation of test sample:
1. Samples of recently manufactured products in which no appreciable separation of components
may be expected:
Open the container, transfer all material adhering to the lid into the container and thoroughly mix
by an up and down movement of a spoon in such a way that the top layers and the contents of the
lower corners are moved and mixed. When the product is in a can, transfer the contents to a jar
with a well-fitting lid. When the product is in a collapsible tube, transfer as much as possible of
the contents to a jar with a well-fitting lid; then cut open the tube, scrape out all material adhering
to the interior and transfer this also to the jar. Mix the contents of the jar as described above.
2. Samples of older products and samples in which separation of components may be expected
Heat in the water bath at approximately 40°C until the sample has nearly reached this
temperature; then open the container. When the product is in a can or tube, transfer the contents
to a jar, scrape out all material adhering to the walls (in the case of a collapsible tube, after
cutting open the tube) and continue the mixing until the whole mass is homogeneous, reducing
the size of any large crystals by crushing them with a glass rod. Close the jar with a well-fitting
lid. Allow it to cool.
Procedure:
1. Connect the water bath-circulating pump with tubing to the jacketed polarimeter tube in
its position in the polarimeter. Check that the water temperature in the jacket and in the
return tube is 20°C. Mark the polarimeter tube in such a way that its placing in the
instrument is always the same (and not reversed)
2. Fill the polarimeter tube with water at 20°C, place it in the instrument and take the
reading. If the reading is not 0.00±0.02 angular degrees, dismantle the tube, clean the
various parts, reassemble and retest with water.
Note: over tightening of the tube and fitting can cause strain in the glass windows and is a
common cause of incorrect zero setting.
3. Connect the jacket of the certified quartz plate to the water circulator the water being at
20°C. Place the plate in the instrument and allow 5 min for thermal equilibration. Take
the reading. If the reading does not agree with the standard rotation given on the quartz
control plate to within the tolerance stated in the certificate, adjust the instrument, making
reference to the instrument manual.
4. Calibration with sucrose standard solution: Place the flask containing the sucrose solution,
together with a flask containing water, in the water bath at 20°C and allow them to attain
this temperature. Remove the flask containing the water and rinse the polarimeter tube 3
or 4 times with small quantities of water. Then fill the tube with water, place it in the
polarimeter and take the reading.
Remove the polarimeter tube and empty it. Remove the flask containing the sucrose standard
solution from the water bath, dry the outside of the flask and rinse the polarimeter tube with small
quantities of the sucrose solution 3 or 4 times. Then fill the tube with the sucrose solution, place
it in the polarimeter and take the reading.
218
The optical rotation of the correctly prepared standard sucrose solution is 3.4620 angular degrees:
if the reading is not within ± 0.010 angular of this value, recheck the zero setting for water at
20ºC, prepare fresh sucrose standard solution and recalibrate.
5. Weigh, to the nearest 0.01g, approximately 40g of the prepared test sample into a beaker.
Determination
6. Add 50ml of hot water (80-90ºC) and mix well. Transfer the mixture quantitatively to a
200ml volumetric flask, rinsing the beaker with successive quantities of water at 60ºC,
until the total volume is between 120 and 150ml.
7. Mix, allow it to cool to room temperature and then add 5ml of the ammonia solution. Mix
again and allow it to stand for 15 min. add a sufficient quantity of the acetic acid solution
to neutralize the ammonia and again mix.
8. Add, mixing gently by rotating the tilted flask, 12.5ml of the zinc acetate solution and
then 12.5ml of the potassium hexacyanoferrate solution. Stopper the flask, place in the
water bath and allow it to attain a temperature of 20ºC. Make up to the mark with water at
20ºC.
9. Close the flask with a dry stopper and mix thoroughly by vigorous shaking. Allow it to
stand for 15 min and filter through a fluted filter paper, rejecting the first few milliliters of
filtrate.
10. Collecting the clear filtrate in a clean, dry conical flask, stopper the flask, place in the
water bath at such a depth that the contents are below the level of the water in the bath,
and allow it to attain a temperature of 20ºC.
11. Place a similar, stoppered flask containing water in the water bath and allow to attain a
temperature of 20ºC. Using this water, carry out a check on the zero setting.
Direct polarization
12. Remove the flask containing the filtrate from the water bath and wipe the outside of the
flask dry. Empty the polarimeter tube, rinse it 3 or 4 times with small quantities of the
filtrate and then fill the tube. Place the tube in the instrument and take the reading at
20±0.5ºC.
Inversion
13. Pipette 40ml of the filtrate into a 50ml volumetric flask. Add 6.0ml of the hydrochloric
acid. Place the flask in the water bath at 60ºC, taking care that the entire bulb of the flask
is immersed. Mix by rotating the flask for 5 min, allow the contents to attain the
temperature of the water bath. After a further 10 min, place the contents to attain the
temperature of the water bath at 20ºC and allow to cool to that temperature. Make up to
the mark with water at 20ºC. Mix and allow to stand for 1 hr at this temperature.
Invert polarization
14. Remove the flask containing the inverted filtrate from the water bath and wipe the outside
of the flask dry. Empty the polarimeter tube and rinse it 3 or 4 times with small quantities
of the inverted filtrate and then fill the tube. Place the tube in the instrument and take the
reading at 20±0.5ºC. Record the exact temperature of measurement.
219
Expression of the Results:
Calculation
The sucrose content of the sample, expressed as a percentage by mass, is equal to:
A-1.25B V-UV V
× × ------- (1)
Q V L×m
Where,
m = mass, in grams, of the test portion
A = direct polarimeter reading before inversion
B = polarimeter reading after inversion
L = length, in decimeters, of the polarimeter tube
Q = the inversion factor
V = volume, in ml, to which the sample is diluted before filtration
UV = the correction, in ml, for the volume of the precipitate formed during the clarification:
V = m/100 (1.08F + 1.55P)
m = mass as above
F = percentage of fat in the sample
P = percentage of protein (6.38 times the nitrogen content) in the sample
Note – If the invert polarization is measured at a temperature, t, other than 20±0.2ºC, the value of
B should be multiplied by (1 + 0.0037{t-20}) and this corrected value used in the calculation.
Value of the inversion division factor Q: The following formula gives an accurate value for Q,
where the light source is sodium light (wavelength 559.44nm, sodium D-line) and rotation is
measured in angular degrees:
Q = 0.8825 (C-9) – 0.0033(t-20) -------- (2)
Where,
C = percentage of total sugars in the inverted solution according to the polarimetric reading
t = the temperature, in degrees Celsius, of the inverted solution during the polarimetric reading.
[Source: International Dairy Federation IDF Standard 35A: 1992]
220
17.7 Determination of Sucrose Content
(Polarimetric Method, IS)
Aim: To determine sucrose content.

Two methods, one polarimetric and the other volumetric are prescribed for the determination of
sucrose. The polarimetric method shall be used, for reference purposes, and for routine analysis
the volumetric (Lane-Eynon) method may be used where facilities for polarimetric analysis are
not available.
1. POLARIMETRIC METHOD
Principle: The method is based on the principle of Clerget inversion. The method involves a
milk treatment of the material with acid, which produces complete hydrolysis of sucrose but
almost none of lactose or other sugars. The sucrose content is derived from the change in rotating
power of the solution. A clear filtrate of the sample, without mutarotation by lactose is prepared
by treatment of the solution with ammonia followed by neutralization – and clearing by the
successive addition of zinc acetate and potassium ferrocyanide solution. In a part of the filtrate
the sucrose is hydrolyzed in a way which meets special case of this milk filtrate. From the
rotation of the filtrate before and after inversion, the sucrose content is calculated.
Chemicals:
Zinc acetate solution (2N): Dissolve in water 21.9g of zinc acetate dehydrate
(CH3COO)2Zn.2H2O) and 3ml of glacial acetic acid, dilute to 100ml and mix.
Potassium ferrocyanide solution (1N): Dissolve in water 10.6g of potassium ferrocyanide
(K4[Fe(CN)6]3H2O) in distilled water and make up to 100ml.
Hydrochloric acid solution (20-22%, 6.35±0.2N)
Aqueous ammonia solution (3.5%, 2±0.2N)
Acetic acid (12%, 2±0.2N)
Apparatus:
Glass beaker (capacity 100ml), volumetric flasks (200ml and 50ml), pipettes (40ml), graduated
cylinders (25ml), measuring pipettes (10ml),, filter funnel (dia 8-10cm and provided with folder
filter papers of dia 15cm), polarimeter tube (length 2dm, exactly calibrated length), polarimeter
or saccharimeter (polarimeter with sodium light or mercury green light, mercury vapor lamp with
prism or the special Wratten screen No 77A, to be readable with an accuracy of at least 0.05º
angular degrees, saccharimeter with international sugar scale, using white light passing through a
filter of 15 mm of 6% solution of potassium dichromate, or using sodium light, to readable with
an accuracy of at least 0.1º international sugar scale degrees), water bath (regulated at 60±1ºC).
Procedure:
1. Preparation of sample:
(i) Samples of recently manufactured products in which no appreciable separation of the
components may be expected:
221
Open the container, transfer all material adhering to the lid into the container and thoroughly mix
by an up and down movement of a spoon in such a way that the top layers and the contents of the
lower corners are moved and mixed. Transfer the contents of a can to a jar with a well-fitting lid.
(ii). Samples of older products and samples in which separation of components may be expected:
Heat in a water bath at about 40ºC until the sample has nearly reached this temperature, open the
container and proceed as above. When the product is in a can, transfer the contents to a jar, scrap
out all material adhering to the walls and continue the mixing until the whole mass is
homogeneous. Close the jar with a well-fitting lid and allow the contents to cool.
2. Control determination
In order to standardize the procedure, the reagents and the apparatus, make a control
determination as described under determination in duplicate on a mixture of 100g of milk or 110g
of skim milk and 18.00f of pure sucrose corresponding to 40g of condensed milk containing 45%
sucrose. Calculate the sugar content by means of the formulas given below, using in formula (1)
for m, F and P respectively the quantity of milk weighed and the fat and protein content of this
milk and in formula (2) for m, the value of 40.00.
The mean of values found shall not differ by more than 0.1% from the actual 45.0%.
3. Determination
Weigh approximately 40g of the well mixed sample to an accuracy of 10mg, into glass beaker.
Add 50ml of hot distilled water 80 to 90ºC and mix well. Transfer the mixture quantitatively to
the 200ml volumetric flask, rinsing the beaker with successive quantities of distilled water at
60ºC, until the total volume is between 120 and 150ml. mix and cool to room temperature. Add
5ml of the ammonia solution. Mix again and then allow it to stand for 15 min. neutralize the
ammonia by adding an equivalent quantity of the acetic acid solution, having determined
beforehand the exact number of milliliters by titration of the ammonia solution with
bromothymol blue as indicator. Mix, and add with gentle mixing by rotating the tilted flask
15.5ml of zinc acetate solution. In the same manner as for zinc acetate solution, add 12.5ml of the
potassium ferrocyanide solution. Bring the contents of the flask to 20ºC and add distilled water
(at 20ºC) up to the 200ml mark.
Note: up to this stage, all additions of water or reagents should be made in such a manner to
avoid formation of air bubbles, and, with the same object in view, all mixing should be done by
rotation of the flask rather than by shaking. If air bubbles are found to be present before
completion of dilution to 200ml, their removal can be assisted by temporarily connecting the
flask to a vacuum pump, and rotating the flask.
Close the flask with a dry stopper and mix thoroughly by vigorous shaking. Allow it to stand for
a few minutes and then filter through a dry filter paper, rejecting the first 25ml of the filtrate.
Direct polarization: Determine the optical rotation of the filtrate at 20+_2ºC.
Inversion: Pipette 40ml of the filtrate obtained above into the 50ml volumetric flask and add
6.0ml of hydrochloric acid. Place the flask in water bath at 60ºC for 15 min, the entire bulb of the
flask being immersed. Mix by a rotatory movement during the first 5 min, in which time the
contents of the flask should have attained the temperature of the bath. Cool to 20ºC and make up
to the 50ml mark with distilled water at 20ºC. Mix and allow it to stand for one hour at this
temperature.
222
Invert polarization: Determine the rotation of the inverted solution at 20±2ºC.
Expression of results:
Calculation
Calculate the sucrose content S, percent by mass by means of the following formulas:
m
V= (1.08F + 1.55P ) ---------(1)
100
D − 5 / 4I V − v V
S= × × ---------(2)
Q V e× m
Where,
v = correction in ml for the volume of the precipitate formed during clarification
m = mass in g of the weighed sample
F = percentage of fat in the sample
P = percentage of protein (N×6.38) in the sample
D = direct polarimeter reading (polarization before inversion)
I = Polarimeter reading after inversion
Q = inversion factor
V = volume in ml to which the sample is diluted before filtration
e = length in dm of the polarimeter tube
223
2. VOLUMETRIC (LANE-EYNON) METHOD
Chemicals:
Sodium hydroxide solution (approx. 0.1N): Prepare from sodium hydroxide, analytical reagent
grade
Stock solutions of invert sugar: Weigh accurately 9.5g of pure sucrose on a watch glass and
transfer it to 1 liter volumetric flask with 100ml of water. Add 5ml of concentrated HCl. Allow it
to stand for 3 days at 20 to 24ºC and then make up to the volume; this solution is stable for
several months.
Standard solution of invert sugar: Neutralize a known aliquot of the stock solution of invert
sugar with sodium hydroxide solution using litmus paper and dilute with water to a known
volume, so that more than 15ml but less than 50ml of it shall be required to reduce all the copper
in the Fehling’s solution taken for titration. Note the concentration of invert sugar in this solution
as milligrams per 100ml. Prepare this solution fresh every day.
Methylene blue indicator solution: Dissolve 0.2g of methylene blue indicator solution in water
and dilute to 100ml.
Fehling’s solution A: Dissolve 34.639g of copper sulfate (CuSO4.5H2O) in water, add 0.5ml conc
H2SO4 of sp grav 1.84, and dilute to 500ml in a volumetric flask. Filter the solution through
prepared asbestos.
Fehling’s solution B: Dissolve 173g of Rochelle salt (potassium-sodium tartrate,
KNaC4H4O6.4H2O), and 50g NaOH (AR grade) in water. Dilute to 500ml in a volutmetric flask.
Allow the solution to stand for a few days. Filter this solution through prepared asbestos.
Mix Fehling’s solution A and B in equal amounts just before titration.
Standardization of Fehling’s solution:
Pour standard invert sugar into a 50ml burette. Pipette 10ml of Fehling’s solution (mixture of A
and B) into a 300ml flask and run in from the burette almost the whole of the standard invert
sugar solution required to affect reduction of all the copper, so that not more than one ml will be
required later to complete the titration. Heat the flask containing the mixture over wire gauze.
Gently boil the contents of the flask for two minutes. At the end of two minutes of boiling, add,
without interrupting boiling, 1ml of methylene blue indicator solution. While the contents of the
flask continue to boil, begin to add standard invert sugar solution (one or two drops at a time)
from the burette until the blue color of the indicator just disappears. The titration should be
completed within one minute so that the contents of the flask boil altogether for 3 min without
interruption. Note the titer used for the reduction of all the copper in 10ml of Fehling’s solution.
Note 1: In adding sugar solution to the reaction mixture the burette may be held in hand over the
flask. The burette may be fitted with a small outlet tube bent twice at right angles, so that the
body of the burette can be kept out of the steam while adding sugar solution. Burette with glass
taps are unsuitable for this work, as the taps become heated by the steam and are liable to jam.
Note 2: It should be observed that with both incremental and standard methods of titration, the
flask containing the reaction mixture is left on the wire gauze over the flame throughout the
titration, except when it may be removed for a few seconds to ascertain if the end point is
reached.
224
Zinc acetate solution: Dissolve 21.9g of crystallized zinc acetate {Zn(C2H3O2)2.2H2O} in water
and add 3ml of glacial acetic acid. Make up to 100ml.
Potassium ferrocyanide solution: Dissolve 10.6g of crystalline potassium ferrocyanide in water
and make up to 100ml.
Concentrated HCl: Specific gravity 1.16
Concentrated ammonia solution: Specific gravity 0.88
Dilute ammonia: 10ml of concentrated ammonia solution diluted to 100ml with water.
Dilute acetic acid: Approx equal to dilute ammonia solution in strength.
Procedure:
1. Preparation of the solution: Weigh accurately 40g of the well-mixed sample and transfer
to a 100ml flask.
2. Add 50ml of hot water at 80-90ºC
3. Mix and transfer to a 200ml measuring flask, washing it with successive quantities of
distilled water to 150ml. mix, cool to room temperature and add 5ml of dilute ammonia
solution
4. Mix again and allow it to stand for 15 min.
5. Add the exact equivalent of dilute acetic acid to neutralize the ammonia added
6. Mix and add 12.5ml of zinc acetate solution followed by 12,5ml of potassium
ferrocyanide solution
7. Mix again and make up to 200ml mark
8. Allow the sediments to settle and filter. Mark this as B-1.
9. Pipette 50ml of solution B-I into a 100ml volumetric flask, add 5ml concentrated HCl and
heat at 68ºC for 5 min.
10. Cool the solution and neutralize with sodium hydroxide solution. Mark the as solution A-I
11. Make up to 100ml. Dilute B-I and A-I so that the volume of solution required to react
with 10ml of Fehling’s solution is between 15 to 50ml. mark them as prepared solution B-
II and solution A-II, respectively.
12. Incremental method of titration:
a. Pour the prepared solution B-II into a 50ml burette.
b. Pipette 10ml of Fehling’s solution into a 300ml conical flask and run in from the
burette 15ml of the solution.
c. Without further dilution, heat the contents of the flask over a wire gauze, and boil
(after the liquid has been boiling for about 15 sec, it will be possible to judge if the
copper is almost all reduced by the bright red color imparted to the boiling liquid
by the suspended cuprous oxide). When it is judged that nearly all the copper is
reduced, add 1ml of methylene blue indicator solution.
225
d. Continue boiling the contents of the flask for 1-2 min from the commencement of
ebullition, and then add the prepared solution in small quantities (1ml or less at a
time), allowing the liquid to boil for about 10 sec between successive additions,
till the blue color of the indicator just disappears.
e. In case there appears to be still much unreduced copper, after the mixture of
Fehling’s solution with 15ml prepared solution has been boiling for 15 sec, add
the prepared solution from the burette in larger increments (more than 1ml at a
time, according to judgment), allow the mixture to boil for a quarter of a minute
after each addition.
f. Repeat the addition of the prepared solution at intervals of 15 sec until it is
considered unsafe to add a large increment of the prepared solution.
g. At this stage, continue boiling for all additional one to two min add 1ml of
methylene blue solution and complete the titration by adding the prepared solution
in small quantities (less than 1ml at a time).
13. Standard method of titration: Pipette 10ml of Fehling’s solution into a 300ml conical flask
and run in from the burette almost the whole of the prepared solution B-II required to
affect the reduction of all the copper, so that, if possible, not more than 1ml shall be
required later to complete titration.
a. Gently boil the contents of the flask for 2 min. at the end of 2 min of boiling, add
without interrupting boiling, 1ml of methylene blue solution.
b. While the contents of the flask continue to boil, begin to add the prepared solution
(1 or 2 drops at a time) from the burette until the blue color of the indicator just
disappears.
c. The titration should be completed within 1 min, so that the contents of the flask
boil altogether for 3 min without interruption. Repeat the titration using solution
A-II.
Calculation
20m  2f 2 f1 
Sucrose percent by mass =  − 
M  v 2 v1 
Where,
m = mass in mg of sucrose corresponding to 10ml Fehling’s solution
M = mass in g of the material taken for the determination
f2 = dilution factor for solution A-II from A-I
v2 = volume in ml of solution A-I corresponding to 10ml Fehling’s solution
f2 = dilution factor for solution B-II from B-I
v1 = volume in mal of solution B-II corresponding to 10ml Fehling’s solution
226
17.8 Determination of Titrable Acidity
Aim: To determine titrable acidity in condensed milk.

Principle: Titrable acidity can be determined by titration of sample with standard alkali solution.
Chemicals: Standard sodium hydroxide (approximately 0.1N), phenolphthalein indicator
solution (dissolve 0.5g of phenolphthalein in 100ml of 50% ethyl alcohol).
Procedure:
Weigh accurately about 10g of the material in a suitable dish or basin.
Add 30ml lukewarm water
Add one ml of phenolphthalein indicator solution.
Shake well and titrate against standard sodium hydroxide solution.
Stir vigorously throughout. Complete titration within 20 sec.
Keep in another dish or basin about 10g of the material diluted with 30ml of lukewarm water as a
blank for comparison of color.
The persistence of a slight pinkish tinge for 30 sec indicates the end point. The titration shall be
preferably made in north light or under illumination from a daylight lamp.
Calculation
Titrable acidity (as lactic acid), percent by mass = 9AN/M
Where,
A = volume in ml of standard sodium hydroxide required for titration
227
228
Chapter 18
BUTTERMILK
229
230
18.1 Determination of Fat content in Buttermilk – Rose Gotlieb
Gravimetric Method (Reference Method)
Aim: To determine fat content in milk.

Principle: The Rrose-Gotlieb procedure can be used for the determination of fat content of milk
and a number of milk products.
This is a gravimetric method for the determination of the fat content of liquid skimmed mil, whey
and buttermilk, especially for the purpose of establishing the operating efficiency of cream
separators. It is expressed as a percentage by mass.
The process involves extraction of an ammoniacal ethanolic solution of two test portions with
diethyl ether and light petroleum, combination of the two extracts, removal of the solvents by
distillation or evaporation, and determination of the mass of the substances extracted which are
soluble in light petroleum ether
Chemicals: Ammonia solution (containing approx. 25%, m/m, of NH3; ρ20 = 910g/lit); ethanol or
ethanol denatured by methanol (at least 94%, v/v); Congo-red solution (dissolve 1g of Congo-red
in water and dilute to 100ml); diethyl ether (recently distilled, free from peroxides and
antioxidants); Light petroleum (recently distilled, having any boiling range between 30 and
60ºC); mixed solvent (prepared shortly before use by mixing equal volumes of the diethyl ether
and the light petroleum).
Apparatus: Analytical balance; Centrifuge (in which the fat-extraction flasks or tubes can be
spun at a rotational frequency of 500 to 600 rpm to produce a gravitational field of 80g to 90g at
the outer end of the flasks or tubes); Distillation or evaporation apparatus (to enable the solvents
and ethanol to be distilled from the flasks or to be evaporated from beakers and dishes at a
temperature not exceeding 100ºC); drying oven (at a temperature of 102±2ºC); Water bath
(capable of being maintained at 35 to 40ºC); Mojonnier-type fat extraction flasks (flasks or tubes,
provided with good quality bark corks or stoppers of other material, e.g., silicon rubber,
unaffected by reagents used: Bark corks will by extracted with the diethyl ether, kept in water at
60ºC or more for at least 15 min, and shall then be allowed to cool in the water so that they are
saturated when used); Fat-collecting vessels (for example, flat-bottomed boiling flasks of 125 to
250ml capacity, or conical flasks of capacity 250ml, metal dishes; the metal dishes shall
preferably be of stainless steel, shall be flat-bottomed, preferably with a spout, and shall have a
diameter of 80 to 100mm and a height of approx. 50mm); Boiling aids (fat-free, non-porous
porcelain or silicon carbide); Measuring cylinders (5 and 25ml capacities); Pipettes (graduated,
10ml capacity); Tongs (made of metal, suitable for holding flasks, beakers, or dishes).
Procedure:
1. Preparation of the test sample:
Adjust the temperature of the laboratory sample to 35-40ºC by means of the water bath if
necessary. Mix the sample thoroughly, but gently, by repeatedly inverting the sample bottle
without causing frothing or churning, and cool quickly to approx. 20ºC.
- 229 -
2. Test portion:
Mix the test sample by gently inverting the bottle 3 or 4 times and immediately weigh, to the
nearest 1mg, 10-11g of the test sample, directly or by difference, into each of two extraction
flasks.
3. Blank test:
Carry out a blank test simultaneously with the determination, using the same procedure and same
reagents, but replacing each of the two test portions by 10ml of water.
4. Preparation of fat-collecting vessel:
Dry a vessel with a few boiling aids in the oven for 1 hr, allow the vessel to cool (protected from
dust) to the temperature of the weighing room (glass vessel for at least 1.5 hr, metal dish for at
least 1 hr). Using tongs (to avoid, in particular, temperature variations), place the vessel on the
balance and weigh to the nearest 0.1mg.
5. Determination:
1. Add 2ml of the ammonia solution, or an equivalent volume of a more concentrated
ammonia solution, and mix thoroughly with the test portions in the small bulbs of the
flasks. After the addition of the ammonia, carry out the determination without delay.
2. Add 10ml of the ethanol and mix gently but thoroughly by allowing the contents of the
flasks to flow backward and forward between the two bulbs; avoid bringing the liquid too
near to the neck of the flasks. If desired, add 2 drops of the Congo-red solution.
3. Add 25ml of the diethyl ether, close the flasks with corks wetted with water, and shake
the flasks vigorously, but not excessively (in order to avoid formation of persistent
emulsions), for 1 min with the flasks in a horizontal position and the small bulb extending
upwards, periodically allowing the liquid in the large bulb to run into the small bulb. If
necessary, cool the flasks in running water, then carefully remove the corks or stoppers
and rinse them and the necks of the flasks with a little of the mixed solvent using the wash
bottle so that the rinsings run into the flasks.
4. Add 25ml of the light petroleum, close the flasks with the rewetted corks or rewetted
stoppers (by dipping in water), and shake the flasks gently for 30 sec.
5. Centrifuge the closed flasks for 3-5 min at a rotational frequency of 500-600 min-1. If a
centrifuge is not available, allow the closed flasks to stand in the rack for at least 1 hr
until the supernatant layers are clear and distinctly separated from the aqueous layers. If
necessary, cool the flasks in running tap water.
6. Carefully remove the corks or stoppers and rinse them and the inside of the necks of the
flasks with a little of the mixed solvent so that the rinsings run into the flasks.
If the interface is below the bottom of the stem of the flask, raise it slightly above this level by
gently adding water down the side of the flask to facilitate the decapitation of solvent.
7. Holding each extraction flask by the small bulb, carefully decant as much as possible of
the supernatant layers into the prepared fat-collecting vessel containing a few boiling aids
in the case of flasks (optional with metal dishes), avoiding decapitation of any of the
aqueous layers.
- 230 -
8. Rinse the outside of the neck of each extraction flask with a little of the mixed solvent,
does not spread over the outside of the extraction flasks.
9. Rinse the inside of the boiling flask with a little of the mixed solvent and carefully distil
the solvent as much as possible, or evaporate carefully from the beaker or dish.
10. Add 5ml of the ethanol to the contents of the extraction flasks, using the ethanol to rinse
the inside of the neck of each flask and mix.
11. Carry out a second extraction by repeating the operation, but using only 15ml of the
diethyl ether and 15ml of the light petroleum; use the ether to rinse the inside of the neck
of each extraction flask.
If necessary, raise the interface to the middle of the stem of each extraction flask to enable the
final decantation of solvent to be as complete as possible.
12. Repeat the operations described in 7 to 9. Remove the solvent (including ethanol) as
completely as possible by distillation or evaporation.
1 hr in the drying oven, maintained at 102±2ºC. Remove the fat-collecting vessels from
the oven, allow to cool (not in desiccator, but protected from dust) to the temperature of
the weighing room (glass vessel for at least 1.5 hr, metal dish for at least 1 hr) and weigh
to the nearest 0.1mg.
14. Repeat the operations described in 13 until the mass of the fat-collecting vessel decreases
by 0.5mg or less, or increases, between two successive weighings. Record the minimum
mass as the mass of the fat-collecting vessel and extracted matter.
15. Add 25ml of the light petroleum to the fat-collecting vessel in order to verify whether or
not the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the
fat is dissolved.
If the extracted matter is wholly soluble in the light petroleum, take the mass of fat as the
difference between the final mass of the vessel containing the extracted matter and its initial
mass.
16. If the extracted matter is not wholly soluble in the light petroleum, or in case of doubt,
extract the fat completely from the vessel by repeatedly washing with warm light
petroleum.
17. All any trace of insoluble material to settle and carefully decant the light petroleum
without removing any insoluble material. Repeat this operation three more times using the
light petroleum to rinse the inside of the neck of the vessel.
18. Finally, rinse the outside of the top of the vessel with mixed solvent so that the solvent
does not spread over the outside of the vessel. Remove light petroleum vapor from the
vessel by heating the vessel for 1 hr in the drying oven, controlled at 102±2ºC, allow it to
cool and weigh, as described in13.
19. Take the mass of fat as the difference between the mass determined in 14 and this final
mass.
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Expression of results:
Calculation
( m1 − m 2 ) − ( m3 − m 4 ) ×100
m0
Where,
m0 = the mass, in g, of the two test portions
m1 = the mass, in g, of the fat-collecting vessel and the extracted matter
m2 = the mass, in g, of the prepared fat-collection vessel, or, in the case of undissolved material,
of the fat-collecting vessel and insoluble residue
m3 = the mass, in g, of the fat-collecting vessel used in the blank test and any extracted matter
m4 = the mass, in g, of the fat-collecting vessel used in the blank test, or, in the case of
undissolved material, of the fat-collecting vessel and insoluble residue.
[Source: International Dairy Federation IDF Standard 22B: 1987]
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Chapter 19
MICROBIOLOGICAL ANALYSIS OF MILK AND MILK

PRODUCTS
233
234
19.1 Diluent, Culture Medium and Reagent for Bacteriological Tests
A: Diluents
1. Peptone/saline solution
Note: Peptone/saline solution is the diluent selected by the International Organization for
Standardization (ISO) for general use.
Composition
Peptone 1.0g
Sodium chloride (NaCl) 8.5g
Water 1000ml
Preparation
Dissolve the components in water, heating if necessary. Adjust the pH so that, after sterilization it
is 7.0±0.1 at 25ºC.
2. Quarter-strength Ringer’s solution
Composition
Potassium chloride (KCl) 0.105g
Calcium chloride anhydrous (CaCl2) 0.06g
Sodium hydrogen carbonate (NaHCO3) 0.05g
Water 1000ml
Preparation
Dissolve the salts in water. Adjust the pH so that after sterilization it is 6.9±0.1 at 25ºC.
3. Peptone solution
Composition
Peptone 1.0g
Water 1000ml
Preparation
Dissolve the peptone in water. Adjust pH so that, after sterilization the pH is 7.0±0.1 at 25ºC.
4. Phosphate buffer
Composition
Potassium hydrogen orthophosphate (KH2PO4) 42.5g
Water 1000ml
Preparation
Dissolve the salt in 500ml of water. Adjust the pH so that after sterilization it is 7.2±0.1 at 25ºC.
Dilute to 1000ml. Add 100ml of this solution to 1000ml of water.
- 234 -
5. Buffered peptone water
Composition
Peptone 10.0g
Disodium hydrogen phosphate (Na2HPO4.12H2O) 9.0g
Potassium dihydrogenphosphate (KH2PO4) 1.5g
Water 1000ml
Preparation
Dissolve the components in the water by heating if necessary. Adjust pH so that after sterilization
it is 7.0±0.1 at 25ºC.
B. Culture Medium
1. Total Plate Count Medium
Composition
Yeast extract 2.5g
Tryptone 5.0g
Glucose (C6H12O6.H2O) 1.0g
Skim milk powder (free from inhibitory substances) 1g
Agar 12-18g
Water 1000ml
The pH after sterilization should be 6.9±0.1 at 25ºC.
Preparation from commercial dehydrated medium: Follow the manufacturer’s instructions
but, in all cases, add the skim milk powder even if the manufacturer considers such an addition
unnecessary. Adjust the pH if necessary to 7.0-7.1 using sodium hydroxide or hydrochloric acid
(1N) to obtain the required pH after sterilization in autoclave.
Preparation from individual ingredients: Dissolve and disperse in the following order: yeast
extract, Tryptone, glucose and finally, skim milk powder in water. Heating the water will assist in
this procedure. Add the agar and heat to boiling, stirring frequently, until the agar is completely
dissolved, or steam for about 30 min. filter through filter paper if necessary. Adjust the pH like
above.
Distribution of medium: Distribute the substrate prepared in 100 to 150ml quantities into flasks.
Sterilize in the autoclave for 15 min at 121±1ºC. Check the pH of the substrate. It should be
6.9±0.1ºC at 25ºC.
Reagents for adjusting pH
Sodium hydroxide solution, approximately 1 mol/liter
Hydrochloric acid, approximately 1 mol/liter
- 235 -
2. Coliform Plate Count (Solid selective medium): Violet Red Bile (VRB) Agar
Composition (for 1 liter substrate):
Yeast extract 3.0g
Peptone 7.0g
Bile salts 1.5g
Lactose 10.0g
Sodium chloride 5.0g
Neutral red 0.03g
Crystal violet 0.002g
Agar 12-18g
Water 1000ml
According to manufacturer’s instructions.
Preparation
Dissolve the components or the dehydrated complete medium in the water. Mix thoroughly and
leave to stand for several minutes. Adjust the pH so that after boiling it is 7.4±0.1 at 25ºC, if
necessary. Bring to boil, swirling from time to time. Allow it to boil for 2 min. Immediately cool
the medium in a water bath set at 45ºC. Avoid overheating the medium or heating it for too long
or reheating it. Consequently do not sterilize in the autoclave and check the sterility of the
medium at the time of use. Use the medium within 3 hr of its preparation.
3. Confirmation of medium for coliforms: Lactose Bile Brilliant Green Broth (BGLB)
Composition
Peptone 10g
Lactose (C22H22O11.H2O) 10g
Dehydrated ox bile 20g
Brilliant green 0.0133g
Water 1000ml
Preparation
Dissolve the components of the dehydrated complete medium in the water by boiling in the water
bath. If necessary, adjust the pH so that after sterilization it is 7.2±0.1 at 25ºC. Dispense the
medium in quantities of 10ml in test tubes containing Durham tubes. Sterilize in an autoclave at
121ºC for 15 min. the Durham tubes shall not contain air bubbles after sterilization.
4. Yeast and Mold Plate Count Medium: Potato Dextrose Agar (PDA):
Potato extract 4.0g
Dextrose 20.0g
Agar 12-15g
water 1000ml
The substrate is sterilized for 15 min. Acidify the molten dextrose with 10% tartaric acid (sterile)
to pH 3.5±0.1 immediately before pouring to the inoculated dishes. Never heat the medium after
addition of the acid. For commercial instant medium, follow manufacturer’s instructions.
236
19.2 Methylene Blue Reduction Test for Milk
Aim: To measure the microbial activities in the milk in terms of time required to reduce the color
of a dye.
Principle: The method is used to grade raw milk, especially for manufacturing purposes. The
method indirectly measures bacterial densities in milk in terms of the time interval required, after
starting incubation, for a dye-milk mixture with a characteristic blue color to become white.
Different “Methylene Blue Reduction Time” intervals permit rapid grouping of samples into
classes or grades. The method depends upon the ability of bacteria in milk, when incubation is
started, to grow and utilize the oxygen dissolved in the mixture, which in turn lowers the
oxidation-reduction potential of the mixture. To measure this bacteriological activity and to
demonstrate visibly the rate of oxygen utilization, a solution of methylene blue thiocyanate is
added. The mixture is incubated at 37±1ºC to hasten oxygen utilization and to shorten the
required period of observation. Certified tablets of proper strength for the preparation dye
solutions are available commercially.
Apparatus: Water bath at 37±1ºC, sterile reductase tubes, sterile 1ml pipettes and sterile rubber
stoppers for the test tubes.
Chemicals: Methylene blue dye solution (Prepare a standard solution of methylene blue by
dissolving one of the good quality methylene blue tablets in 200ml of cold, sterile, glass distilled
water in a sterile flask. It is preferable to allow the mixture to stand several hours to ensure
complete solution. The stock solution is further diluted to make 800ml solution. A concentration
of 1 part of methylene blue in 300,000 parts of milk is used to obtain satisfactory results. Store in
light resistant bottle in a cold, dark place preferably a refrigerator.
Procedure
1. Thoroughly mix the sample and pour aseptically into a sterile reductase tube to the 10ml
mark, wetting only one side of the tube.
2. Add 1ml of methylene blue solution.
3. Close the test tube with a sterile rubber stopper and invert the tube gently twice to insure
complete mixture.
4. Place the tube in the 37ºC water bath. The level of the water in the bath should be slightly
higher than the milk in the tube. If possible, close the lid of the water bath to exclude light
and note the time.
5. Set up a control tube consisting of 10ml milk in a sterile reductase test tube with 1ml of
tap water. Place it in boiling water for 3 min, cool, and place in the water bath.
6. Examine the tube after half an hour. The milk is regarded decolorized when the whole
milk column is completely decolorized to within 5mm of the surface.
7. When the test is to proceed beyond the half-hour period, tubes should be examined for
decolorization at half hourly intervals inverting the tubes where decolorization not started.
Leave the tubes where decolorization has begun. Inversion should be gently so as not to
distort the test.
237
8. Record the time required to reduce the color of the dye in hours.
Methylene Blue Reduction time (hr) Quality of milk
5 and above Very good
3–4 Good
1–2 Fair
½ Poor
Methylene Rubber stopper

Blue GOOD QUALITY MILK
Methylene Blue is Not
Reduced Within 6 Hours
35oC
Water Bath
MILK POOR QUALITY MILK

Methylene Blue is
Reduced Within 2 Hours
1ml Methylene Blue Tube is inverted Three
is Added to 10ml Milk Times After Plugging with
Stopper
Fig. 10: Outline of MBRT test
238
19.3 Preparation of Samples and Dilutions for Microbiological
Examination (Dilution Techniques)
Aim: To prepare an appropriate dilution of a sample so that 1ml will obtain 30-300 colonies
when inoculated on a Petri dish and grown on agar medium.
Principle: In determining the microbial content of milk, milk products, water and rinse and swab
from dairy utensils and equipment, serial dilutions of the samples are prepared. The measured
amount of the prepared dilution when inoculated in a Petri dish and grown on agar medium
should contain 30-300 colonies. The ten-fold serial dilution (1ml of sample diluted with 9ml of
diluent or 1.1ml of sample with 10ml diluent) can be prepared by the dilution technique.
Primary dilution (initial suspension): The suspension, solution or emulsion is prepared from a
weighed or measured quantity of the product under examination (or of a test sample prepared
from the product) by mixing (blender may be used if necessary) aseptically with nine-fold
quantity of dilution fluid, allowing large particles (if present) to settle.
Further decimal dilutions: The primary dilution is mixed aseptically with nine-fold volume of
diluent to get a total of 100-fold dilution. This operation is repeated taking aliquot from the
preceding dilution until a decimal dilution series, suitable for the inoculation of the culture media
is obtained.
Apparatus: 1ml (or 1.1ml) sterile blow-out pipettes in canister; test tubes with cap or a plug of
cotton wool; test tube rack; oven or autoclave (170-175ºC for not less than 1 hr in an oven,
121±1ºC for not less than 20 min in an autoclave); blending equipment; mixer (capable of 1ml or
2ml of the test sample in case of liquid products, or the decimal dilutions in a tube of adequate
dimensions with 9ml diluent in order to obtain a homogeneous suspension, e.g., vortex mixer);
flasks or bottles; test tubes; 10ml graduated pipettes; pH meter; balance; water bath (45ºC).
Chemicals: quarter-strength Ringer’s solution sterilized in test tubes: 9ml when 1ml pipettes are
used or 10ml when 1.1ml pipettes are used.
Procedure
A: Preparation of the sample and primary dilution
1. Milk and liquid milk products
Agitate the test sample thoroughly so that the microorganisms are distributed as evenly as
possible by rapidly inverting the sample container 25 times. Avoid foaming or allow foam to
disperse. The interval between mixing and removing the test portion shall not exceed 3 min.
Remove 1ml of the test sample with a sterile pipette and add to 9ml of diluent (or 10ml of test
sample to 90ml of diluent, or 11ml of test sample to 99ml of diluent). Shake this primary dilution
manually or in vortex mixer. A 10-1 dilution is thus prepared.
2. Dried milk, sweet whey powder, acid whey powder, dried buttermilk, lactose
Warm a bottle containing 90ml of the diluent to 45±1ºC in the water bath. Weigh 10g of the test
sample directly into the bottle with the diluent.
239
3. Cheese
Weigh 10g of the test sample is a dish and transfer it to the container of a rotary blender and add
90ml of diluent at pH 7.5±0.1. Blend until the cheese is thoroughly dispersed (1-3 min).
4. Butter
Weigh 10g of the test sample into a container and place the container in the water bath at 45±1ºC.
Keep it at the same temperature until the whole test portion has just been melted. Add 90ml of
the diluent and mix.
5. Frozen milk products (including edible ices)
Proceed as in butter
6. Fermented milk products
Weigh 10g of the test sample into a flask containing 90ml of diluent and shake to disperse.
B. Further decimal dilutions
Transfer by means of a fresh pipette, 1ml of the primary dilution into another tube containing 9ml
of sterile diluent, avoiding contact between the pipette and the diluent. Use a fresh pipette for
each dilution.
If large volumes are required, transfer 10ml of the primary dilution to a bottle containing 90ml of
sterile diluent or 11ml of primary dilution to 99ml of sterile diluent. In a routine procedure, if a
10-3 dilution is required, transfer 1ml of the primary dilution to 99ml of sterile diluent.
Mix carefully, either by aspirating 10 times with a fresh pipette or in the mechanical mixer for 5
to 10 sec to obtain 10-2 dilution. The rotational frequency of the mixer shall be chosen so that the
liquid, as it swirls, rises to within 2-3cm of the rim of the vessel.
If necessary, repeat these operations with sterile diluent using 10-2 and further dilutions to obtain
10-3, 10-4, etc, dilutions until the appropriate number of microorganisms has been obtained.
[Source: International Dairy Federation IDF 122C: 1996]
240
19.4 Enumeration of Microorganisms by Colony Count Technique
(Total Plate Count)
Aim: To determine the estimated number of total viable bacterial colonies per ml or gram of milk
and milk products.
Principle: The bacteria gain entrance to the milk and milk products from surfaces of dairy
utensils and equipment, air and water supply of the milk barn and processing plant, and feed. The
number of total bacteria present in a measured amount of sample (milk, milk product, rinses and
swabs from dairy equipment, etc.) can be estimated by growing the bacteria on media containing
Tryptone, glucose, yeast extract, skim milk powder, agar and water for 72 hours at about 30ºC.
this determination is carried out in the standard plate count, by inoculating 1ml of the diluted
sample into a Petri dish and incubate for 72 hours at 30ºC on standard plate count agar (with skim
milk powder). The growing bacterial colonies are counted and reported as “Total plate count per
ml or gram”.
Apparatus: Incubator (30±1ºC); Petri dishes (glass of 90-100mm dia); graduated pipettes
(plugged with cotton wool, 1ml, 10ml), water bath (45±1ºC); colony counting equipment
(mechanical or electric digital counter); pH meter; test tubes (20ml capacity); bottles and flasks
(150-250ml capacity).
Chemical: Culture medium (plate count agar) – see composition in the preceding topics (19.1).
Procedure:
Inoculation and incubation
1. Take two sterile Petri dishes
2. Transfer to each dish by means of a sterile pipette, 1ml of the test sample, if liquid or 1ml
of the initial suspension in the case of other products.
3. Take two further sterile Petri dishes.
4. Transfer to each dish by means of another sterile pipette, 1ml of the 10-1 dilution (liquid
product) or 1ml of the 10-2 dilution (other products).
5. If necessary, repeat this operation using further decimal dilutions.
6. Pour 12-15ml of the culture medium into each Petri dish.
7. Carefully mix the prepared dishes and allow the mixture to solidify by leaving the Petri
dishes to stand on a cool horizontal surface.
8. Invert the prepared dishes and place them in the incubator at 30±1ºC for 72±3hr.
Do not stack the dishes more than six high. Stacks of dishes should be separated from one
another and from the walls and top of the incubator.
Counting the colonies
1. Count the colonies on the plates using the colony counting equipment.
241
2. Examine the dishes in subdued light. It is important that pinpoint colonies should be
included in the count but it is essential that the operator avoid mistaking particles of
undissolved or precipitated matter in dishes for pinpoint colonies.
3. Examine doubtful objects carefully, using higher magnification where required to
distinguish colonies from foreign matter.
4. If less than one quarter of the dish is overgrown by spreading colonies, count the colonies
on the unaffected part of the dish and calculate the corresponding number of the entire
dish.
5. If more than one quarter of the dish is overgrown by spreading colonies, discard the
count.
Calculation
Calculate N, the number of colony forming units of bacteria per gram or per ml of sample using
the following equation:
N=
∑c
( n1 + 0.1n2 ) d
Where,
Σc = the sum of colonies counted on all dishes retained
n1 = the number of dishes retained in the first dilution
n2 = the number of dishes retained in the second dilution
d = dilution factor corresponding to the first dilution.
Example of calculation
A count of the colonies gave the following results:
At 10-2 dilution: 168 and 215 colonies
At 10-3 dilution: 14 and 25 colonies
168 + 215 + 14 + 25
N= = 19182
 2 + ( 0.1× 2 )  10−2
Round the result obtained to two significant figures. When the number to be rounded is 5, with
no further significant figures, round the number immediately to the left of the 5 to give an even
figure. For example, 28500 is rounded to 28000 and 11500 is rounded to 12000.
In the above example, rounding the results gives 19000 or 1.9×104 cfu of total count per gram or
per mil of the product.
242
19.5 Enumeration of Coliforms (Colony Count Technique)
Aim: To detect the presence of coliform organisms in milk and milk products.
Principle: Coliforms are bacteria which at 30ºC form characteristic colonies in crystal violet
neutral red bile lactose agar (VRBA) and which in the confirmation test cause fermentation of
lactose with the production of gas under the test conditions specified in this method.
The group of coliform microorganism includes the general Escherichia and Aerobacter. The
widespread distribution of coliform in nature, feeding itself on available food of its habitat,
permits a minimal occurrence of these microorganisms in milk, milk products, surfaces of dairy
equipment, air and water supply. A large number of these bacteria indicate poor hygiene and
negligence. Proper heat treatment is sufficient to destroy the coliform bacteria and its presence in
pasteurized milk and milk products serves as index of recontamination or post pasteurization
contamination, largely due to poor hygienic practices.
Coliform bacteria are Gram-negative rods, and their ability to produce acid and gas as products of
lactose fermentation is the prime basis of laboratory tests for detecting their presence in a sample.
The medium used contains a bile salt that prevents the growth of the Gram-positive organisms.
One ml of diluted sample is inoculated in a Petri dish and grown on Violet Red Bile Agar
substrate. The coliform colonies appear purplish red with a zone of precipitated bile adjacent to
the colony.
Apparatus: Autoclave (121±1ºC); oven for dry sterilization (170-175ºC for 1 hr); Incubator
(30±1ºC); Petri dishes (90-100mm dia); pipettes (1ml and 10ml); water bath (45±1ºC); colony
counting equipment (mechanical or electronic digital counter); pH meter; bottles or flasks (for
boiling and storage of culture media); test tubes (16mm×160mm); Durham tubes (for use with the
test tubes).
Chemical: Violet Red Bile Agar medium, Lactose Bile Brilliant Green broth medium.
Procedure:
Test portion, initial suspension and further dilutions
Prepare the test portion, initial suspension (primary dilution) and further dilutions in accordance
with dilution technique method.
1. Mix the sample thoroughly and prepare 1 dilution following the “Dilution Technique A”
for liquid samples and “Dilution technique B” for solid samples.
2. Prepare two dishes from the liquid and/or from each dilution chosen.
3. Transfer with sterile pipette 1ml of liquid product or the appropriate dilutions to the center
of each dish. Touch the tip of the pipette on to a dry area in the Petri dish. Use another
sterile pipette to inoculate each dilution (10-1, 10-2, etc.) into the dishes.
4. Inoculate a sterile Petri dish with 1ml of sterile quarter strength Ringer solution and 15ml
of the medium for checking its sterility as “control”.
243
5. Pour 15ml molten VRB agar at 45ºC to each inoculated Petri dish and mix well. Allow
the agar to set and after complete solidification overlay another 5ml of the molten VRB
agar onto the surface of the inoculated medium so as to restrict surface growth (or to
maintain anaerobic condition for coliforms). Allow solidifying as described above.
6. Invert the prepared dishes and incubate the inoculated dishes and “control” in the
incubator set at 30ºC for 24±2 hr.
Enumeration
1. After the specified period of incubation select the Petri dishes with more than 10 and fewr
than 150 colonies.
2. Count using the colony counting equipment, the purplish red colonies a diameter of at
least 0.5mm and sometimes surrounded by a reddish zone of precipitated bile.
3. Confirm doubtful colonies, which can in particular e expected in the case of milk products
containing sugars other than lactose, immediately after the incubation period, according to
confirmation method described below.
Confirmation
1. Inoculate five colonies of each doubtful type, if available, into tubes of lactose bile
brilliant green broth.
2. Incubate the tubes in the incubator set at 30ºC for 24±2 hr
3. Consider colonies, which show gas formation in the Durham tubes as coliforms.
4. Take the results into account in the calculation.
Calculation
Calculate N, the number of colony forming units of coliforms per gram or per ml of sample using
the following equation:
N=
∑c
( n1 + 0.1n2 ) f
Where,
Σc = the sum of colonies counted on all dishes retained
f = the mass in g or the volume in ml of the undiluted test sample present in the dish with the first
dilution.
A count of the colonies of coliforms gave the following results:
At the 10-2 dilution: 130 and 125
At the 10-3 dilution: 20 and 18 colonies
244
138 + 125 + 20 + 18 301
N= = = 13680
 2 + ( 0.1× 2 )  10 −2
0.022
figure. For example, 28500 is rounded to 28000 and 11500 is rounded to 12000. In the above
example, rounding the results gives 13680 or 1.3×104 cfu of coliforms per g or per ml of the
product.
Initial suspendion (solid products) or
Test sample (liquid product)
1ml
10ml D*
3 tubes (5.3.1a) Note - Proceed in the 3 tubes (5.3.1b)

9.2.1 same way for further 9.2.1
dilutions (9.2.3)
Incubation 24h
9.2.4
D+1
Incubation 48h
Inoculation
9.3.1
D+2
Examination
Incubation 48h Inoculation

9.3.2
(+) (-)
D+3 1st case 2nd case 3rd case
Interpretation 9.4
Incubation 48h
(+) (-)
1st case 2nd case

D+4
Interpretation 9.4
(+) (-)
1st case 2nd case

Before incubation
After incubation
Key Gas Opacity No gas

formation formation
(+) (-)
Loop
(6.13)
Tubes containing single- Tubes containing single-

strength medium (5.3.1a) strength medium (5.3.1b)
Fig. 11: Coliform test

245
19.6 Enumeration of Yeasts and Molds by Colony Count Technique
(Yeast and Mold Count)
Aim: To detect the presence of yeast and mold in dairy products.

Principle: In the dairy industry, the presence of yeast and mold in dairy products as yogurt and
butter is an indicator of low hygienic level of the plant and/or poor quality raw materials.
The number of yeasts and molds in a measured amount of a dairy product can be estimated by
plating these organisms on a potato dextrose agar. The acidity of this substrate is adjusted to pH
3.5 and this pH favors both growth of yeast and mold but inhibits almost all bacteria.
Yeasts are unicellular, oval to elliptical cells, Gram-positive, non-motile, large in comparison
with bacteria, and commonly reproduce by budding – appearing like a cactus plant under the
microscope.
Molds are multicellular composed of aggregate of branching of thread – mycelium producing
spores. Mold colonies appear cottony or wooly, white, cream, green, black or brownish due to
pigmentation.
Procedure:
1. Mix the sample thoroughly and prepare 1 dilution following the “Dilution Technique A”
for liquid samples and “Dilution Technique B” for solid samples.
2. Inoculate sterile Petri dishes with 1ml per dish of each dilution (normally only 10-1).
Inoculate a sterile Petri dish with 1ml sterile quarter strength Ringers solution as
“control”.
3. Pour molten Potato Dextrose Agar at 45ºC to each inoculated dish and mix well.
Remember to add tartaric acid just before pouring to the inoculated dishes.
4. Incubate the inoculated dishes and “control” upside down at 25ºC for 48 hr.
5. Count the colonies selecting the dishes with 10-150 colonies.
Expression or Result:
Take the average count of all the yeast and mold colonies on the dishes counted and multiply
with the dilution inoculated. Report as “Yeast and mold per ml or per grams”.
[Source: Manual of Food Quality Control, Microbiological analysis, FAO, Rome, Vol. 4]
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19.7 Enumeration of Yeast and Mold by Colony Count Technique
Aim: To determine yeasts and molds in milk and milk products

Principle: Yeasts and molds are microorganisms which at 25ºC form colonies in a selective
medium under the conditions specified in this method. Plates are poured with a specified
selective culture medium and a specified quantity of the test sample if the initial product is liquid,
or of an initial suspension in the case of other products. Similar technique is followed for other
plates under the same conditions using decimal dilutions of the test sample. It should be
incubated aerobically at 25ºC for 5 days. Calculation of the number of yeasts and molds per gram
or per ml of sample from the number of colonies is done on plates chosen at dilution levels so as
to give a significant result.
Apparatus: usual microbiological laboratory apparatus; incubator (25±1ºC); water bath
(45±1ºC); pH meter; culture bottles or flasks; graduated pipettes (10ml and 1ml); Petri dishes.
Chemicals: Yeast extract-dextrose-chloramphenicol-agar medium
Composition:
Yeast extract 5g
Dextrose (C6H12O6) 20g
Chloramphenicol (C11H12C12N2O5) 0.1g
Agar 12-15g
Water 1000ml
Preparation
Dissolve the components in the water by boiling. If necessary, adjust the pH so that after
sterilization it is 6.6 at 25ºC. Dispense the agar medium into suitable containers. Sterilize at
121±1ºC for 15 min.
Procedure:
1. Take two sterile Petri dishes. Transfer to each dish by means of a sterile pipette 1ml of the
test sample if liquid or 1ml of the initial suspension in the case of other products.
2. Take further sterile Petri dishes. Transfer to each dish by means of another sterile pipette
1ml of the 10-1 dilution (liquid product) or 1ml of the 10-2 dilution (other product). If
necessary, repeat this operation using further decimal dilutions.
3. Pour about 15ml of yeast extract-dextrose-chloramphenicol-agar medium previously
melted and maintained at 45±1ºC in the water bath from a culture bottle or flask into each
Petri dish.
4. Carefully mix the inoculum with the medium and allow the mixture to solidify by leaving
the Petri dishes to stand on a cool horizontal surface.
5. Prepare a control plate with 15ml of the medium to check its sterility.
6. Invert the prepared dishes and place them in the incubator at 25±1ºC for 5 days.
247
Interpretation
After the specified period of incubation, count the colonies on each plate except the occasional
bacterial colony that may have grown.
Calculation
Use counts from plates containing more than 10 and fewer than 150 colonies. The number of
yeasts and molds per gram or per ml is equal to:
N=
∑c
( n1 + 0.1n2 ) d
Where,
Σc = the sum of colonies counted on the plates retained
d = the value corresponding with the dilution from which the first counts were obtained (for
example, 10-2).
A count of the colonies of yeast and mold gave the following results:
83 + 97 + 33 + 28 241
N= = = 10954
2 + ( 0.1× 2 )  10 −2
0.022
figure. For example, 28500 is rounded to 28000 and 11500 is rounded to 12000.
In the above example, rounding the results gives 11000 or 1.1×104 cfu of yeasts and molds per
gram or per ml of the product.
[Source: International Dairy Federation IDF: 94: 1990]
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19.8 Method for Yeast and Mold Count of Foodstuffs
Aim: To determine yeast and mold count in foodstuffs.

Principle: Most of the foods provide good cultural media for the growth of microorganisms.
Food laboratories and food processing industries, therefore, often conduct, among other rests,
microbiological analysis to check whether the food is fit for human consumption. Moreover,
microbiological examination of foods provides reliable information regarding the quality of raw
foods, the sanitary conditions under which the food was processed, and the effectiveness of the
method of preservation.
High yeast and mold count in a food product is not desirable and indicates that the product is
manufactured under improper plant sanitary control, improper packing and faulty storage.
Apparatus: Screw-cap or glass-stoppered glass bottles (suitable sizes, 25ml size); Petri dishes
with covers (100×15mm); pipettes (1.1ml, 10ml and 11ml); water bath (maintained at 43 to
45ºC); incubator (maintained at 25±1ºC or at 30±1ºC); autoclave (working at 121ºC); pH
measuring equipment.
Chemicals:
Buffered water blank (99ml or 90ml, sterilized), which can be prepared as follows:
Prepare stock phosphate buffer solution by dissolving 34g of KH2PO4 in 500ml distilled water,
adjust to pH 7.2 with 1N NaOH solution and make up to 1 liter with distilled water. For use as
dilution water, take 1.25ml of stock phosphate buffer solution and make up to 1 liter with
distilled water. Prepare stock Ringers solution by dissolving 9.0g NaCl, 0.42g KCl, 0.48g
crystalline CaCl2.6H2O (or 0.42g anhydrous CaCl2), and 0.2g sodium bicarbonate in 1000ml
distilled water. For use as diluents, take 250ml of the solution and make up to 1000ml with
distilled water.
1. Potato Glucose Agar (acidified)
Infusion from 200g 1000ml Boil 200g white, peeled and sliced potatoes in about
white potato 500ml water for 15 min or until soft. Filter through cotton
and make up to 1000ml
Glucose 20g
Agar 15g
Final pH 3.5±0.1
2. Alternative media (Salt Dextrose Agar)
Ammonium nitrate 1g
Ammonium sulfate 1g
K2HPO4 4g
KH2PO4 2g
NaCl 1g
Dextrose 10g
Yeast extract 1g
Water 1000ml
Final pH 3.5±0.1
249
3. Wort Agar Medium (particularly suitable for canned fruits, sugar and sugar syrups)
Malt extract (difco or equivalent) 15g
Peptone 0.78g
Maltose 1275g
Dextrin 2.75g
Glycerol 2.35g
K2HPO4 1.0g
Agar 20.0g
Distilled water 1000ml
Final pH 4.8±0.1
Preparation of medium – Heat the above mixture to boiling to dissolve the ingredients.
Distribute into tubes, flasks and autoclave for 15 min at 121ºC. Melt in flowing steam/boiling
water, cool and acidify to the required pH with a sterile 10% tartaric or lactic acid or citric acid
solution. Mix thoroughly and pour into plates. To preserve solidifying properties of the agar, do
not heat the medium after addition of the acid.
Tartaric acid – 10% solution, sterilized
Lactic acid – 10% solution, sterilized
Citric acid – 10% solution, sterilized
Bromophenol pH disc and solution – pH 2.8 to 4.4
Procedure:
1. Preparation of dilution blanks – Transfer 10g (using sterile spatula) or 10ml (using sterile
pipetted) of the sample, under aseptic condition to a 90-ml sterile buffered water blank
(11g or 11ml of sample may be added to 99ml of buffered water to give the same 1 to 10
dilution). Shake this dilution 25 times in the usual manner just before inoculating the Petri
dishes with the different dilutions given below in duplicate: 1:2 (5ml of the 1:10 dilution);
1:10 (1ml of the 1:10 dilution); and 1:100 (0.1ml of the 1:10 dilution).
2. Pouring plates, incubation and colony counting – Prior to pouring, adjust the reaction of
the melted medium in each container (preferable electrometrical) to pH 3.5±0.1 or
4.8±0.1 (depending upon the medium used) with sterile 10% tartaric or lactic or citric
acid. Because remelting of acidified medium may destroy its solidifying properties, adjust
only the amount needed for immediate plating. Amount of acid required for adjustment in
any one flask of same batch of medium ordinarily will establish amount needed for the
others containing equal quantities thereof.
3. For colorimetric adjustments, use bromophenol blue and titrate 5ml of medium with
dilute acid prepared by adding 1ml of sterile 10% stock acid solution to 19ml of water.
The number of ml of dilute acid used to titrate to pH 3.5 or 4.8 will represent the amount
of stock solution that should be added to 100ml of the medium. The amount of 10% acid
required will vary, depending on buffering properties of the medium.
4. The Petri dishes containing different dilutions are flooded with the melted and adjusted
medium. Not more than 30 min should elapse from the time of preparing dilution to
pouring of the medium on the plates. After solidification, the agar plates are to be
incubated for 5 days at 24±1ºC in case of meat and meat products and at 30±1ºC in other
cases.
250
5. At the end of the incubation period, count the colonies of yeast and mold in the same
manner as counting bacterial colonies in the plate count if interested only in the total yeast
and mold count. Generally, it is desirable to differentiate between molds and yeasts. It is
advisable to examine the plates at the end of three days for yeast colonies as they are
likely to be overgrown by molds. Make a separate count of the yeast colonies, which
usually will be characterized as smooth, moist, elevated or surface colonies. Mold
colonies are easily recognized by their profuse growth of hyphae. If only yeast counts are
required, add 0.25% of sterile sodium propionate solution to the plate at the time of
pouring to inhibit the growth of molds.
6. Although the acidity of the medium is supposed to inhibit the growth of bacterial
colonies, some may develop in spite of the acid. Usually these may be distinguished from
the yeast colonies because they are smaller. If there is doubt regarding the identity of
yeast, or bacterial colonies, the colonies in question should be confirmed by microscopic
observation of the stained smears.
The number of yeast and mold colonies per ml or per g of the material should be reported as the
total yeast and mold count, although in control work the separate yeast and mold count are
sometimes informative. To give the actual colony counts per ml or per g of the sample, the
colony counts obtained from 1:2 dilution (5ml of 1:10 dilution) should be multiplies by the factor
2; those from 1:10 dilution (1ml of the 1:10 dilution) by the factor 10; those from 1:100 dilution
(0.1ml of the 1:10 dilution) by the factor 100.
[Source: Bureau of Indian Standard IS: 5403-1969]
251
19.9 Detection of penicillin in milk by disk assay technique
Aim: To detect penicillin in milk by disk assay technique

Principle: the procedure is for the qualitative detection of penicillin in excess of 0.0025IU/ml in
raw milk, processed liquid milk, partially skimmed milk, skimmed milk and reconstituted dried
milk.
Penicillin in milk
The milk contains penicillin when the sample gives a clear zone of inhibition and a corresponding
sample to which penicillinase has been added gives no clear zone or a clear zone of smaller
diameter when both are test by the procedure described.
The procedure is based on the method of T. E. Galesloot and F. Hassing. A disk of absorbent
paper impregnated with the milk to be examined is place on the surface of an agar medium
inoculated with Bacillus stearothermophilus var calidolactis, which is particularly sensitive to
inhibition by penicillin. Incubation resulting in normal growth of the organism causes the agar to
become cloudy. The presence in the milk of substances, which are inhibitory to the growth of the
organism, is indicated by a clear zone around the disk. When a positive result for inhibitory
substances is obtained as indicated by a clear zone, penicillin is present. A clear zone, or a zone
of smaller diameter is obtained around a disk impregnated with a corresponding sample of the
milk in which penicillin has been inactivated by the addition of penicillinase.
The size of the clear zone depends, among other things, on the concentration and type of
inhibitory substance in the milk.
Materials
Test organism: Bacillus stearothermophilus var calidolactis strain C953 ((Netherlands Institute
for Dairy Research)
Slant agar
Yeast extract 2.0g
Peptone 5.0g
Meat extract 1.0g
Sodium chloride 5.0g
Agar 15g
Sterilized at 120ºC for 15 min
Final pH: 7.4±0.1ºC
Culture medium
Yeast extract 1.0g
Tryptone 2.0g
Glucose 0.05g
252
Agar medium
Yeast extract 2.5g
Tryptone 5.0g
Glucose 1.0g
Agar 15g
Standard penicillin solution
Prepare a standard solution of penicillin of 100 IU/ml by dissolving sodium or potassium benzyl
penicillin in sterile distilled water in a suitably stoppered sterile bottle. This solution should be
used only on the day of preparation keeping it in a refrigerator at 5ºC in the meantime.
Penicillinase solution
Prepare penicillinase solution of 1000 units/ml by dissolving penicillinase in sterile distilled
water. Store in a refrigerator at 5ºC for a maximum period of two weeks.
Milk free from inhibitory substances
Prepare 10% solution of inhibitor-free milk by the reconstitution in sterile distilled water of
skimmed milk powder previously tested and found to be free from inhibitory substances.
Alternatively, a sufficient quantity of fresh bulk milk test and found to be free from inhibitory
substances may be dispensed in bottles, heated for 1 hr at 100ºC and thereafter stored in a
refrigerator at 5ºC for a maximum period of 1 week.
Antibiotic assay paper disks
12-13mm diameter
A blank test should be carried out on nine disks from each batch of paper disks using sterile
distilled water to verify that the disks do not give a clear zone when incubated at 55±1ºC for 5 hrs
on the test plates.
Distilled water
Prepared using a glass still.
Apparatus having special characteristics
Sterile sample bottles with suitable closures (note that some rubber stoppers may deposit
inhibitory substances on the neck of the bottle); Petri dishes (good quality, with flat bottom of
uniform thickness, minimum internal diameter of 110mm); fine pointed forceps; incubator
(thermostatically controlled at 55±1ºC).
Samples
1. Samples should be taken in accordance with IDF standard 50 – “Standard methods for
sampling milk and milk products”.
2. Dried milk samples should be reconstituted in sterile distilled water prior to testing.
253
3. Liquid samples should be tested as soon as possible and preferably within 10 hrs of
sampling, maintaining the samples at as low a temperature as possible and at not more
than 5ºC in the meantime.
4. If it is not possible to test the samples within 10 hr, they should be maintained in deep-
freeze (-30 to -15ºC) to minimize inactivation of penicillin and should be tested within 24
hr.
Procedure
Maintenance of test culture
1. Keep the test culture on slant agar
2. Inoculate a tube of the slant agar in streaks, using a loop of the test culture, and then place
for 48 hr in an incubator at 55±1ºC.
3. After incubation, flame the wadding stopper of the tube, then force a little way into the
tube, which is then closed by a sterile rubber stopper. The culture obtained can be kept
several months in a refrigerator at 5ºC.
Propagation of the test culture
1. Transfer 10ml of the culture medium aseptically into a sterile 150ml Erlenmeyer flask.
2. Inoculate the culture medium in the flask with a loop of the culture from a tube of the
slant agar.
3. Alternatively, keep 0.1ml of the preceding liquid culture and this may be used provided it
is no more than 36 hr old and has been in a refrigerator at 5ºC.
4. Incubate at 55±1ºC for 16-18 hr.
5. When using a culture from slant agar or a liquid culture which is more than 36 hr old, the
above subculturing procedure should be carried out at least twice with no more than an
interval of 36 hr between subculturing.
Preparation of test plates
1. Melt the agar medium and cool to 55ºC, for example, by placing the medium in a
incubator at 55±1ºC for 30 min.
2. Add 1 part of freshly prepared liquid culture to 5 parts of the agar medium at 55±1ºC in a
test tube or bottle, mix thoroughly.
3. Transfer a quantity calculated to give a layer of 0.8 to 1.0mm thick of the test medium to
a sterile Petri dish previously heated to 55ºC.
4. Transfer the Petri dishes to a cold horizontal surface previously checked with a spirit level
and allow the agar medium to solidify.
5. When the medium has solidified, the lids are replaced on the dishes, which are then
inverted to minimize condensation on the surface of the agar medium.
6. The test plates thus prepared are used preferably on the same day, but they may be kept
several days provided they are cooled immediately after preparation and kept in a sealed
polythene bag in a refrigerator at 5ºC.
254
7. For reference purposes, mark off and number squares of 25-mm side on the bottom of the
test plate using a ruler and wax pencil or other suitable means.
Penicillin controls
1. Prepare a working solution of penicillin by making up 1.25ml of the standard penicillin
solution to 1000ml with sterile distilled water. This working solution contains 0.125
IU/ml.
2. Prepare 50ml of a solution of penicillin by making up 1.25ml of the standard penicillin by
adding inhibitor-free milk to 2ml of the working solution and mixing.
3. Prepare 50ml of a solution containing 0.005 IU/ml penicillin by adding inhibitor-free milk
to 2ml of the working solution and mixing.
4. The penicillin solutions are to be prepared on the day the test is carried out.
Penicillinase control
1. Mix thoroughly the sample of milk to examined and transfer approximately 10ml to a
suitable sterile wide-mouthed bottle.
2. Add about 0.4ml of the penicillinase solution and mix.
Test method
1. Mix thoroughly the sample of milk to be examined. Dip a paper disk into the milk sample
by means of a clean dry pair of forceps.
2. Remove any excess milk by touching the disk against the side of the sample bottle.
3. Place the disk flat on the surface of the agar at the center of a marked square and press
down gently with the forceps.
4. Repeat with another disk.
5. Repeat the operation in triplicate using the penicillin control containing 0.0025 IU/ml
penicillin, instead of the milk sample.
6. Repeat the operation in duplicate using the penicillin control containing 0.005IU/ml
penicillin, instead of the milk sample.
7. Repeat the operation in duplicate using the penicillinase control instead of the milk
sample.
8. When all nine disks have been placed on the agar medium in random fashion, the plates
are incubated with the cover downward at 55±1ºC for 0.5-5 hr.
9. After incubation the plates are examined in front of a suitable light source for clear zones
of inhibition around the paper disks.
10. The average diameters of the clear zones of inhibition for the milk sample, the penicillin
controls and the penicillinase control should be determined.
255
Interpretation of Results
1. Clear zones around the disks containing the penicillin control corresponding to 0.0025
IU/ml penicillin should just be perceptible; there should be somewhat large zones around
the disks containing the penicillin control corresponding to 0.005IU/ml penicillin.
2. The presence of clear zones of inhibition around the disks containing the sample of milk
indicates the presence of milk substances inhibitory to the test organism.
3. If there are no clear zones around the disks containing the penicillinase control, but there
are clear zones around the disks containing the milk sample, the inhibitory substance in
the milk is penicillin in excess of 0.0025 IU/ml.
4. If the average diameter of the clear zones around the disks containing the penicillinase
control is equal to the average diameter of the clear zones around the disks containing the
milk sample, the milk does not contain penicillin.
5. In there are clear zones around the disks containing the penicillinase control which are
smaller in average diameter than that of clear zones around the disks, the milk contains
penicillin together with other inhibitory substances.
6. The clear zones away from the disks are attributable to inhibitory substances other than
penicillin, while those around the disks are attributable to penicillin and other inhibitory
substances.
General note
The growth of the microorganism Bacillus stearothermophilus var. calidolactis is inhibited
particularly by penicillin and to a lesser extent by other antibiotics and inhibitors including those
which occur naturally in milk. Consequently, because antibiotics or inhibitory substance (other
than penicillin) are not detected by the test procedure it cannot be concluded that they are absent
in the sample.
Synthetic penicillins, such as sodium cloxacillin, may not be inactivated by penicillinase under
the conditions of the test and may therefore be classified with inhibitors other than penicillin.
Bacterial growth in the sample may produce penicillinase, which is partially, but not necessarily
completely, inactivated by heat treatment. Heat treatment may result in some slight destruction of
penicillin (and incidentally destruction of some, but not all, of the naturally occurring inhibitors
to which the organism is sensitive). Bearing in mind that the procedure is for qualitative detection
of penicillin, the samples should not be heat-treated, but liquid samples should be tested as soon
as possible after sampling and should be kept at low temperature in the meantime.
256
19.10 Detection of Mastitis in Milk
Aim: To detect mastitis in milk

Principle: Mastitis-infected milk can be detected by several chemical and other methods as
follows:
A: California Mastitis Test (CMT)
Apparatus: Glass dish, beaker, pipette.
Chemicals: CMT solution (15g NaOH, 5.0g Teepol, 0.1g bromothymol blue, 1000ml distilled
water).
Procedure:
1. Place 3 drops of CMT solution and 2 drops of milk sample in a glass dish and mix with a
glass rod.
2. Read the result within 15-30 sec.
The CMT is scored according to the amount of sediment or gel that is formed by the reaction of
milk and reagent. The scored is graded from 0 to 3.
0 scroe or CMT-negative: The mixture remains liquid with no evidence of formation of a gel.
CMT 1: This is weakly positive, indicated by distinct sediment but no tendency towards gel
formation. This indicates possibility of subclinical mastitis.
CMT 2: The mixture thickens quickly with some gel formation, indicating a possibility of
advanced subclinical mastitis.
CMT 3: The strong positive reaction in which gel is formed to produce a lump, ant to adhere to
the bottom of the dish, indicating possibility of clinical mastitis.
[Source: Standard of Performance of Mastitis Control Programs, Meller, D. D. and Kearns, J. V.,
1973]
B: Mastrip Test Method:
1. Put a Mastrip Test strip into a beaker containing milk sample.
2. Note the change of color in Mastrip test strip within few seconds.
257
The change of color should be noted down as follows:
Yellow: Negative
Green yellow: Subclinical mastitis
Green: Advanced subclinical mastitis
Blue: Clinical mastitis
[Source: Mastitis Control, Breakthrough in herbal research, the right way, the right time.
Ayurvet, Dabur Ayurvet Liminted]
C: Whiteslide Test:
Apparatus: Glass plate, glass rod.
Chemical: 1N NaOH.
Procedure:
1. Place 3 drops of milk and 2 drops of 1N NaOH solution on a glass plate and mix with a
glass rod for 20 sec. Examine against a black background.
2. Note the results within 20 sec.
Ropiness, flocs or slimes are indications of mastitis milk.
258
259
Chapter 20
HYGIENIC CONDITIONS IN DAIRY PLANTS
260
261
Hygienic Conditions in Dairy Plant
(General Guide on Sampling and Inspection Procedures)
Aim: To improve hygienic conditions in dairy plant.

Principle: the principal reason for checking plant hygiene is to insure that the plant will not
contaminate the product; however, if contamination has taken place, it is possible to discover
where in the circuit bacteriological infection, chemical contamination or contamination from filth
has taken place. Such checks will be necessary not only to ensure quality control requirements
within the plant but also to insure compliance of products with legal requirements. Also the
checks give information on the checking and sampling procedures used to endorse the practices
adopted to insure cleanliness of the plant.
There are three types of checks on the effectiveness of cleaning and disinfection for which
sampling might be performed.
1. Checking all contact surfaces which have to be cleaned after and shortly before the
production process and checking reusable product containers (bottles, molds, etc.) which
have to be cleaned and which will hold the finished product intended for sale.
2. Indirect checking on solutions or methods used for cleaning. Such checks will concern
principally the different operations carried out to insure that optimum cleanliness is
maintained.
3. Checking the raw materials or semi-finished products in the course of preparation or of
finished products. In practice such checks give a good idea of the quality of cleaning but
they are ancillary to the quality of raw material used and in some cases to the standard of
hygiene of the operators of the plants.
Scope and field of application
This topic chapter specifies methods of inspection and sampling to be employed to check the
effectiveness of cleaning and disinfection methods used in dairy plants and receiving stations
including milk collection tankers.
It deals with:
• Visual inspection
• Sampling from plant surfaces (product line, bottle washing equipment, containers, etc.)
• Reusable product containers
• Air
• Sampling of water and aqueous solutions other than those added to the product
• Sampling of raw materials and products
It does not deal with equipment normally installed in farms for example milking machinery or
refrigerated bulk milk tanks, nor does it deal with the equally important areas of health and
hygiene of personnel, factory environment, internal arrangement of the factory, methods of
cleaning, packaging materials brought in new from outside (paper, cardboard, plastic, new
- 260 -
bottles, etc.), food ingredients and additives, selection of number of units and treatment of the
sample in the laboratory.
The need for sampling should have been considered in the design of plant. It is important that any
devices which are included to enable samples to be taken are so designed and fitted that their use
results in representative samples being obtained without any adverse effect on the hygienic
condition of the plant (for example by introducing dead spots in cleaning circuits). Such design is
outside the scope of this chapter.
General instructions
1. The demands for effectiveness of cleaning operations vary from plant to plant depending
on management supervision, quality control requirements and type of production
undertaken.
2. A cleaning control should not be based solely on the results of microbiological tests even
if such checks are clearly of prime importance; other checks such as visual inspection,
smell and touch, chemical and/or physical analysis and intelligent interpretation of
records are important in order not to overlook such factors as visible residues,
malfunction of equipment, cleaning residues and corrosion.
3. Sampling for microbiological examination should only be carried out by personnel trained
in sampling for this purpose.
4. The frequency of sampling depends essentially on the type of manufacture, the means of
checking available to the organization and the costs acceptable to the organization in
carrying out the checking. In theory a check should be carried out after each cleansing or
at a stated interval when cleansing is continuous during a production run (for example, the
case of a bottle washer) or just prior to recommencement of production. However, in
practice, a number of checks are carried out to ensure the quality of the product and these
checks provide an indirect check on the efficiency of cleansing. Thus in practice, the
checking of effectiveness of cleansing depends upon the quality assurance for the product,
bearing in mind that deterioration in quality is often due to failure in cleaning.
5. Generally it can be said that the frequency of sampling should be determined by
measuring the variability of the process and comparing this with the risk of making non
standard product. The optimum solution to this problem requires a good quantitative
knowledge of the process, an understanding of statistical quality control and deliberate
management decisions on the degree of risk which is acceptable.
6. It is essential that samples should be accompanied by a report which identifies the place,
date and time of sampling including any batch details and the name and designation of the
sampling personnel. Where appropriate, the report must include any relevant conditions or
circumstances (for example the condition of the product containers and their
surroundings, temperature and humidity of the atmosphere, method of sterilization of the
sampling equipment, the location of the sampling point on the equipment, whether a
preservative substance has been added to the samples) and any special information
relating to the product being sampled (for example difficulty in achieving homogeneity of
the product).
- 261 -
Procedure:
Inspection and sampling procedure
1. Visual inspection
An immediate and important impression of the cleanliness of a production line in a dairy
plant can be obtained by visual inspection of the accessible parts of a plant. Included in this are
all open containers and those closed with a lid, pipe fittings with theirs washers and gaskets,
powder transport lines, air filters, parts which are operated by mechanical means (for example,
homogenizer, pistons, counting devices, stirrers and pumps) and reusable product containers.
Visual inspection should allow detection of damage due to corrosion or erosion.
Visual inspection may be carried out using good natural or artificial light. Use of ultraviolet
light should be resorted to only rarely because of the hazards involved. If UV light is used, it is
more effective when the plant has been flushed with a fluorescent dye. It is essential to cleanse
the plant fully after use of such dyes. Among many other confirmatory tests the following may be
applied to the surface under examination.
a. A clean spatula may be used to scrape a surface carefully to demonstrate the presence of a
film or residue on improperly cleaned equipment.
b. A piece of clean disposable muslin or tissue paper (moist if desired) wiped over the inside
of a can or over metal surfaces of other equipment will be soiled if the surface is
improperly cleaned.
c. No sign of fluorescence shall be detectable when the surface is carefully inspected with
long wavelength (340 to 380nm) UV light.
Stains, greasy residues, powder or thin, hard films are indicative of inadequate cleaning
conditions (for example inadequate times, chemical concentrations, flow velocities, etc.).
More substantial residues of product indicate poor training or discipline of cleaning personnel
and/or inadequate circulation and/or leaking valves. Incomplete drainage of equipment increases
the risk of contamination of product with chemicals and microorganisms.
At intervals, based on past observations and experience, product pumps and valves should be
opened and seals and rubbers inspected, especially if products with a high viscosity are
processed. This is important even when cleaning in place (CIP) is fully automated.
It is equally important to inspect at regular intervals the spray cleaning devices of the CIP system
to ascertain whether they are working correctly. If plant must be dismantled for checking, a rinse
and disinfection cycle of the section of plant involved should follow reassembly.
When visible residues are found in the equipment, it is essential that the cause be traced and
measures be taken to remedy the fault. There is only limited value in a microbiological check of
visually dirty equipment. Even if a sample happened to be satisfactory from microbiological
viewpoint, all other consequences of inadequate cleaning should be considered. However,
determination by chemical means of the main composition of the residue is often more helpful
when it comes to trouble-shooting.
Because visual inspection is the most rapid, cheapest and easiest method of examination it
should be carried out as often as possible (that is, daily).
- 262 -
2. Sampling procedures for equipment
2.1 Contact surfaces
Although all product contact surfaces should be checked, may such surfaces are inaccessible and
only limited facilities are available for sampling and checking the samples. Therefore a rigorous
selection will be necessary in practice. Particular attention should be given to those places which
are difficult to clean, for example recesses, elbows, valves, shafts, stirrer paddles, gauges, probes.
2.2 Times of checking
Appropriate times of checking will be after the cleansing and disinfection of the processing
equipment and shortly before reuse of the processing equipment to check that it has not become
contaminated while out of operation.
2.3 Direct methods
The methods for the examination of contact surface infection are numerous, but in a dairy plant
where it is essential that all surfaces are disinfected if not sterilized, rinse and swab tests are
preferred. Swab tests are used for plant and equipment where the rinsing technique is not
applicable. Also used is the impression method, where a sterile medium is pressed on the contact
surface, then placed and held in a sterile container and later incubated.
2.4 Indirect methods
As an alternative to the above mentioned direct methods, indirect methods can be used, either by
sampling and examination of the final rinse water (this may include a check for absence of
disinfecting agents) before the start of production or by sampling the initial processed product
passed through the plant and considering that as a ‘rinse’ test of the process line.
In many manufacturing plants there are large sections of process plant which operate as a unit
and which is desirable not to dismantle during routine operation and cleaning. Such plant is
frequently cleaned automatically and may operate under automatic control (for example, a UHT
production line, tank filling, allocation and emptying). In these cases, dismantling for rinsing,
swabbing or other direct methods can cause contamination and such methods should only be
attempted when other evidence makes a special investigation necessary.
The preferred method for this type of plant is to sample the first product emerging from the
process. However, periodic examination of equipment remains necessary.
2.5 Rinse methods
For rinsing purposes, sterile rinsing solutions (for example peptone/saline solution, quarter
strength Ringers solution) are normally dispensed in 500ml quantities. This quantity will suffice
for rinsing most pieces of equipment.
Do not use quantities less than 500ml. if the utensils to be tested will not contain this quantity
leave the surplus rinsing solution in the bottle.
Thoroughly wet, as far as possible, the whole surface of the piece of equipment to be rinsed with
the rinse solution. Agitation of the solution by rotary or other movement is necessary for
dislodging organisms.
Return the rinse to the bottle. The solution should be examined immediately. If this is not
possible any delay shall be kept to a minimum and the sample shall be cooled quickly to not more
- 263 -
than 4ºC and maintained between 0 and 4ºC until examined in any case within 24 hr of sampling
but preferably within 6 to 10 hr.
2.6 Swab technique
General: this technique is applicable to tanks, heat exchangers, large open surface coolers,
butter churns, cheese vats, cocks, agitators, air vents, bottle fillers, etc., which cannot
conveniently be rinsed. Swab tests can also provide useful local information in addition to the
overall picture given by the rinse test.
Apparatus: Test tubes (length 250mm and diameter 25mm, of heavy borosilicate or
polypropylene; stainless steel wire of suitable length and stiffness (approx. 350mm length and
2.6mm diameter) formed into a loop at one end and notched at the other end to hold the ribbon
gauze; ribbon gauze (non-medicated, 50mm wide).
Preparation of swab: The swab shall by 50mm in length and shall consist of 175mm of the
gauze wound round the notched end of the wire and secured with thread.
Sterilization of swab: Place the swab in 25ml of rinsing solution in a test tube, plug with
cotton wool or a suitable rubber closure, cover the plug with greaseproof paper and sterilize by
autoclaving at 121±1ºC for 15 min. To obtain a final quantity of 25ml of solution it is necessary
to start initially with a larger quantity to allow for evaporation during autoclaving. The actual
quantity shall be determined by trial and error with each individual autoclave.
Procedure: Where possible, examine an area 900cm2. Press the swab with a rolling motion
against the side of the test tube to remove excess liquid. Remove the swab and with heavy
pressure rub back and forth over the area to be examined so that all parts of the surface are treated
twice. The second rubbing should be at an angle of 90º to the first. Rotate the swab so that all
parts of it make contact with the surface under test. Return the swab to the test tube and insert the
cotton plug or rubber closure. The swab should be examined immediately. If this is not possible
any delay shall be kept to a minimum and the tube with the swabs shall be cooled quickly to not
more than 4ºC and maintained between 0 and 4ºC until examined in any case within 24 hr of
sampling but preferably within 6 to 10 hr.
3. Sampling procedures for reusable product containers
For reusable product containers a representative sample taken from each batch should be
checked.
Techniques: usually the rinse technique is used for sampling for reusable product containers
but depending on the material, impression and swabbing methods can be used.
Washed cans (or churns)
General: the examination is designed to give information on the condition of washed cans.
When the examination is carried out at the premises where the cans were washed, examine within
an interval of not less than 30 min and not more than 1 hr after washing.
Cans with open seams or containing milky water or easily removable milk solids or
milkstone layers shall be regarded as unsatisfactory.
Rinse method: Pour 500ml of sterile rinsing solution into the lid and then into the can.
Replace the lid. Lay the can on its side on a clean floor or on a can roller and roll it so that it
makes 12 complete revolutions. Allow the can to stand upright for 5 min and then repeat the
- 264 -
rolling. Pour the rinse solution from the can into the lid and then into the original bottle. In
transferring the original solution collect as much as possible of the 500ml. the solution should be
examined immediately. If this is not possible any delay shall be kept to a minimum and the
sample shall be cooled quickly to not more than 4ºC and maintained between 0 and 4ºC until
examined (in any case within 24 hr of sampling, but preferably within 6 to 10 hr).
Washed milk collection tankers
General: The method outlined is designed to give information on the condition of tankers
immediately after washing and immediately prior to reuse. Areas to be examined include lid,
hose, valves, and surrounding pipework as well as the barrel itself.
The examination should be undertaken not less than 30 min and within 1 hr after
washing.
Swab method: See above
Washed milk bottles
General: The method outlined is designed to give information on the condition of bottles
immediately after washing and immediately before reuse.
Sampling: Select bottles for examination immediately after washing and immediately before
reuse. Close with suitable sterile closures. After closing, take the bottles to the laboratory as soon
as possible.
Rinse method: Add 20ml of sterile rinsing solution to each of the bottles and replace the
closure. Use this quantity irrespective of the size of the bottle. Where bottles are taken from hot
section of machine for special purposes fit them with a suitable sterile closure and allow to cool
before rinsing. Hold the bottle horizontally in the hands and rotate gently 12 times in one
direction so that the whole of the internal surface is thoroughly wetted. Allow the bottle to stand
for not less than 15 min and not more than 30 min and again gently rotate 12 times so that the
whole internal surface is thoroughly wetted.
Return the rinse to its previous container. The solution should be examined
immediately. If this is not possible any delay shall be kept to a minimum and the
sample shall be cooled quickly to no more than 4ºC and maintained between 0 and
4ºC until examined (in any case within 24 hr of sampling, but preferably within 6 to
10hr).
4. Sampling procedures for water and aqueous solutions other than those added to the
product
Sampling of water and aqueous solutions should be carried out while the material is in motion
and care shall be exercised to ensure that the samples are representative. Not less than 2 liters
shall be taken. The sample shall be placed in a suitable clean airtight container, reserved solely
for water samples, which shall be of such a size that it is completely filled by the sample. Care
shall be taken to avoid the risk of contaminating the content in any way.
- 265 -
All cleaning solutions should be tested before use in order either to confirm that the
concentration is correct or to calculate any adjustments needed to obtain this concentration.
Routine monitoring of the amount of detergent which is necessary to add will indicate any
abnormal demand, which could be the result of heavy deposits on the plant surfaces.
Temperature of cleaning solutions should be checked during each cleaning cycle. Moreover
the duration of the cleaning cycles should be checked at regular intervals.
The varieties of conditions under which collection of water and solutions is performed make
it impossible to prescribe a fixed procedure of sampling. In general, the sampling procedure
should take account of the tests to be performed.
The sample container should be rinsed two or three times with the material being collected
before filling.
[Source: International Dairy Federation IDF-121A: 1987]
- 266 -
267
Chapter 21
RECOMMENDATION FOR ESTABLISHMENT OF

LABORATORY
268
269
Recommendation for Establishment of Laboratory
Aim: To establish dairy laboratory

Definition: The dairy quality-testing laboratory is established according to dairy plant capacity.
Considering the difference in milk handling process in dairy plants with different capacity the
dairy plants are categorized into three categories, viz., Large, Medium, and Small. Large-scale
processing plants are having capacity more than 20,000 liters per day. The medium-scale plants
are those having capacity between 10,000 and 20,000 liters per day. The medium-scale plants are
those have capacity less than 10,000 liters per day.
The process of categorization of the dairy plants and quality-testing laboratory is to give idea for
the investment to establish such type of laboratory.
1. Small scale laboratory
2. Middle scale laboratory
3. Large scale laboratory
Standardization and Calibration of Equipment and Chemicals
Standardization and calibration of equipment, glassware and chemicals is very important. It
affects in the final result of the test if the equipment and glassware are not properly standardized.
All the equipment and glassware and chemicals should be controlled and calibrated. Calibration
and control of equipment and glassware should be done from certified institutions. The chemicals
should be standardized with standard chemicals.
While analyzing milk and milk products, all the chemicals, equipment, and glassware used in the
laboratory must be standardized and calibrated.
Small scale laboratory
Definition: Small scale laboratory conducts only the routine tests that entail simple equipment
and readily available chemicals. The number of tests to be done depends on the variety of product
produced. However, microbiological or other complicated tests (such as penicillin residues) are
not done. The tests are applied for raw and pasteurized milk, yogurt, ghee, paneer, etc. The tests
are important for purchase (milk payment), adulteration, and compliance with the mandatory
standard. Some of the common tests carried out in small scale laboratory are given in page 269
and 270. Those for medium scale and large laboratory are given through pages 271 to 280.
- 268 -
Product Analysis Name of equipment Name of chemicals
Raw milk Alcohol test Test tubes (15ml) Ethyl alcohol (60%,
Pipettes (1ml) 68%)
Alcohol hydrometer (0-100%)
Measuring cylinder (250-500ml)
Acidity test Glass burette with stand (25-50ml) 0.1N NaOH

Erlenmeyer flask (50ml) 1% phenolphthalein
Pipettes (10ml)
Adulteration tests
Sugar Test tubes (15ml) Conc. H2SO4
Pipettes (1 and 10ml) Resorcinol (pure)
Beaker
Heater
Sodium bicarbonate Test tubes (15ml) Alcohol (95%)
Pipettes (2ml) Rosalic acid (1%)
Starch test Test tubes Iodine solution
Pipettes
Milk payment tests

Fat test Gerber centrifuge H2SO4 (Sp. Gr. 1.820)
Butyrometer for milk Amyl alcohol (>60%)
Lock stopper
Stopper key
Butyrometer racks
Pipettes (10.75ml)
Pipette stand
Butyrometer brushes
Automatic tilt measure (10ml)
Hydrometer for sulfuric acid
Hydrometer for amyl alcohol
Water bath
Milk sample bottle (100ml)
Rack for sample bottle
Cleaning brushes for sample bottles
Plunger
Sample dipper
SNF test Lactometer

Thermometer (0-100ºC)
Lactometer jar
269
Pasteurized Fat test Gerber centrifuge H2SO4 (Sp. Gr. 1.820)
milk Butyrometer for milk Amyl alcohol (>60%)
Lock stopper
Stopper key
Butyrometer racks
Pipettes (10.75ml)
Pipette stand
Butyrometer brushes
Water bath
Plunger
Sample dipper
SNF test Lactometer

Lactometer jar
Yogurt Fat test Gerber centrifuge H2SO4 (Sp. Gr. 1.820)

Butyrometer stoppers
SNF test of yogurt Lactometer
milk Thermometer (0-100ºC)
Lactometer jar
Butter/ghee Moisture test Electronic balance

Hot air oven/hot plate
Glass rod
Aluminum dish
Desiccator
Paneer Moisture test Electronic balance Distilled water

Hot air oven/hot plate
Glass rod
Aluminum dish
Water bath
Desiccator
270
Medium scale laboratory
Definition: In medium scale laboratory, the number and types of tests are done are larger and
varied. This type of laboratory should have following pieces of equipment:
Raw milk Platform test
COB test Test tubes (15ml)
Bunsen burner
Pipettes (2ml)
Alcohol test Test tubes (15ml) Ethyl alcohol (60%,
Pipettes (1ml) 68%)
Measuring cylinder (250-500ml)
Acidity test Glass burette with stand 0.1N NaOH
Erlenmeyer flasks (50ml) 1% phenolphthalein
Pipettes (10ml)
Adulteration/ Test tubes Conc. H2SO4
Preservative/ Pipettes Resorcinol (pure)
Neutralizer/ Sugar Beaker
Heater
Salt Test tubes 1.69% AgNO3
Pipettes 5% pot. chromate
Urea Pipettes Sod. Hypochlorite
Test tubes Trichloroacetic acid
Filter paper 2% NaOH
Filtration set Phenol solution (pure)
NaHCO3 Test tubes Alcohol (95%)
pipettes Rosalic acid
starch Test tubes Iodine solution
Pipettes
Milk payment test
Stopper key
Pipettes (10.75ml)
Automatic tilt measure (10ml, 1ml)
Water bath
Plunger
Sample dipper
271
SNF test Lactometer
Lactometer jar
Milk quality test
(MBRT test) Water bath with cover Methylene blue
Reductase tubes tablets
Pipettes (1ml, 10ml)
Autoclave
Pasteurized Compositional test
milk
Fat test Same as for raw milk
SNF test Same as for raw milk
Microbiological test
Coliform test Incubator VRBA medium
Petri dish (glass) Disinfectants (Lysol,
Autoclave (22 lit) cresol, etc.)
Colony counter
Glassware (pipettes, flasks)
Bunsen burner (or spirit lamp)
Precision balance
Total count test Same as for coliform test PCA medium
Cream Fat test Butyrometer for cream Same as for raw milk
Pipettes (5ml)
Others same as for raw milk
Butter/ghee Fat test Butyrometer for butter Same as for raw milk
Others same as for raw milk
Moisture Balance
Oven
Salt test Balance 5% pot. Chromate
Conical flask (250ml) Silver nitrate solution
Beaker
Measuring cylinder (100ml)
Pipette (2ml)
Burette (50ml)
272
Yogurt Fat test Gerber centrifuge H2SO4 (Sp. Gr. 1.82)
Butyrometer for milk Amyl alcohol
Water bath
Tilt measure (1ml and 10ml)
Total solids test Balance Zinc oxide
Desiccator Distilled water
Drying oven
Dishes
Water bath
Blender
Paneer/ Moisture test Balance
Chhana Oven
Khoa Moisture test Balance
Oven
Fat test Butyrometer H2SO4
Centrifuge Amyl alcohol
Water bath
Lock stoppers
Misti doi Fat test Butyrometer H2SO4
Water bath
Lock stoppers
Total solids Aluminum dish
Oven
Balance
Ice cream Fat test Butyrometer H2SO4
Water bath
Lock stoppers
Oven
Balance
Overrun Weighing balance
Sugar Filter paper Fehlings solution A
Balance Fehlings solution B
Pipettes Hydrochloric acid
Burette Alumina cream
Conical flask Neutral lead acetate
Heating arrangements Sodium oxalate solution
Invert sugar solution
273
Large scale laboratory
Definition: Large scale laboratory is found in large scale dairy plant. There are different sections
for chemical laboratory and microbiological laboratory. All the chemical and microbiological
analyses are carried out in microbiological laboratory. Many of the equipment and chemicals
necessary for large scale laboratory are as follows:
Raw milk Platform test
COB test Test tubes (15ml)
Bunsen burner
Pipettes (2ml)
Alcohol test Test tubes Ethanol (60%, 68%)
Pipettes (1ml)
Measuring cylinder
Acidity test Glass burette with stand 0.1N NaOH
Erlenmeyer flask (50ml) 1% phenolphthalein
Pipette (10ml)
Adulteration tests
Sugar Test tube (15ml) Conc. H2SO4
Pipettes (1ml, 10ml) Resorcinol (pure)
Beaker
Heater
Salt Test tubes (15ml) 1.69% AgNO3
Pipettes (1ml) 5% pot. chromate
Urea Pipettes p-dimethylamino-
Test tubes benzaldehyde
Ethanol
Conc. HCl
NaHCO3 Test tubes (15ml) Alcohol (95%)
Pipettes (2ml) Rosalic acid
Starch Test tubes Iodine solution
Pipettes
Formaldehyde Test tubes Chromotropic acid
Pipettes H2SO4
Hydrogen peroxide Test tubes Paraphenylenediamine
Pipettes solution
Caustic soda Pipettes 0.1N HCl
Test tubes
Burettes
Porcelain dish
Milk payment test
274
Stopper key
Butyrometer racks
Pipettes (10.75ml)
Pipette stand
Butyrometer brushes
Automatic tilt measure (1ml, 10ml)
Water bath
Plunger
Sample dipper
SNF test Lactometer
Lactometer jar
Fat/ Protein/ Freezing Infrared electronic milk analyzer
point Sampling bottle
Milk quality test
Methylene Blue Test Water bath with cover Methylene blue tablets
Reductase tube
Rubber stopper
Autoclave
Other glassware
Pasteurized Chemical test
milk
Phosphatase test Test tubes Lactognost tablets (I,
Pipettes (1ml) II, and III)
Comparison chart
Acidity test Burette (25ml) 0.1N NaOH
Conical flask (50ml) 1% phenolphthalein
Pipette (10ml)
Compositional test
Fat/ Protein Same as for raw milk Same for raw milk
SNF test Same as for raw milk Same as for raw milk
Bacteriological test
Coliform test Incubator Ringers tablets
Petri dish (glass) VRBA medium
Sterilizing box for Petri dish Diluents
Pipettes (1ml)
275
Sterilizing box for pipettes
Test tubes (20ml, for dilution)
Autoclave (22 lit)
Colony counter
Bunsen burner
Precision balance
Total Plate Count Same as for coliform test Plate count agar
Cream Chemical test
pH Same as for pasteurized milk Same as for Milk
Acidity test Burette 0.1N NaOH
Porcelain dish 1% phenolphthalein
Conical flask
Pipette (10ml)
Compositional test
Fat test Cream Butyrometer H2SO4 (Sp. Gr. 1.525)
Others (same as for milk) Amyl alcohol
Coliform count Incubator Ringers tablets
Petri dish VRBA medium
Pipettes (1ml)
Racks for test tubes
Autoclave (for test tubes)
Colony counter
Bunsen burner
Precision balance
Butter Chemical tests
pH test Same as for milk Same as for milk
Acidity Same as for milk Same as for milk
Peroxide value Graduated pipette (1ml) Acetic acid-Chloroform
Erlenmeyer flask (250ml) Potassium iodide
Measuring cylinder Sod. Thiosulfate
Balance Starch indicator
Compositional test
Fat test Butyrometer (for cream) Gerber H2SO4
Others (same as for milk) Amyl alcohol
Moisture test Precision balance
Dish (aluminum)
276
Bunsen burner
Tongs
Double ended spatula
Salt content in butter Balance 5% potassium chromate
Erlenmeyer flask (250ml) 0.1N AgNO3
Measuring cylinder (100ml)
Burette
Solid not fat content Gooch crucible Petroleum hydrocarbon
Glass funnel solvent
Flask (250ml)
Desiccator
Asbestos sheet
Coliform count Same as for milk Ringers tablets
VRBA medium
Diluents
Ghee Chemical test
Acidity test Glass burette (50ml) 95% alcohol
Porcelain dish (100ml) 0.1N NaOH
Conical flask (250ml) 1% phenolphthalein
Pipette (50ml)
Peroxide value Graduated pipette (1ml) Acetic acid-Chloroform
Erlenmeyer flask (250ml) Potassium iodide
Measuring cylinder (100ml) Sod. thiosulfate
Balance Starch indicator
RM value Condenser Glycerol (98%)
Still head H2SO4
Receiving flask 9110ml) Phenolphthalein
Polenske flask Sodium hydroxide
Stand for condenser Filter paper
Graduated cylinder (10ml, 25ml)
Gas burner
Glass funnel
Vegetable fat in ghee Conical flask (250ml) Potassium hydroxide
Microfiltering device) Digitonine solution
Melting point apparatus Ethanol
Melting point tubes Diethyl ether
Microscope and slides Acetic anhydride
Cover slips
Thermometer
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Compositional test Precision balance
Beaker (or aluminum dish)
Bunsen burner
Tongs
Double ended spatula
Yogurt Chemical test
pH test pH meter Buffer solutions
Beaker
Acidity test Burette 0.1N NaOH
Porcelain dish 1% phenolphthalein
Compositional test
Butyrometer for milk Amyl alcohol
Water bath
Tilt measure (1ml, 10ml)
Total solids Balance Zinc oxide
Desiccator Distilled water
Drying oven
Dishes
Water bath
Coliform Incubator Ringers tablets
Pipettes (1ml)
Colony counter
Bunsen burner
Precision balance
Yeast/Mold count Same as for coliform test Potato Dextrose Agar
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Ice cream Compositional test
Fat test Ice cream butyrometer Gerber H2SO4
Pipette (1ml, 5ml) Amyl alcohol
Water bathe
Gerber centrifuge
Balance
Water bath
Oven
Balance
Overrun 100ml aluminum dish
Precision balance
Sugar test Filter paper Fehling’s solution A
Balance Fehling’s solution B
Pipettes Hydrochloric acid
Burette Alumina cream
Conical flask Neutral lead acetate
Heating arrangements Sodium oxalate solution
Invert sugar solution

Pipettes (1ml)
Colony counter
Bunsen burner
Precision balance
Total plate count Same as for coliform test Plate count agar
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Cheese/ Chemical test
Paneer
pH test Same as for pasteurized milk Same as for milk
Acidity test on whey Same as for milk Same as for milk
Compositional test
Fat test Gerber centrifuge H2SO4 (Sp. Gr. 1.53-1.60)
Butyrometer for cheese Amyl alcohol
Pipette (1ml, 10ml)
Water bath
Moisture Analytical balance
Desiccator
Oven
Aluminum dish
Salt content (cheese) Erlenmeyer flask
Burette
Pipette
Pipettes (1ml)
Colony counter
Bunsen burner
Precision balance
Yeast and mold count Same as for coliform test Potato Dextrose Agar
Milk Chemical test
powder
pH test Same as for milk Same as for milk
Acidity Same as for milk Same as for milk
Compositional test
Fat test Same as for milk Same as for milk
Moisture test Same as for butter Same as for butter
Physical test
Solubility index Solubility index mixer Water
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Chapter 22
GOOD LABORATORY PROCEDURES
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General requirements for the competence of Testing and Calibration
Laboratories
1. Scope
1.1 This International Standard specifies the general requirements for the competence to carry out
tests and/or calibrations, including sampling. It covers testing and calibration performed using
standard methods, non-standard methods, and laboratory-developed methods.
1.2 This International Standard is applicable to all organizations performing tests and/or
calibrations. These include, for example, first-, second-, and third-party laboratories, and
laboratories where testing and/or calibration forms part of inspection and product certification.
This International Standard is applicable to all laboratories regardless of the number of personnel
or the extent of the scope of testing and/or calibration activities. When a laboratory does not
undertake one or more of the activities covered by this International Standard, such as sampling
and the design/development of new methods, the requirements of those clauses do not apply.
1.3 The notes given provide clarification of the text, examples and guidance. They do not contain
requirements and do not form an integral part of this International Standard.
1.4 This International Standard is for use by laboratories in developing their quality,
administrative and technical systems that govern their operations. Laboratory clients, regulatory
authorities and accreditation bodies may also use it in confirming or recognizing the competence
of laboratories.
1.5 Compliance with regulatory and safety requirements on the operations of the laboratories is
not covered by this International Standard.
1.6 If testing and calibration laboratories comply with the requirements of this International
Standard thy will operate a quality system for their testing and a calibration activity that also
meets the requirements of ISO 9001 when they engage in the design/development of new
methods, and/or develop test programs combining standard and non-standard test and calibration
methods and ISO 9002 when they only use standard methods.
Annex A provides nominal cross-references between this International Standard and ISO 9001
and ISO 9002. ISO/IEC 17025 covers several technical competence requirements that are not
covered by ISO 9001 and ISO 9002.
Note1: It might be necessary to explain or interpret to certain requirements in this International
Standard to insure that the requirements are applied in consistent manner. Guidance for
establishing applications for specific fields, especially for accreditation bodies (see ISO/IEC
Guide 58: 1993, 4.1.3) is given in Annex B.
Note 2: If a laboratory wishes accreditation for part or all of its testing and calibration activities,
it should select an accreditation body that operates in accordance with ISO/IEC Guide 58.
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2. Normative references
The following normative documents contain provisions, which, through reference in this text,
constitute provisions of this International Standard. For dated references, subsequent amendments
based on this International Standard are encouraged to investigate the possibility of applying the
most recent editions of the normative documents indicated below. For undated references, the
latest edition of the normative document referred to applies. Members of ISO and IEC maintain
registers of currently valid International Standards.
ISO 9001: 1994, Quality systems – Model for quality assurance in design, development,
production installation and servicing.
ISO 9002: 1994, Quality systems – Model for quality assurance in production, installation and
servicing.
ISO/IEC Guide 2, General terms and their definitions concerning standardization and related
activities.
VIM, International Vocabulary of basic and general terms in metrology, issued by BIPM, IEC,
IFCC, ISO, IUPAC, and OIML.
Note 1: Further related standards, guides, etc., on subjects included in this International Standard
are given in the bibliography.
Note 2: It should be noted that when this International Standard was being developed, the
revisions of ISO 9001 and ISO 9002 were anticipitated to be published in late 2000 as a merged
ISO 9001:2000. This is no longer the case.
3. Terms and definitions
For the purposes of this International Standard, the relevant terms and definitions given in the
ISO/IEC Guide 2 and VIM apply.
Note: General definitions related to quality are given in ISO 8402, whereas ISO/IEC Guide 2
gives definitions specifically related to standardization, certification and laboratory accreditation.
Where different definitions are given in ISO 8402, the definitions in ISO/IEC Guide 2 and VIM
are preferred.
4. Management requirements
4.1 Organization
4.1.1 The laboratory or the organization of which it is part shall be an entity that can be held
legally responsible.
4.1.2 It is the responsibility of the laboratory to carry out its testing and calibration activities in
such a way as to meet the requirements of this International Standard and to satisfy the needs of
the client, the regulatory authorities or organizations providing recognition.
4.1.3 The laboratory management system shall cover work carried out in the laboratory’s
permanent facilities, at sites away from its permanent facilities, or in associated temporary or
mobile facilities.
4.1.4 if the laboratory is part of an organization performing activities other than testing and/or
accreditation, the responsibilities of key personnel in the organization that have an involvement
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or influence on the testing and/or calibration activities of the laboratory shall be defined in order
to identify potential conflicts of interest.
Note 1: Where a laboratory is part of a larger organization, the organizational arrangements
should be such that departments having conflicting interests, such as production, commercial
marketing or financing do not adversely influence the laboratory’s compliance with the
requirements of this International Standard.
Note 2: If the laboratory wishes to be recognized as a third-party laboratory, it should be able to
demonstrate that it is impartial and that it and its personnel are free from any undue commercial,
financial and other pressures which might influence their technical judgment. The third-party
testing or calibration laboratory should not engage in any activities that may endanger the trust in
its independence of judgment and integrity in relation to its testing or calibration activities.
4.1.5 The laboratory shall
a) have the managerial and technical personnel with the authority and resources needed to
carry out their duties and to identify the occurrence of departures from the quality system
or from the procedures for performing tests and/or calibrations, and to initiate actions to
prevent or minimize such departures (see also 5.2);
b) have arrangements to insure that its management and personnel are free from any undue
internal and external commercial, financial and other pressures and influences that may
adversely affect the quality of their work;
c) have policies and procedures to insure the protection of its clients’ confidential
information and proprietary rights, including procedures for protecting the electronic
storage and transmission of results;
d) have policies and procedures to avoid involvement in any activities that would diminish
confidence in its competence, impartiality, judgment or operational integrity;
e) define the organization and management structure of the laboratory, its place in any parent
organization, and the relationships between quality management, technical operations and
support services;
f) specify the responsibility, authority and interrelationships of all personnel who manage,
perform or verify work affecting the quality of the tests and/or calibrations;
g) provide adequate supervision of testing and calibration staff, including trainees, by
persons familiar with methods and procedures, purpose of each test and/or calibration, and
with the assessment of the test or calibration results;
h) have technical management which has overall responsibility for the technical operations
and the provision of the resources needed to insure the required quality of laboratory
operations;
i) appoint a member of staff as quality manager (however named) who, irrespective of other
duties and responsibilities, shall have defined responsibility and authority for insuring that
the quality system is implemented and followed at all times; the quality manager shall
have direct access to the highest level of management at which decisions are made on
laboratory policy or resources;
j) appoint deputies for key managerial personnel (see note).
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Note: Individuals may have more than one function and it may be impractical to appoint deputies
for every function.
4.2 Quality system
4.2.1 The laboratory shall establish, implement and maintain a quality system appropriate to the
scope of its activities. The laboratory shall document its policies, systems, programs, procedures
and instructions to the extent necessary to assure the quality of the test and/or calibration results.
The system’s documentation shall be communicated to, understood by, available to, and
implemented by the appropriate personnel.
4.2.2 The laboratory’s quality system policies and objectives shall be defined in a quality manual
(however named). The overall objectives shall be documented in a quality policy statement. The
quality policy statement shall be issued under the authority of the chief executive. It shall include
at least the following:
a) the laboratory management’s commitment to good professional practice and to the quality
of its testing and calibration in servicing its clients;
b) the management’s statement of the laboratory’s standard of service;
c) the objectives of the quality system;
d) a requirement that all personnel concerned with testing and calibration activities within the
laboratory familiarize themselves with the quality documentation and implement and
policies and procedures in their work; and
e) the laboratory management’s commitment to compliance with this International Standard.
Note: the quality policy statement should be concise and may include the requirement that tests
and/or calibrations shall always be carried out in accordance with stated methods and clients’
requirements. When the test and/or calibration laboratory is part of a larger organization, some
quality policy elements may be in other documents.
4.2.3 The quality manual shall include or make reference to the supporting procedures including
technical procedures. Is shall outline the structure of the documentation used in the quality
system.
4.2.4 The roles and responsibilities of technical management and the quality manager, including
their responsibility for insuring compliance with this International Standard, shall be defined in
the quality manual.
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4.3 Document control
4.3.1 General
The laboratory shall establish and maintain procedures to control all documents that form part of
its quality system (internally generated or from external sources), such as regulations, standards,
other normative documents, test and/or calibration methods, as well as drawings, software,
specifications, instructions and manuals.
Note 1: In this context “document” could be policy statements, procedures, specifications,
calibration tables, charts, text books, posters, notices, memoranda, software, drawings, plans, etc.
These may be on various media, whether hard copy or electronic, and they may be digital,
analog, photographic or written.
Note 2: the control of data related to testing and calibration is covered in 5.4.7. The control or
records is covered in 4.12.
4.3.2 Document approval and issue
4.3.2.1 All documents issued to personnel in the laboratory as part of the quality system shall be
reviewed and approved for use by authorized personnel prior to issue. A master list or an
equivalent document control procedure identifying the current revision status and distribution of
documents in the quality system shall be established and be readily available to preclude the used
of invalid and/or obsolete documents.
4.3.2.2 The procedures adopted shall insure that:
a) authorized editions of appropriate documents are available at all locations where
operations essential to the effective functioning of the laboratory are performed;
b) documents are periodically reviewed and, where necessary, revised to insure continuing
suitability and compliance with applicable requirements;
c) invalid or obsolete documents are promptly removed from all points of issue or use, or
otherwise assured against unintended use;
d) obsolete documents retained for either legal or knowledge preservation purposes are
suitably marked.
4.3.2.3 Quality system documents generated by the laboratory shall be uniquely identified. Such
identification shall include the date of issue and/or revision identification, page numbering, the
total number of pages or a mark to signify the end of the document, and the issuing authority
(ies).
4.3.3 Document changes
4.3.3.1 Changes to documents shall be reviewed and approved by the same function that
performed the original review unless specifically designated otherwise. The designated personnel
shall have access to pertinent background information upon which to base their review and
approval.
4.3.3.2 Where practicable, the altered or new text shall be identified in the document or the
appropriate attachments.
4.3.3.3 If the laboratory’s documentation control system allow for the amendment of documents
by hand pending the re-issue of the documents, the procedures and authorities for such
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amendments shall be defined. Amendments shall be clearly marked, initialized and dated. A
revised document shall be formally re-issued as soon as practicable.
4.3.3.4 Procedures shall be established to describe how changes in documents maintained in
computerized systems are made and controlled.
4.4 Review of requests, tenders and contracts
4.4.1 The laboratory shall establish and maintain procedures for the review of requests, tenders
and contracts. The policies and procedures for these reviews leading to a contract for testing
and/or calibration shall insure that:
a) the requirements, including the methods to be used, are adequately defined, documented
and understood (see 5.4.2);
b) the laboratory has the capability and resources to meet the requirements;
c) the appropriate test and/or calibration method is selected and capable of meeting the clients’
requirements (see 5.4.2).
Any differences between the request or tender and the contract shall be resolved before any work
commences. Each contract shall be acceptable both to the laboratory and the client.
Note 1: The request, tender and contract review should be conducted in a practical and efficient
manner, and the effect of financial, legal and time schedule aspects should be taken into account.
For internal clients, reviews of requests, tenders and contracts can be performed in a simplified
way.
Note 2: The review of capability should establish that the laboratory possesses the necessary
physical, personnel and information resources, and that the laboratory’s personnel have the skills
and expertise necessary for the performance of the tests and/or calibrations in question. The
review may also encompass results of earlier participation in inter-laboratory comparisons or
proficiency testing and/or the running of trial test or calibration programs using samples or items
of known value in order to determine uncertainties of measurement, limits of detection,
confidence limits, etc.
Note 3: A contract may be any written oral agreement to provide a client with testing and/or
calibration services.
4.4.2 Records of reviews, including any significant changes, shall be maintained. Records shall
also be maintained of pertinent discussions with a client relating to the client’s requirements or
the results of the work during the period of execution of the contract.
Note: For review of routine and other simple tasks, the date and the identification (e.g., the
initials) of the person in the laboratory responsible for carrying out the contracted work are
considered adequate. For repetitive routine tasks, the review need be made only at the initial
enquiry stage or on granting of the contract for on-going routine work performed under a general
agreement with the client, provided that the client’s requirements remain unchanged. For new,
complex or advanced testing and/or calibration tasks, a more comprehensive record should be
maintained.
4.4.3 The review shall also cover any work that is subcontracted by the laboratory.
4.4.4 The client shall be informed of any deviation from the contract.
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4.4.5 If a contract needs to be amended after work has commenced, the same contract review
process shall be repeated and any amendments shall be communicated to all affected personnel.
4.5 Subcontracting of tests and calibrations

4.5.1 When a laboratory subcontracts work whether because of unforeseen reasons (e.g.,
workload, need for further expertise or temporary incapacity) or on a continuing basis (e.g.,
through permanent subcontracting, agency or franchising arrangements), this work shall be place
with a competent subcontractor. A competent subcontractor is one that, for example, complies
with this International Standard for the work in question.
4.5.2 The laboratory shall advise the client of the arrangement in writing and, when appropriate,
gain the approval of the client, preferably in writing.
4.5.3 The laboratory is responsible to the client for the subcontractor’s work, except in the case
where the client or a regulatory authority specifies which subcontractor is to be used.
4.5.4 The laboratory shall maintain a register of all subcontractors that it uses for tests and/or
calibrations and a record of the evidence of compliance with this International Standard for the
work in question.
4.6 Purchasing services and supplies

4.6.1 The laboratory shall have a policy and procedure(s) for the selection and purchasing of
services and supplies it uses that affect the quality of the tests and/or calibrations. Procedures
shall exist for the purchase, reception and storage of reagents and laboratory consumable
materials relevant for the tests and calibrations.
4.6.2 The laboratory shall insure that purchased supplies and reagents and consumable materials
that affect the quality of tests and/or calibrations are not used until they have been inspected or
otherwise verified as complying with standard specifications or requirements defined in the
methods for the tests and/or calibrations concerned. These services and supplies used shall
comply with specified requirements. Records of actions taken to check compliance shall be
maintained.
4.6.3 Purchasing documents for items affecting the quality of laboratory output shall contain data
describing the services and supplies ordered. These purchasing documents shall be reviewed and
approved for content prior to release.
Note: The description may include type, class, grade, precise identification, specifications,
drawings, inspection instructions, other technical data including approval of test results, the
quality required and the quality system under which they were made.
4.6.4 The laboratory shall evaluate suppliers of critical consumables, supplies and services which
affect the quality of testing and calibration, and shall maintain records of these evaluations and
list those.
4.7 Service to the client

The laboratory shall afford clients for their representatives’ cooperation to clarify the client’s
request and to monitor the laboratory’s performance in relation to the work performed, provided
that the laboratory insures confidentiality to other clients.
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Note 1: Such cooperation may include:
a) providing the client or the client’s representative reasonable access to relevant areas of the
laboratory for the witnessing of tests and/or calibrations performed for the client;
b) preparation, packaging, and dispatch of the test and/or calibration item needed by the client
for verification purposes.
Note 2: Clients value the maintenance of good communication, advice and guidance in technical
matters, and opinions and interpretations based on results. Communication with the client,
especially in large assignment, should be maintained throughout the work. The laboratory should
inform the client of any delays or major deviations in the performance of the tests and/or
calibrations.
Note3: Laboratories are encouraged to obtain other feedback, both positive and negative, from
their clients (e.g., client surveys). The feedback should be used to improve the quality system,
testing and calibration activities and client service.
4.8 Complaints
The laboratory shall have a policy and procedure for the resolution of complaints received from
clients or other parties. Records shall be maintained of all complaints and of the investigations
and corrective actions taken by the laboratory (see also 4.10).
4.9 Control of non-conforming testing and/or calibration work
4.9.1 The laboratory shall have a policy and procedures that shall be implemented when any
aspect of its testing and/or calibration work, or the results of this work, do not conform to its own
procedures or the agreed requirements of the client. The policy and procedures shall insure that:
a) the responsibilities and authorities for the management of non-conforming work are
designated and actions (including halting of work and withholding of test reports and
calibration certificates, as necessary) are defined and taken when non-conforming work is
identified;
b) an evaluation of the significance of the non-conforming work is made;
c) corrective actions are taken immediately, together with any decision about the acceptability
of the non-conforming work;
d) where necessary, the client is notified and work is recalled;
e) the responsibility for authorizing the resumption of work is defined.
Note: Identification of non-conforming work or problems with the quality system or with testing
and/or calibration activities can occur at various places within the quality system and technical
operations. Examples are costumer complaints, quality control, instrument calibration, checking
of consumable materials, staff observations or supervision, test report and calibration certificate
checking, management reviews and internal or external audits.
4.9.2 Where the evaluation indicates that the non-conforming work could recur or that there is
doubt about the compliance of the laboratory’s operations with its own policies and procedures,
the corrective action procedures given in 4.10 shall be promptly followed.
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4.10 Corrective action
1.10.1 General
The laboratory shall establish a policy and procedure and shall designate appropriate authorities
for implementing corrective action when non-conforming work or departures from the policies
and procedures in the quality system or technical operations have been identified.
Note: A problem with the quality system or with the technical operations of the laboratory may
be identified through a variety of activities, such as control of non-conforming work, internal or
external audits, management reviews, and feedback from clients or staff observations.
4.10.2 Cause analysis
The procedure for corrective action shall start with an investigation to determine the root cause(s)
of the problem.
Note: Cause analysis is the key and sometimes the most difficult part in the corrective action
procedure. Often the root cause is not obvious and thus a careful analysis of all potential causes
of the problem is required. Potential causes could include client training, consumables, or
equipment and its calibration.
4.10.3 Selection and implementation of corrective actions
Where corrective action is needed, the laboratory shall identify potential corrective actions. It
shall select and implement the action(s) most likely to eliminate the problem and to prevent
recurrence. Corrective actions shall be to a degree appropriate to the magnitude and the risk of
the problem. The laboratory shall document and implement any required changes resulting from
corrective action investigations.
4.10.4 Monitoring of corrective actions
The laboratory shall monitor the results to insure that the corrective actions taken have been
effective.
4.10.5 Additional audits
Where the identification of non-conformances or departures cast doubts on the laboratory’s

compliance with it own policies and procedures, or on its compliance with this International
Standard, the laboratory shall insure that the appropriate areas of activity are audited in
accordance with 4.13 as soon as possible.
Note: Such additional audits often follow the implementation of the corrective actions to confirm
effectiveness. An additional audit should be necessary only when a serous issue or risk to the
business is identified.
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4.11 Preventive action
4.11.1 Needed improvements and potential sources of non-conformances, either technical or

concerning the quality system shall be identified. If preventive action is required, action plans
shall be developed, implemented and monitored to reduce the likelihood of the occurrence of
such non conformances and to taken advantage of the opportunities for improvement.
4.11.2 Procedures for preventive actions shall include the initiation of such actions and
application of controls to ensure that they are effective.
Note 1: Preventive action is a pro-active process to identify opportunities for improvement rather
than to the identification of problems or complaints.
Note 2: Apart from the review of the operational procedures, the preventive action might involve
analysis of data, including trend and risk analyses and proficiency-testing results.
4.12 Control of records
4.12.1 General
4.12.1.1 The laboratory shall establish and maintain procedures for identification, collection,
indexing, access, filing, storage, maintenance and disposal of quality and technical records.
Quality records shall include reports from internal audits and management reviews as well as
records of corrective and preventive actions.
4.12.1.2 All records shall be legible and shall be stored and retained in such a way that they are
readily relievable in facilities that provide a suitable environment to prevent damage or
deterioration and to prevent loss. Retention times of records shall be established.
Note: Records may be in any media, such as hard copy or electronic media.
4.12.1.3 All records shall be held secure and in confidence.
4.12.1.4 The laboratory shall have procedures to protect and back-up records stored electronically
and to prevent unauthorized access to or amendment of these records.
4.12.2 Technical records

4.12.2.1 The laboratory shall retain records of original observations, derived data and some
information to establish an audit trail, calibration records, staff records and a copy of each test
report or calibration certificate issued, for a defined period. The records for each test or
calibration shall contain sufficient information to facilitate, if possible, identification of factors
affecting the uncertainty and to enable the test or calibration to be repeated under conditions as
close as possible to the original. The records shall include the identity of personnel responsible
for the sampling, performance of each test and/or calibration and checking of results.
Note 1: In certain fields it may be impossible or impractical to retain records of all original
observations.
Note 2: Technical records are accumulations of data (see 5.4.7) and information which result
from carrying out tests and/or calibrations and which indicate whether specified quality or
291
process parameters are achieved. They may include forms, contracts, worksheets, workbooks,
check sheets, work notes, control graphs, external and internal test reports and calibration
certificates, clients’ notes, papers and feedback.
4.12.2.2 Observations, data and calculations shall be recorded at the time they are made and shall
be identifiable to the specific task.
4.12.2.3 When mistakes occur in records, each mistake shall be crossed out, not erased, made
illegible or deleted, and the correct value entered alongside. All such alterations to records shall
be signed or initialed by the person making the correction. In the case of records stored
electronically, equivalent measures shall be taken to avoid loss or change of original data.
4.13 Internal audits
4.13.1 The laboratory shall periodically, and in accordance with a predetermined schedule and
procedure, conduct internal audits of its activities to verify that its operations continue to comply
with the requirements of the quality system and this International Standard. The internal audit
program shall address all elements of the quality system, including the testing and/or calibration
activities. It is the responsibility of the quality manager to plan and organize audits as required by
the schedule and requested by management. Such audits shall be carried out by trained and
qualified personnel who are, wherever resources permit, independent of the activity to be audited.
Note: The cycle for internal auditing should normally be completed in one year.
4.13.2 When audit findings cast doubt on the effectiveness of the operations or on the correctness
or validity of the laboratory’s test or calibration results, the laboratory shall take timely corrective
action, and shall notify clients in writing if investigations show that the laboratory results may
have been affected.
4.13.3 The area of activity audited, the audit findings and corrective actions that arise from them
shall be recorded.
4.13.4 Follow-up audit activities shall verify and record the implementation and effectiveness of
the corrective action taken.
4.14 Management reviews
4.14.1 In accordance with a predetermined schedule and procedure, the laboratory’s executive
management shall periodically conduct a review of the laboratory’s quality system and testing
and/or calibration activities to ensure their continuing suitability and effectiveness, and to
introduce necessary changes or improvements. The review shall take account of:
- the suitability of policies and procedures;
- reports from managerial and supervisory personnel;
- the outcome of recent internal audits
- corrective and preventive actions;
- assessments by external bodies;
- the results of inter-laboratory comparisons or proficiency tests;
- changes in the volume and type of the work;
- client feedback;
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- complaints;
- other relevant factors, such as quality control activities, resources and staff training.
Note 1: A typical period for conducting a management review is once every 12 months.
Note 2: Results should feed into the laboratory planning system and should include the goals,
objectives and action plans for the coming year.
Note 3: A management review includes consideration of related subjects at regular management
meetings.
4.14.2 Findings from management reviews and the actions that arise from them shall be recorded.
The management shall ensure that those actions are carried out within an appropriate and agreed
timescale.
5. Technical requirements
5.1 General
5.1.1 Many factors determine the correctness and reliability of the tests and/or calibrations
performed by a laboratory. These factors include contributions from:
- human factors (5.2);
- accommodation and environmental conditions (5.3);
- test and calibration methods and method validation (5.4);
- equipment (5.5)
- measurement traceability (5.6)
- sampling (5.7);
- the handling of test and calibration items (5.8).
5.1.2 The extent to which the factors contribute to the total uncertainty of measurement differs
considerably between (types of) tests and between (types of) calibrations. The laboratory shall
take account of these factors in developing test and calibration methods and procedures, in the
training and qualification of personnel, and in the selection and calibration of the equipment it
uses.
5.2 Personnel
5.2.1 The laboratory management shall insure the competence of all who operate specific
equipment, perform tests and/or calibrations, evaluate results, and sign test reports and calibration
certificates. When using staff who are undergoing training, appropriate supervision shall be
provided. Personnel performing specific tasks shall be qualified on the basis of appropriate
education, training, experience and/or demonstrated skills, as required.
Note 1: In some technical areas (e.g., non-destructive testing) it may be required that the
personnel performing certain task hold personnel certification. The laboratory is responsible for
fulfilling specified personnel certification requirements. The requirements for personnel
certification might be regulatory, included in the standards for the specific technical field, or
required by the client.
Note 2: The personnel responsible for the opinions and interpretation included in test reports
should, in addition to the appropriate qualifications, training, experience and satisfactory
knowledge of the testing carried out, also have:
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- relevant knowledge of the technology used for the manufacturing of the items, materials,
products, etc., tested, or the way they are used or intended to be used, and of the defects or
degradations which may occur during or in service.
- knowledge of the general requirements expressed in the legislation and standards; and
- an understanding of the significance of deviations found with regard to the normal use of
the items, materials, products, etc. concerned.
5.2.2 The management of the laboratory shall formulate the goals with respect to the education,
training and skills of the laboratory personnel. The laboratory shall have a policy and procedures
for identifying training needs and providing training of personnel. The training program shall be
relevant to the present and anticipated tasks of the laboratory.
5.2.3 The laboratory shall use personnel who are employed by, or under contract to, the
laboratory. Where contracted and additional technical and key support personnel are used, the
laboratory shall insure that such personnel are supervised and competent and that they work in
accordance with the laboratory’s quality system.
5.2.4 The laboratory shall maintain current job descriptions for managerial, technical and key
support personnel involved in tests and/or calibrations.
Note: Job descriptions can be defined in many ways. As a minimum, the following should be
defined:
- the responsibilities with respect to performing tests and/or calibrations;
- the responsibilities with respect to the planning of tests and/or calibrations and evaluation of
results;
- the responsibilities for reporting opinions and interpretations;
- the responsibilities with respect to method modification and development and validation of
new methods;
- expertise and experience required;
- qualifications and training programs;
- managerial duties
5.2.5 The management shall authorize specific personnel to perform particular types of sampling,
test and/or calibration, to issue test reports and calibration certificates, to give opinions and
interpretations and to operate particular types of equipment. The laboratory shall maintain records
of the relevant authorization(s), competence, educational and professional qualifications, training,
skills and experience of all technical personnel, including contracted personnel. This information
shall be readily available and shall include the date on which authorization and/or competence is
confirmed.
5.3. Accommodation and environmental conditions
5.3.1 Laboratory facilities for testing and/or calibration, including but not limited to energy
sources, lighting and environmental conditions, shall be such as to facilitate correct performance
of the tests and/calibrations.
The laboratory shall insure that the environmental conditions do not invalidate the results or
adversely affect the required quality of any measurement. Particular care shall be taken when
sampling and tests and/or calibrations are undertaken at sites other than a permanent laboratory
facility. The technical requirements for accommodation and environmental conditions that can
affect the results of tests and calibrations shall be documented.
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5.3.2 The laboratory shall monitor, control and record environmental conditions as required by
the relevant specifications, methods and procedures or where they influence the quality of the
results. Due attention shall be paid, for example, to biological sterility, dust, electromagnetic
disturbances, radiation, humidity, electrical supply, temperature, and sound and vibration levels,
as appropriate to the technical activities concerned. Tests and calibrations shall be stopped when
the environmental conditions jeopardize the results of the tests and/or calibrations.
5.3.3 There shall be effective separation between neighboring areas in which there are
incompatible activities. Measures shall be taken to prevent cross-contamination.
5.3.4 Access to and use of areas affecting the quality of the tests and/or calibrations shall be
controlled. The laboratory shall determine the extent of control based on its particular
circumstances.
5.3.5 Measures shall be taken to ensure good housekeeping in the laboratory. Special procedures
shall be prepared where necessary.
5.4 Test and calibration methods and method validation
5.4.1 General
The laboratory shall use appropriate methods and procedures for all tests and/or calibrations
within its scope. These include sampling, handling, transport, storage and preparation of items to
be tested and/or calibrated, and, where appropriate, an estimation of the measurement uncertainty
as well as statistical techniques for analysis of test and/or calibration data.
The laboratory shall have instruction on the use and operation all relevant equipment, and on the
handling and preparation of items for testing and/or calibration, or both, where the absence of
such instructions could jeopardize the results of tests and/or calibrations. All instructions,
standards, manuals and reference data relevant to the work of the laboratory shall be kept up to
date and shall be made readily available to personnel (see 4.3). Deviation from test and
calibration methods shall occur only if the deviation has been documented, technically justified,
authorized, and accepted by the client.
Note: International, regional or national standards or other recognized specifications that contain
sufficient and concise information on how to perform the tests and/or calibrations do not need to
be supplemented or rewritten as internal procedures if these standards are written in a way that
they can be used as published by the operating staff in the laboratory. It may be necessary to
provide additional documentation for optional steps in the method or additional details.
5.4.2 Selection of methods
The laboratory shall use test and/or calibration methods, including methods for sampling, which
meet the needs of the client and which are appropriate for the tests and/or calibration it
undertakes. Methods published in international, regional or national standards shall preferably be
used. The laboratory shall insure that it uses the latest valid edition of a standard unless it is not
appropriate or possible to do so. When necessary, the standard shall be supplemented with
additional details to insure consistent application.
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When the client does not specify the method to be used, the laboratory shall select appropriate
methods that have been published either in international, regional or national standards, or by
reputable technical organizations, or in relevant scientific texts or journals, or as specified by the
manufacturer of the equipment. Laboratory-developed methods of methods adopted by the
laboratory may also be used if they are appropriate for the intended use and if they are validated.
The client shall be informed as to the method chosen. The laboratory shall confirm that it can
properly operate standard methods before introducing the tests or calibrations. If the standard
method changes, the confirmation shall be repeated.
The laboratory shall inform the client when the method proposed by the client is considered to be
inappropriate or out of date.
5.4.3 Laboratory-developed methods
The introduction of test and calibration methods developed by the laboratory for it own use shall
be a planned activity and shall be assigned to qualified personnel equipped with adequate
resources.
Plans shall be updated as development proceeds and effective communication amongst all
personnel involved shall be insured.
5.4.4 Non-standard methods
When it is necessary to use methods not covered by standard method, these shall be subject to
agreement with the client and shall include a clear specification of the client’s requirements and
the purpose of the test and/or calibration. The method developed shall have been validated
appropriately before use.
Note: For new test and/or calibration methods, procedures should be developed prior to the tests
and/or calibrations being performed and should contain at least the following information:
a) appropriate identification;
b) scope;
c) description of the type of item to be tested or calibrated;
d) parameters or quantities and ranges to be determined;
e) apparatus and equipment, including technical performance requirements;
f) reference standards and reference materials required;
g) environmental conditions required and any stabilization periods needed;
h) description of the procedure including:
- affixing of identification marks, handling, transporting, storing and preparation of
items,
- checks to be made before the work is started,
- checks that the equipment is working properly and, where required, calibration and
adjustment of the equipment before each use,
- the method of recording the observations and results,
- any safety measures to be observed.
i) criteria and/or requirements for approval/rejection;
j) data to be recorded and method of analysis and presentation;
k) the uncertainty or the procedure for estimating uncertainty.
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5.4.5 Validation of methods
5.4.5.1 Validation is the confirmation by examination and the provision of objective evidence
that the particular requirements for a specific intended use are fulfilled.
5.4.5.2 The laboratory shall validate non-standard methods, laboratory-designed/developed
methods, standard methods used outside their intended scope, and amplifications and
modifications of standard methods to confirm that the methods are fit for the intended use. The
validation shall be as extensive as is necessary to meet the needs of the given application or field
of application. The laboratory shall record the results obtained, the procedure used for the
validation, and a statement as to whether the method is fit for the intended use.
Note 1: Validation may include procedures for sampling, handling and transportation.
Note 2: the techniques used for the determination of the performance of a method should be one
of, or a combination of, the following:
- calibration using reference standards or reference materials;
- comparison of results achieved with other methods;
- inter-laboratory comparisons;
- systematic assessment of the factors influencing the result;
- assessment of the uncertainty of the results based on scientific understanding of the
theoretical principles of the method and practical experience.
Note 3: When some changes are made in the validated non-standard methods, the influence of
such changes should be documented and, if appropriate, a new validation should be carried out.
5.4.5.3 The range and accuracy of the values obtainable from validated methods (e.g., the
uncertainty of the results, detection limit, selectivity of the method, linearity, limit of
repeatability and/or reproducibility, robustness against external influences and/or cross-
sensitivity against interference from the matrix of the sample/test object), as assessed for the
intended use, shall be relevant to the clients’ needs.
Note 1: Validation includes specification of the requirements, determination of the characteristics
of the methods, a check that the requirements can be fulfilled by using the method, and a
statement on the validity.
Note 2: As method-developed proceeds, regular review should be carried out to verify that the
needs of the clients are still being fulfilled. Any change in requirements requiring modifications
to the development plan should be approved and authorized.
Note 3: Validation is always a balance between costs, risks and technical. There are many cases
in which the range and uncertainty of the values (e.g., accuracy, detection limit, selectivity,
linearity, repeatability, reproducibility, robustness and cross-sensitivity) can only be given in a
simplified way due to lack of information.
5.4.6 Estimation of uncertainty of measurement
6.4.6.1 A calibration laboratory, or a testing laboratory performing its own call shall have and
shall apply a procedure to estimate the uncertainty of measurement for all calibrations and types
of calibrations.
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5.4.6.2 Testing laboratories hall have and shall apply procedures for estimating uncertainty of
measurement. In certain cases the nature of the test method may preclude rigorous, metrology
and statistically valid, calculation of uncertainty of measurement. In these cases the laboratory
shall at least attempt to identify all the components of uncertainty and make a reasonable
estimation, and shall insure that the form of reporting of the result does not give a wrong
impression of the uncertainty. Reasonable estimation shall be based on knowledge of the
performance of the method and on the measurement scope and shall make use of, for example,
previous experience and validation data.
Note 1: The degree of rigor needed in an estimation of uncertainty of measurement depends on
factors such as:
- the requirements of the test method;
- the requirements of client;
- the existence of narrow limits on which decisions on conformance to a specification
are based.
Note 2: In those cases where a well-organized test method specifies limits to the values of the
major sources of uncertainty of measurement and specifies the form of presentation of calculated
results, the laboratory is considered to have satisfied this clause by following the test method and
reporting instructions (see 5.10).
5.4.6.3 When estimating the uncertainty of measurement, all uncertainty components which are
of importance in the given situation shall be taken into account using appropriate methods of
analysis.
Note 1: Sources contributing to the uncertainty include, but are not necessarily limited to, the
reference standards and reference materials used, methods and equipment used, environmental
conditions, properties and condition of the item being tested or calibrated, and the operator.
Note 2: The predicted long-term behavior of the tested and/or calibrated item is not normally
taken into account when estimating the measurement of uncertainty.
Note 3: For further information, see ISO 5725 and the Guide to the Expression of Uncertainty in
Measurement (see bibliography).
5.4.7 Control of data
5.4.7.1 Calculations and data transfers shall be subject to appropriate checks in a systematic
manner.
5.4.7.2 When computers or automated equipment are used for the acquisition, processing,
recording, reporting, storage or retrieval of test or calibration data, the laboratory shall insure
that:
a) computer software developed by the user is documented in sufficient detail and suitably
validated as being adequate for use;
b) procedures are established and implemented for protecting the data; such procedures shall
include, but not limited to, integrity and confidentiality of data entry or collection, data
storage, data transmission and data processing;
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c) computers and automated equipment are maintained to insure proper functioning and are
provided with the environmental and operation conditions necessary to maintain the
integrity of test and calibration data.
Note: Commercial off-the-shelf software (e.g., word processing, database and statistical
programs) in general use within their designed application range may be considered to be
sufficiently standard. However, laboratory software configuration/modifications should be
validated as in 5.4.7.2a).
5.5 Equipment
5.5.1 The laboratory shall be furnished will all items of sampling, measurement and test
equipment required for the correct performance of the tests and/or calibrations (including
sampling, preparation of test/or calibration items, processing and analysis of test and/or
calibration data). In those cases where the laboratory needs to use equipment outside its
permanent control, it shall insure that the requirements of this International Standard are met.
5.5.2 Equipment and its software used for testing, calibration and sampling shall be capable of
achieving the accuracy required and shall comply with specifications relevant to the tests and/or
calibrations concerned. Calibration programs shall be established for key quantities or values of
the instruments where these properties have a significant effect on the results. Before being
placed into service, equipment (including that used for sampling) shall be calibrated or checked
to establish that it meets the laboratory’s specification requirements and complies with the
relevant standard specifications. It shall be checked and/or calibrated before use (see 5.6).
5.5.3 Equipment shall be operated by authorized personnel. Up-to-date instructions on the use
and maintenance of equipment (including any relevant manuals provided by the manufacturer of
the equipment) shall be readily available for use by the appropriate laboratory personnel.
5.5.4 Each item of equipment and its software used for testing and calibration and significant to
the result shall, when practicable, be uniquely identified.
5.5.5 Records shall be maintained of each item of equipment and its software significant to the
tests and/or calibrations performed. The records shall include at least the following:
a) the identity of the item of equipment and its software;

b) the manufacturer’s name, type identification, and serial number or other unique
identification;
c) checks that equipment complies with the specification (see 5.5.2);
d) the current location, where appropriate;
e) the manufacturer’s instructions, if available, or reference to their location;
f) dates, results and copies of reports and certificates of all calibrations, adjustments,
acceptance criteria, and the due date of next calibration;
g) the maintenance plan, where appropriate, and maintenance carried out to date;
h) any damage, malfunction, modification or repair to the equipment.
5.5.6 The laboratory shall have procedures for safe handling, transport, storage, use and planned
maintenance of measuring equipment to insure proper functioning and in order to prevent
contamination or deterioration.
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Note: Additional procedures may be necessary when measuring equipment is used outside the
permanent laboratory tests, calibrations or sampling.
5.5.7 Equipment that has been subjected to overloading or mishandling, gives suspect results, or
has been shown to be defective or outside specified limits, shall be taken out of service. It shall
be isolated to prevent its use or clearly labeled or marked as being out of service until it has been
repaired and shown by calibration or test to perform correctly. The laboratory shall examine the
effect of the defect or departure from specified limits on previous tests and/or calibrations and
shall institute the “Control of non-conforming work” procedure (see 4.9).
5.5.8 Whenever practicable, all equipment under the control of the laboratory and requiring
calibration shall be labeled, coded or otherwise identified to indicate the status of calibration,
including the date when last calibrated and the date or expiration criteria when recalibration is
due.
5.5.9 When, for whatever reason, equipment goes outside the direct control of the laboratory, the
laboratory shall insure that the function and calibration status of the equipment are checked and
shown to be satisfactory before the equipment is returned to the service.
5.5.10 When intermediate checks are needed to maintain confidence in the calibration status of
the equipment, these checks shall be carried out according to a defined procedure.
5.5.11 Where calibrations give rise to a set o correction factors, the laboratory shall have
procedures to insure that copies (e.g., in computer software) are correctly updated.
5.5.12 Test and calibration equipment, including both hardware and software, shall be
safeguarded from adjustments which would invalidate the tests and/or calibration results.
5.6 Measurement traceability

5.6.1 General
All equipment used for tests and/or calibrations, including equipment for subsidiary
measurements (e.g., for environmental conditions) having a significant effect on the accuracy or
validity of the result of the test, calibration or sampling shall be calibrated before being put into
service. The laboratory shall have an established program and procedure for the calibration of its
equipment.
Note: Such a program should include a system for selecting, using, calibrating, checking,
controlling and maintaining measurement standards, reference materials used as measurement
standards, and measuring and test equipment used to perform tests and calibrations.
Specific requirements
5.6.2.1 Calibration
5.6.2.1.1 For calibration laboratories, the program for calibration of equipment shall be designed
and operated so as to insure that calibrations and measurements made by the laboratory are
traceable to the International System of Units (SI) (Systeme intenational d’unites).
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A calibration laboratory establishes traceability of its own measurement standards and measuring
instruments to the SI by means of an unbroken chain of calibrations or comparison linking them
to relevant primary standards of the SI units of measurement. The link to SI units may be
achieved by reference to national measurement standards. Nation measurement standards may be
primary standards, which are primary realization of the SI units or agreed representations of SI
units based on fundamental physical constants, or they may be secondary standards which are
standards calibrated by another national metrology institute. When using external calibration
services, traceability of measurement shall be assured by the use of calibration services from
laboratories that can demonstrate competence, measurement capability and traceability. The
calibration certificates issued by these laboratories shall contain the measurement results,
including the measurement uncertainty and/or a statement of compliance with an identified
metrological specification (see also 5.10.4.2).
Note 1: Calibration laboratories fulfilling the requirements of this International Standard are
considered to be competent. A calibration certificate bearing an accreditation body logo from a
calibration laboratory accredited to this International Standard, for the calibration concerned, is
sufficient evidence of traceability of the calibration data reported.
Note 2: Traceability to SI units of measurement may be achieved by reference to an appropriate
primary standard (see VIM: 1993, 6.4) or by reference to a natural constant, the value of which in
terms of the relevant SI unit is known and recommended by the General Conference of Weights
and Measures (CGPM) and the International Committee for Weights and Measures (CIPM).
Note 3: Calibration laboratories that maintain their own primary standard or representation of SI
units based on fundamental physical constants can claim traceability to the SI system only after
these standards have been compared, directly or indirectly, with other similar standards of a
national metrology institute.
Note 4: The term “identified metrological specification” mean that it must be clear from the
calibration certificate which specification the measurements have been compared with, by
including the specification or by giving and unambiguous reference to the specification.
Note 5: When the terms “international standard” or “national standard” are used in connection
with traceability, it is assumed that these standards fulfill the properties of primary standards for
the realization of SI units.
Note 6: Traceability to national measurement standards does not necessarily require the use of
the national metrology institute of the country in which the laboratory is located.
Note 7: If a calibration laboratory wishes or needs to obtain traceability from a national
metrology institute other than in its own country, this laboratory should select a national
metrology institute that actively participates in the activities of CIPM either directly or indirectly
though regional groups.
Note 8: The unbroken chain of calibrations or comparisons may be achieved in several steps
carried out by different laboratories that can demonstrate traceability.
5.6.2.1.2 There are certain calibrations that currently cannot be strictly made in SI units. In these
cases calibration shall provide confidence in measurements by establishing traceability to
appropriate measurement standards such as:
- the use of certified reference materials provided by a competent supplier to give a
reliable physical or chemical characterization of a material;
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- the use of specified methods and/or consensus standards that are clearly described and
agreed by all parties concerned. Participation in a suitable program of the inter-
laboratory comparisons is required where possible.
5.6.2.2 Testing
5.6.2.2.1 For testing laboratories, the requirements given in 5.6.2.1 apply for measuring and test
equipment with measuring functions used, unless it has been established that the associated
contribution from the calibration contributes little to the total uncertainty of the test result. When
this situation arises, the laboratory shall insure that the equipment used can provide the
uncertainty of measurement needed.
Note: The extent to which the requirements in 5.6.2.1 should be followed depends on the relative
contribution of the calibration uncertainty to the total uncertainty. If calibration is the dominant
factor, the requirements should be strictly followed.
5.6.2.2.2 Where traceability of measurements to SI units is not possible and/or not relevant, the
same requirements for traceability to for example, certified reference materials, agreed methods
and/or consensus standards, are required as for calibration laboratories (see 5.6.2.1.2).
5.6.3 Reference standards and reference materials
5.6.3.1 Reference standards

The laboratory shall have a program and procedure for the calibration of its reference standards.
Reference standards shall be calibrated by a body that can provide traceability as described in
5.6.2.1. Such reference standards of measurement held by the laboratory shall be used for
calibration only and for no other purpose, unless it can be shown that their performance as
reference standards would not be invalidated. Reference standards shall be calibrated before and
after any adjustment.
5.6.3.2 Reference materials
Reference materials shall, where possible, be traceable to SI units of measurement, or to certified
reference materials. Internal reference materials shall be checked as far as is technically and
economically practicable.
5.6.3.3 Intermediate checks
Checks needed to maintain confidence in the calibration status of reference, primary, or working
standards and reference materials shall be carried out according to defined procedures and
schedules.
5.6.3.4 Transport and storage
The laboratory shall have procedures for safe handling, transport, storage and use of reference
standards and reference materials in order to prevent contamination or deterioration and in order
to protect integrity.
Note: Additional procedures may be necessary when reference standards and reference materials
are used outside the permanent laboratory for tests, calibrations or sampling.
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5.7 Sampling
5.7.1 The laboratory shall have a sampling plan and procedures for sampling when it carries out
sampling of substances, materials or products for subsequent testing or calibration. The sampling
plan as well as the sampling procedure shall be available at the location where sampling is
undertaken. Sampling plans shall, whenever reasonable, be based on appropriate statistical
methods. The sampling process shall address the factors to be controlled to ensure the validity of
the test and calibration results.
Note 1: Sampling is a defined procedure whereby a part of a substance, material or product is
taken to provide for testing or calibration of a representative sample of the whole. Sampling may
also be required by the appropriate for which the substance, material or product is to be tested or
calibrated. In certain cases (e.g., forensic analysis), the sample may not be representative but is
determined by availability.
Note 2: Sampling procedures should describe the selection, sampling plan, withdrawal and
preparation of a sample or samples from a substance, material or product to yield the required
information.
5.7.2 Where the client requires deviations, additions or exclusions from the documented sampling
procedure, these shall be recorded in detail with the appropriate sampling data and shall be
included in all documents containing test and/or calibration results, and shall be communicated to
the appropriate personnel.
5.7.3 The laboratory shall have procedures for recording relevant data and operations relating to
sampling that forms part of the testing or calibration that is undertaken. These records shall
include the sampling procedure used, the identification of the sampler, environmental conditions
(if relevant) and diagrams or other equivalent means to identify the sampling location as
necessary and, if appropriate, the statistics the sampling procedures are based upon.
5.8 Handling of test and calibration items
5.8.1 The laboratory shall have procedures for the transportation, receipt, handling, protection,
storage, retention and/or disposal of test and/or calibration items, including all provisions
necessary to protect the integrity of the test or calibration item, and to protect the interests of the
laboratory and client.
5.8.2 The laboratory shall have a system for identifying test and/or calibration items. The
identification shall be retained throughout the life of the item in the laboratory. The system shall
be designed and operated so as to insure that items cannot be confused physically or when
referred to in records or other documents. The system shall, if appropriate, accommodate a sub-
division of groups of items and the transfer of items within and from the laboratory.
5.8.3 Upon receipt of the test or calibration item, abnormalities or departures from normal or
specified conditions, as described in the test or calibration method, shall be recorded. When there
is doubt as to the suitability of an item for test or calibration, or when an item does not conform
to the description provided, or the test or calibration required is not specified in sufficient detail,
the laboratory shall consult the client for further instructions before proceeding and shall record
the discussion.
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5.8.4 The laboratory shall have procedures and appropriate facilities for avoiding deterioration,
loss or damage to the test or calibration item during storage, handling and preparation. Handling
instructions provided with the item shall be followed. When items have to be stored or
conditioned under specified environmental conditions, these conditions shall be maintained,
monitored and recorded. Where a test or calibration item or a portion of an item is to be held
secure, the laboratory shall have arrangements for storage and security that protect the condition
and integrity of the secured items or portions concerned.
Note 1: Where test items are to be returned into service after testing, special care is required to
insure that they are not damaged or injured during handling, testing or storing/waiting processes.
Note 2: Sampling procedure and information on storage and transport of samples, including
information on sampling factors influencing the test or calibration result, should be provided to
those responsible for taking and transporting the samples.
Note 3: Reasons for keeping a test or calibration item secure can be for reasons of record, safety
or value, or to enable complementary tests and/or to be performed later.
5.9 Assuring the quality of test and calibration results
The laboratory shall have quality control procedures for monitoring the validity of tests and
calibrations undertaken. The resulting data shall be recorded in such a way that trends are
detectable and, where practicable, statistical techniques shall be applied to the reviewing of the
results. This monitoring shall be planned and reviewed and may include, but not be limited to, the
following:
a) regular use of certified reference materials and/or internal quality control using secondary
reference materials;
b) participation in inter-laboratory comparison or proficiency-testing programs;
c) replicate tests or calibrations using the same or different methods;
d) retesting or recalibration of retained items;
e) correlation of results for different characteristics of an item.
Note: The selected methods should be appropriate for the type and volume of the work
undertaken.
5.10 Reporting the results
5.10.1 General
The results of each test, calibration, or series of tests or calibrations carried out by the laboratory
shall be reported accurately, clearly, unambiguously and objectively, and in accordance with any
specific instructions in the test or calibration methods.
The results shall be reported, usually in a test report or a calibration certificate (see Note 1), and
shall include all the information requested by the client and necessary for the interpretation of the
test or calibration results and all information required by the method used. This information is
normally that required by 5.10.2, and 5.10.3 or 5.10.4.
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In the case of tests or calibrations performed for internal clients, or in the case of a written
agreement with the client, the results may be reported in a simplified way. Any information listed
in 5.10.2 to 5.10.4 which is not reported to the client shall be readily available in the laboratory
which carried out the tests and/or calibrations.
Note 1: Test reports and calibration certificates are sometimes called test certificates and
calibration reports, respectively.
Note 2: Test reports and calibration certificates may be issued as hard copy or by electronic data
transfer provided that the requirements of this International Standard are met.
5.10.2 Test reports and calibration certificates
Each test report or calibration certificate shall include at least the following information, unless
the laboratory has valid reasons for not doing so:
a) a title (e.g., “Test Report” or “Calibration Certificate”);
b) the name and address of the laboratory, and the location where the tests and/or
calibrations were carried out, if different from the address of the laboratory;
c) unique identification of the test report or calibration certificate (such as the serial
number), and on each page an identification in order to insure that the page is recognized
as a part of the test report or calibration certificate, and a clear identification of the end of
the test report or calibration certificate;
d) the name and address of the client;
e) identification of the method used;
f) a description of, the condition of, and unambiguous identification of the items(s) tested or
calibrated;
g) the date of receipt of the test or calibration item(s) where this is critical to the validity and
application of the results, and the date(s) of performance of the test or calibration;
h) reference to the sampling plan and procedures used by the laboratory or other bodies
where these are relevant to the validity or application of the results;
i) the test or calibration results with, where appropriate, the units of measurement;
j) the name(s), function(s) and signature(s) or equivalent identification of person(s)
authorizing the test report or calibration certificate;
k) where relevant, a statement to the effect that the results relate only to the items tested or
calibrated.
Note 1: Hard copies of test reports and calibration certificates should also include the page
number and total number of pages.
Note 2: It is recommended that laboratories include a statement specifying that the test report or
calibration certificate shall not be reproduced except in full, without written approval of the
laboratory.
5.10.3 Test reports
5.10.3.1 In addition to the requirements listed in 5.10.2, test reports shall, where necessary for
interpretation of the test results, include the following:
a) deviations from, additions to, or exclusions from the test method, and information on
specific test conditions, such as environmental conditions;
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b) where relevant, a statement of compliance/non-compliance with requirements and/or
specifications;
c) where applicable, a statement on the estimated uncertainty of measurement; information
on uncertainty is needed in test reports when it is relevant to the validity or application of
the test results, when a client’s instruction so requires, or when the uncertainty affects
compliance to a specification limit;
d) where appropriate and needed, opinions and interpretations (see 5.10.5);
e) additional information which may be required by specific methods, clients or groups of
clients.
5.10.3.2 In addition to the requirements listed in 5.10.2 and 5.10.3.1, test reports containing the
results of sampling shall include the following, where necessary for the interpretation of test
results:
a) the date of sampling;
b) unambiguous identification of the substance, material or product sampled (including the
name of the manufacturer, the model or type of designation and serial numbers as
appropriate);
c) the location of sampling, including any diagrams, sketches or photographs;
d) a reference to the sampling plan and procedures used;
e) details of any environmental conditions during sampling that may affect the interpretation
of the test results;
f) any standard or other specification for the sampling method or procedure, and deviations,
additions to or exclusions from the specification concerned.
5.10.4 In addition to the requirements listed in 5.10.2, calibration certificates shall include the
following, where necessary for the interpretation of calibration results:
a) the conditions (e.g., environmental) under which the calibrations were made that have an
influence on the measurement results;
b) the uncertainty of measurement and/or a statement of compliance with an identified
metrological specification or clauses thereof;
c) evidence that the measurements are traceable (see Note 2 in 5.6.2.1.1).
5.10.4.2 The calibration certificate shall relate only to quantities and the results of functional
tests. If a statement of compliance with a specification is made, this shall identify which clauses
of the specification are met or not met.
When a statement of compliance with a specification is made omitting the measurement results
and associated uncertainties, the laboratory shall record those results and maintain them for
possible future reference.
When statements of compliance are made, the uncertainty of measurement shall be taken into
account.
5.10.4.3 When an instrument for calibration has been adjusted or repaired, the calibration results
before and after adjustment or repair, if available, shall be reported.
306
5.10.4.4 A calibration certificate (or calibration label) shall not contain any recommendation on
the calibration interval except where this has been agreed with the client. This requirement may
be superseded by legal regulations.
5.10.5 Opinions and interpretations
When opinions and interpretations are included, the laboratory shall document the basis upon
which the opinions and interpretations have been made. Opinions and interpretations shall be
clearly marked as such in a test report.
Note 1: Opinions and interpretations should be confused with inspections and product
certifications as intended in ISO/IEC 17020 and ISO/IEC Guide 65.
Note 2: Opinions and interpretations included in a test report may comprise, but not be limited to,
the following:
− an opinion on the statement of compliance/non-compliance of the results with requirements;
− fulfillment of contractual requirements;
− recommendations on how to use the results;
− guidance to be used for improvements.
Note 3: In many cases it might be appropriate to communicate the opinions and interpretations
by direct dialogue with the client. Such dialogue should be written down.
5.10.6 Testing and calibration results obtained from subcontractors
When the test report contains results of tests performed by subcontractors, these results shall be
clearly identified. The subcontractor shall report the results in writing or electronically.
When a calibration has been subcontracted, the laboratory performing the work shall issue the
calibration certificate to the contracting laboratory.
5.10.7 Electronic transmission of results
In the case of transmission of test or calibration results by telephone, telex, facsimile or other
electronic or electromagnetic means, the requirements of this International Standard shall be met
(see also 5.4.7).
5.10.8 Format of reports and certificates
The formal shall be designed to accommodate each type of test or calibration carried out and to
minimize the possibility of misunderstanding or misuse.
Note 1: Attention should be given to lay-out of the test report or calibration certificate, especially
with regard to the presentation of the test or calibration data and ease of assimilation by the
reader.
Note 2: The headings should be standardized as far as possible.
307
5.10.9 Amendments to test reports and calibration certificates
Material amendments to a test report calibration certificate after issue shall be made only in the
form of a further document, or data transfer, which includes the statement:
“Supplement to Test Report [or Calibration Certificate], serial number…..[or as otherwise
identified]” or an equivalent form of wording.
Such amendments shall meet all the requirements of this International Standard.
When it is necessary to issue a complete new test report or calibration certificate, this shall be
uniquely identified and shall contain a reference to the original that it replaces.
308
Guidelines for Establishing Applications for Specific Fields
B.1 The requirements specified in this International Standard are stated in general terms and,
while they are applicable to all test and calibration laboratories, explanations might be needed.
Such explanations on applications are herein referred to as applications. Applications should not
include additional general requirements not included in this International Standard.
B.2 Applications can be thought of as an elaboration of the generally stated criteria
(requirements) of this International Standard for specific fields of test and calibration, test
technologies, products, materials or specific tests or calibrations. Accordingly, applications
should be established by persons having appropriate technical knowledge and experience and
should address items that are essential or most important for the proper conduct of a test or
calibration.
B.3 Depending on the application at hand, it may be necessary to establish applications for the
technical requirements of this International Standard. Establishing applications may be
accomplished by simply providing detail or adding extra information to the already generally
stated requirements in each of the clauses (e.g., specific limitations to the temperature and
humidity in the laboratory).
In some cases the applications will be quite limited, applying only to a given test or calibration
method or to a group of calibration or test methods. In other cases the applications may be quite
broad, applying for the testing or calibration of various products or items or to entire fields of
testing or calibration.
B.4 If the applications apply to a group of test or calibration methods in an entire technical field,
common wording should be used for all of the methods.
Alternatively, it may be necessary to develop a separate document of applications to supplement
this International Standard for specific types or groups of tests or calibrations, products, materials
or technical fields of tests or calibrations. Such a document should provide only the necessary
supplementary information, while maintaining this International Standard as the governing
document through reference. Applications which are too specific should be avoided in order to
limit the proliferation of detailed documents.
B.5 The guidance in this annex should be used by accreditation bodies and other types of
evaluation bodies when the develop applications for their own purposes (e.g., accreditation in
specific areas).
[Source: International Standard ISO/IEC 17025: 1999(E)]
309
Harihar Bhawan, Punchowk Lalitpur, Nepal

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LABORATORY HANDBOOK

FOR DAIRY INDUSTRY

National Dairy Development Board (NDDB)

National Dairy Development Board (NDDB)

The methodologies included follow the standard methodologies of:

• Nepal Bureau of Standard and Metrology (NBSM)

The priority given to the mentioned standards are as follows:

• 1st priority is given to Nepal Bureau of Standard

6. Chapter 6 – PASTEURIZED MILK…………………………………………………. 69

9. Chapter 9 – GHEE……………………………………………………………………... 108

10. Chapter 10 – ICE CREAM……………………………………………………………. 126

11. Chapter 11 – PANEER……………………………………………………………….. 142

12. Chapter 12 – CHHANA………………………………………………………………. 147

13. Chapter 13 – YOGHURT…………………………………………………………….. 151

15. Chapter 15 – KHOA…………………………………………………………………… 181

16. Chapter 16 – SKIM MILK AND WHOLE MILK POWDER……………………… 187

17. Chapter 17 – SWEETENED CONDENSED MILK………………………………… 204

18 Chapter 18 – BUTTERMILK…………………………………………………………. 228

19. Chapter 19 – MICROBIOLOGICAL ANALYSIS………………………………….. 233

20. Chapter 20 – HYGIENIC CONDITIONS IN DAIRY INDUSTRY………………... 259

21. Chapter 21 – RECOMMENDATIO FOR ESTABLISHMENT OF 267

22. Chapter 22 – GOOD LABORATORY PROCEDURES……………………………. 282

Aim: To accurately sample milk and dairy products.

Figure 1: Recommended agitator (plunger) for cans and buckets

Figure 3: Suitable dipper for liquids

[Source: International Dairy Federation (IDF) Standard 50C: 1995]

[Source: International Organization for Standardization (ISO/R) 707-1968 (E)]

DISINFECTION AND STERILIZATION

Equipment for chemical and sensory testing

1. Rinse in cold water

[Source: Quality Control Handbook, Dairy Development Corporation]

1. Exposure to hot air at 170-175°C for not less than 1 hour

1. Exposure to saturated steam at 100°C for 1 hour

[Source: Quality Control Handbook, Dairy Development Corporation]

[Source: Quality Control Handbook, Dairy Development Corporation]

RECOMMENDED SAMPLING PLAN

EXAMPLES OF REGISTRATION &

Aim: To register the observed result of individual products in organized tables.

10. Skim & Whole Milk Powder

Daily/Weekly Reporting Date: .

Daily Reporting Date: .

Daily Reporting Date: .

Daily/Weekly Reporting Date: .

Daily/Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Weekly Reporting Date: .

Aim: To determine the sensory quality of raw milk

[Source: International Dairy Federation (IDF) Standard 99C: 1997]

Aim: To determine the temperature of milk

[Source: Handbook of Food analysis, BIS SP: 18 (Part XI) -1981]

Aim: To determine pH of milk

[Source: Laboratory Guide in Dairy Chemistry Practicals, FAO]

Aim: To determine the suitability of milk for pasteurization by COB test