TESTING METHODS OF FOOD

MICROORGANISM

Food Technology Department

School of Biotechnology
International University -VNU HCMC
Instructor: Nguyễn Vũ Hồng Hà
 OUTLINE
- Preparation of microbial samples for analysis
- Methods to calculate and quantify microorganisms
- Analysis of microbial indicators of food
Preparation of microbial samples for analysis

Term definitions

 Sample: the units (subsamples) taken per lot for analysis.

 Sample unit: the smallest definable part of a lot, also
called a unit.
 Lot: a number of sample units produced in one batch of in
some specified period of time such that units will be about
of the same quality.
 Population: any finite or infinite collection of individuals
(samples or units) on which decisions are to be made.
Each lot or batch should consist of units of product of a
single type, grade, class, size and composition.
Preparation of microbial samples for analysis

Term definitions

 Representative sample: a sample that is considered

typically of a population in respect of certain
characteristics.
 Random sample: a sample that was chosen in such a
way that every sample or unit in the lot had equal
chance of being selected.
Preparation of microbial samples for analysis

Term definitions
Requirements to obtain a truly representative sample:
1. Determine the location of sampling points critical
to the population.
2. Establish a sample method representing the
population characteristics.
3. Select the sample size.
4. Specify the frequency of sampling.
1. Preparation of glassware and other
laboratory materials
 Preparation
 Sterilization/decontamination
 Equipment and materials
 Storage and management of clean and sterile glassware and
materials
 Use of decontamination and disinfection
 Waste management
 Washing
 1. Preparation of glassware and
other laboratory materials
Preparation
 Glassware and other laboratory materials used in microbiology
shall be of suitable design, used properly and prepared in such a
manner as to guarantee its cleanliness and/or sterility up until the
time of use.
 It should be designed to prevent or limit contact between the
operator and infectious material.
 Tubes and bottles should be stoppered by appropriate means.
 1. Preparation of glassware and
other laboratory materials
Sterilization/decontamination
 Sterilization by dry heat
Heat glassware, etc., in a sterilizing oven for at least 1 h at 170°C or
equivalent.
 Sterilization by moist heat (steam)

The temperature of the autoclave chamber shall remain at 121 °C for

at least 15 mins.
 Sterilization by irradiation
Exposure to a sufficient dose of ɣ-radiation.
 1. Preparation of glassware and
other laboratory materials
Sterilization/decontamination:
 Decontamination with chemical compounds
Example: Immersion in ethanol of at least 70 % volume fraction
followed by 5 min drying time.
 Exposure of all working surfaces of the sampling equipment

to a suitable flame.
 Ignition with ethanol of 96 % volume fraction
 1. Preparation of glassware and
other laboratory materials
Equipment and materials:
 may be disposable instead of re-usable things if the
specifications are similar.
For example: glassware, Petri dishes, pipettes, bottles, tubes,
loops, spreaders, etc.
 are suitable for use in microbiology (in particular as regards its

sterility).
 contain no substances that inhibit the growth of microorganisms
 1. Preparation of glassware and
other laboratory materials
Storage and manage of clean glassware and materials
 Protect clean glassware and materials against dust during storage,
in conditions which will maintain its cleanliness.
 Sterilized glassware to be (e.g. pipettes) should be placed in
special containers or wrapped in an appropriate material (special
paper, aluminium foil, etc.).
 Glassware to be autoclaved empty should allow free access of
steam, otherwise sterilization will not be achieved
 When sterilizing equipment is intended for microbiology, put an
expiry date (or date of manufacture) on each package.
 1. Preparation of glassware and
other laboratory materials
Use of decontamination and disinfection
 Decontamination of disposable equipment
 Decontamination of glassware and materials prior to use
 Decontamination of glassware and materials after use
 1. Preparation of glassware and
other laboratory materials
Waste management
 The correct disposal of contaminated materials does not directly affect
the quality of sample analysis, but it is a matter of good laboratory
management.
 Materials contaminated with risk category 3 microorganisms and their
containers shall be autoclaved before they are incinerated.
Washing
 Wash re-usable equipment only after it has been decontaminated.
After washing, rinse all equipment with deionized water.
 After washing, re-usable equipment shall be free of residues that
may affect the subsequent growth of microorganisms.
Examples: pipette washer, dishwasher, ultrasonic trough
 Reminder for the food inspector

Before going sampling

1. Does the laboratory need to know the sample is coming?
2. Have you got your authorization card?
3. Have you got the correct labels, forms, seals and equipment?
4. Is any specialized sampling equipment needed?
5. Do you need any specialized clothing?
When taking sample
1. What information should be taken about the food?
2. What do you need to tell the company that owns the food?
3. Is the security of the sealing procedures adequate?
4. Do you know how to transport the sample?
5. Do you know how to store the sample?
 2. Sampling

 A sample:
- Should be representative of the batch of product and has not
been damaged or changed during transport and storage.
- Should be protected against extraneous contamination.
- Must be identified and recorded information clearly and
completely to permit good traceability.
- Should be submitted in the original, unopened container.
- Should be packed in such a way that breakage or spillage is
avoided.
2. Sampling

 Samples must be clearly and completely identified with the

following information:
- Sample description.
- Collector‘s name.
- Name and address of the manufacturer.
- Lot number.
- Dealer and distributor.
- Date, place, time of collection.
 [Link]

Ensuring proper sample collection

 Training in sample collection.
 Aseptic technique.
 Traceability.
 Temperature control: samples should be chilled at (32º-40ºF)
 Time dependency: should be within 24 or 48 hrs
 Sample handling.
 Negative control
 [Link]
Selecting a sample site
 Product itself samples.
 Product-contact environmental samples
 Non-product contact environmental samples
 Field samples.
 Water samples.

When identifying sample sites, one must consider:

• Does the site reflect the product in its “intended use” state?
• Can a representative sample be obtained at the site with
reasonable control of preventing contamination?
• Is there a more representative site?
 Common sampling problems

 Small sample or sampling method may not be ideal for detection.

Examples-small swab device, small carcass or environmental area
sampled
 Sanitizer or excessive intervention might interfere with the test.
Example: insufficient drip time prior to carcass rinse procedure.
 Temperature abuse for the sample prior to testing. Holding under
refrigeration for long periods allows competing bacteria to grow.
 Freezing can kill some pathogens (e.g., Campylobacter)
 Common sampling problems

 Chilling the samples < -15ºC kill 10% and 50% of the aerobic
bacteria.
 The samples were transported to the laboratory at temperatures < 7°C,
but not below 0°.
 Process the samples in the laboratory within 1 h of collection or store
them at 2 ºC ± 2 °C for a maximum of 24 h
 2. Sampling

 A sterile sample container:

- Should not be more than three-quarters
full
- Should be opened for just long enough
to allow the sample to be transferred and
closed immediately afterwards.
 If the product is bulky or in a container

too large for submission to the laboratory,

transfer a portion aseptically to a sterile
sample container.
 Sample containers may be sealed with an
official seal.
2. Sampling

A sterile sample container:

- Should not be closed with cork stoppers or caps with cork seals
- Containers for solid, semi-solid or viscous products should be
wide-mouthed.
 - Small retail containers are considered as sample containers; the
sample should consist of the contents of one or more intact,
unopened containers
2. Sampling

 Note: the temperature at the time of collection and upon

receipt is useful to the laboratory for the interpretation of
results.
 2. Sampling

 Transport
- Deliver samples to the laboratory promptly with the original
storage conditions maintained as nearly as possible.
- The product label should indicate whether refrigeration is
required.
- Samples not requiring refrigeration or freezing may be packed
in a container using appropriate packing material to avoid breakage.
- Do not use loose ice as this may cause product contamination
if the container breaks or leaks.
- Do not allow the samples to freeze or to come into contact
with the frozen blocks of ice, if used.

 2. Sampling

 Transport
Unless otherwise specified in specific standards, the following
temperatures during transport are recommended:
- Stable products: ambient temperature (below 40°C);
- Frozen or deep-frozen products: below 15°C, preferably below
18°C;
- Other products not stable at ambient temperature: 1°C to 8°C.
When no conditions are specified, it is recommended that the parties
concerned agree on the duration and the temperature of transport.
2. Sampling

Receipt:
 Check the condition of the samples on receipt.

 Check sample containers for evident physical defects.

 Note the following information: date (and time, if

relevant) of receipt; details of sampling (sampling date
and time, if relevant and known, sample condition);
client's name and address.
 2. Sampling

Storage: The following storage temperatures are recommended:

 stable products: ambient temperature (18°C to 27°C);

 frozen or deep-frozen products: below -15°C, preferably -18°C;

 other products not stable at ambient temperature, including spoiled

foods: 3°C ± 2°C.

 For highly perishable products (such as shellfish), testing should

commence within 24 h of sampling.

 For perishable products (such as fish, raw milk), testing should

commence within 36 h
 Temperature recorders or indicators should be included to record in-

transit temperatures
2. Sampling

 Test portion:
- Specific rules for taking test portions
- Conservation and destruction of laboratory samples.
NOTE: It is not normally accepted practice to retest samples,
due to possible changes in microbial status
2. Sampling

 Sampling site:
- Depend on the slaughterhouse practices for different animals.
- Examine the sites with the highest prevalence of contamination.
 [Link]

Examples of sampling sites for raw meats

Pig Beef Lamp
Distal hind limb (trotter) (1) Brisket (2) Abdomen (flank) (3)
Hind limb, lateral (2) Forerib (3) Thorax, lateral (4)
Abdomen, lateral (belly) (3) Flank (4) Crutch (6)
Mid-dorsal region (mid-back) Flank groin (6) Breast, lateral (7)
(4)
Abdomen, medial (10) Round, lateral (8)
 [Link]

 All sampling plans have significant limitations. Therefore, it is need to

be evaluated the relative rigour of the program.
 Best sampling plans provide the opportunity but no guarantee of
detection .i.e., scattered contamination is difficult to detect.
 Frequent sampling and sampling multiple sites/time points provides a
better opportunity for detection.
Examples: Multiple samples per day vs. once per month
One “grab” sample per lot vs. “n60” per lot
 Does the type of sampling meet the intended need?
 Destructive vs. non-destructive sampling
 2. Sampling

Sample collection: representative of lots

- Sponge/Surface samples: carcasses, food contact surfaces.
- Liquid samples
- Solid samples: excision samples, ground samples
- Agar contact methods.
 Sample handling:
- Deliver sample to lab promptly/refrigerate
- Transport in Ice Chest
- Maintain at 0-4 C
- Do NOT Freeze
- Analyze within 36 h
 2. Sampling

Swab method:
 Contaminating bacteria are removed from the surface to be tested
by using a sterile swab.
 Standardization by using a reference square area is needed (e.g.
by sterile metal frame).
 Advantage : Even in case of heavy contamination, the number of
microorganisms can be determined by applying dilution
techniques.
 Disadvantage : Part of the contaminating flora may not be
recovered, in particular in case of uneven rugged surfaces, e.g.
meat
 2. Sampling

Destructive methods
 A standardized sample is cut out (“destructive”) from the surface of
meat or meat products.
Advantage:
 The testing includes all microorganisms present in the sample.

Samples can be exactly standardized according to surface area (cm2)

or weight (g).
 The sample comprises not only superficial contamination, but also

microorganisms from the interior of meat/meat products.

Disadvantages: ??
2. Sampling
Sampling techniques for meat
The purpose of the microbiological analysis may be to detect
and/or enumerate
 deep microbial flora,

 surface microbial flora, or

 surface and deep microbial flora (total).

The preparation of the test sample shall take account of the aim of
the analysis and the nature of the sample
 2. Sampling
Sampling techniques for raw meat
Non-destructive sampling: swabbing for carcass sampling
- Wet and dry swab method.

- Sponge sampling method.

- Gauze tampon method

- Carcass rinsing.
 2. Sampling
Sampling techniques for raw meat
Surface samples (swabs and small cloths)
 Mix the swabs in the same diluent as the one used to saturate them.
 The resulting solution may be diluted afterwards in a decimal
manner.
2. Sampling
Sampling techniques for meat

Specific procedures
 Laboratory sample with a mass equal to or less than 50 g.
 Slice or individual portion of meat or cooked meat: Take a strip
from the middle.
 Meat products in “skins”.
 Pre-cooked meals.
 2. Sampling
Sampling techniques for meat
 Manufactured products stored under refrigeration:
For packaged products, proceed as follows:
a. Soft packaging: to be removed using scissors or a scalpel.
b. Rigid packaging (glass containers, etc.): clean and disinfect the
outside surface thoroughly using alcohol; open under sterile conditions.
+ Clean the surface of rigid or semi-rigid packaging using soap or
detergent with water then dry with a clean towel or fresh absorbent
paper.
+ Disinfect the outside of the packaging to avoid contamination
when opening. The disinfecting process shall be carried out with great
care.
 2. Sampling
Sampling techniques for meat
 Manufactured products stored under refrigeration:
- Sampling from deep within the test material.
- Sampling from the surface of meat.
- Sampling from individual sliced portions
2. Sampling
Sampling techniques for meat
 Sampling of poultry carcasses:
Samples may be taken as follows:
- From the depth of the pectoral muscle;
- As a surface sample from the skin;
- By rinsing the whole carcass in diluent (non-destructive
sampling).
2. Sampling
Specific procedures
 Samples from cuts of poultry meat.
 Sampling rabbit carcasses: for animals presented with hair,
use scalpels and tongs to remove the appropriate area of skin
from cuts of meat.
- With cauterization to take a deep sample within the thigh
muscle.
- Without cauterization, use sterile scalpel and forceps to
take samples from designated surfaces of the carcass and place in
a sterile container for further preparation.
 2. Sampling
Specific procedures
 Procedure for frozen meats.
- Large pieces or blocks from which samples are taken without prior
defrosting.
- Total sample (surface and deep parts): Using the electric drill with he
speed about 900 r/min to avoid fusion or dispersion of the shavings
- Sample at depth: using the wood chisel and the hammer remove a
surface strip with a thickness of around 3 mm from an area of about 6 cm
by 6 cm.
- Surface sample: apply the hot template to the surface of the frozen
meat.
- Small samples likely to be defrosted:
+ Defrost it at ambient temperature should not exceed 2 h to 3 h.
+ Defrost slowly in an enclosed chamber, 2°C ± 2°C, max of 18 h
 2. Sampling
Sampling techniques for frozen meat
Means of sampling a frozen or deep-frozen test piece or block for
non-homogeneous block:

1. Cauterized area
2. Tray
Dimension in millimetres
2. Sampling
Sampling techniques for processed meat
Procedure for partially
dehydrated meat extracts
 Open the packaging.
 Using a spatula/ corkborer take a
sample of a certain product mass and
operate using the same method as for
refrigerated products
 For dehydrated meats, refer to ISO
6887-4.
 2. Sampling
Sampling techniques for milk
1. Milk and liquid milk products
2. Sampling
Sampling techniques for milk
1. Milk and liquid milk products
Collecting milk samples from the cow
 2. Sampling
Sampling techniques for milk
1. Milk and liquid milk products
Collecting a bulk tank milk sample
 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 but avoid foaming.
 Take the sample immediately after mixing
2. Sampling
Sampling techniques for milk
1. Milk and liquid milk products
Collecting a bulk tank milk sample
2. Sampling
Sampling techniques for milk
[Link] and liquid milk products
Shipping bulk tank milk samples
2. Sampling
Sampling techniques for milk

Sampling from flat and

Sampling a spherical cheese, also
cylindrical shape cheese
with flattened sides.

Sampling by cutting out a piece

from a block-shaped or loaf-
Sampling by cut one of pieces
shaped cheese of 10-20 kg mass
 2. Sampling
Fish and fishery products
Raw fish, crustaceans, molluscs and others
a. Whole fresh fish
 The gills, intestinal area and the anus should be covered with sterile

cotton wool, drenched in 70 % alcohol.

 Take a cube-shaped sample of dorsal muscle, dice and grind it up in

the diluent
b. Sliced fish, fillets and steaks.
c. Whole and sliced cephalopods
d. Whole and sliced cephalopods.
e. Shelled crustacean flesh
2. Sampling
Fish and fishery products
f. Crustaceans such as prawns, crayfish, lobsters or Norway
lobsters
g. Live bivalves, gastropods and others:
A representative test sample shall contain at least six
individuals and shall be about 75 g to 100 g
- Bivalves: takes account of both the flesh and intervalvular
water
- Gastropods: it is impossible to extract the animal's flesh
without contaminating it via the shell
h. Sea urchins.
i. Holothurians and tunicates
2. Sampling
Fish and fishery products
Processed products of fish, crustaceans, molluscs and
other products.
a. Salted or pickled products.
b. Dried fish: rehydrate the sample by soaking, if necessary, for 60
min at 18 °C to 27 °C
c. Whole smoked fish
d. Smoked fish fillets and slices, with or without skin
e. Marinated products: treat as low pH/acidified product
f. Breaded fish, surimi; fish, crustacean and mollusc
delicatessen
g. Fish and crustacean and mollusc-based cooked dishes
 2. Sampling
Fish and fishery products
Frozen fish, crustaceans, molluscs and other products:
a. Shelled prawns frozen in blocks
b. Whole prawns frozen in blocks
c. Flaked crab flesh frozen in blocks
d. Whole cephalopods frozen in blocks
e. Precooked shelled snails, molluscs frozen in blocks
f. Fish fillets frozen in blocks: 1- 3 hours.
g. Large fish pieces (e.g. tuna fillets) frozen in blocks
h. Frozen small parts and single portions
i. Whole and large portion frozen fish: defrost in a refrigerator, at
0 °C to 4 °C, for a maximum of 48 h, or drill
 3. Sampling plan
 Sampling plan is a systematic way to assess the microbiological
quality of food lots.
 A “lot” refers to a batch of products manufactured under the same
conditions at the same time. During sampling, the samples should be
taken from the lot independently and randomly.
 In developing a sampling plan, a number of factors should be taken

into consideration including:

+ Properties of food.
+ Production processes.
+ Storage conditions of the final products.
+ Associated risks.
+ Targeted consumers.
+ Practical limitations.
Each food product should be considered individually.
 3. Sampling plan

A comprehensive sampling plan includes the following elements:

(a) The microbe or group of microbes of concern or interest;
(b) Number of samples to be tested (n)
(c) Testing method(s)
(d) Microbiological limit(s), m & M
+ Acceptable (< m)
+ Marginally acceptable (> m and < M)
+ Unacceptable (> M)
(e) Number of samples which fall into each category of
microbiological limit (i.e. acceptable / marginal /unacceptable).
 3. Sampling plan
Type of sampling plan
[Link]-class attributes plan
 For a three-class attributes plan, two microbiological limits, m & M,
are set. The microbiological limit “m” commonly reflects the upper
limit of a good manufacturing practice (GMP).

 The criterion “M” marks the limit beyond which the level of
contamination is hazardous or unacceptable.
 3. Sampling plan
Type of sampling plan
1. Three-class attributes plan
Organism SU RU n c m M comments
Yeast and Mold: 11 gm CFU/gm 5 1 10 100

 SU = sample unit
 n = number of samples analysed
 m = maximum level of target organism(s) or toxins acceptable under
conditions of good manufacturing practice
 M = level of target organism(s) or toxin(s) which, if exceeded, is
considered unacceptable i.e. defective
 c = the number of samples that can fall between m and M without the
batch being considered unacceptable
 RU = reporting units
 3. Sampling plan
Type of sampling plan
1. Three-class attributes plan
 3. Sampling plan
Type of sampling plan
2. Two-class attributes plan
 Under this plan, sample(s) is (are) taken from the lot and tested.

 As only one microbiological limit “m” is involved in this plan,

therefore two classes of attributes, < m & > m, could be identified.

 The maximum allowable number of sample(s) that yielded

unsatisfactory test results is represented by “c”.
 3. Sampling plan
Type of sampling plan
2. Two-class attributes plan
Organism SU RU n c m comments
Salmonella: 25 gm CFU/gm 15 0 0

SU = sample unit

n = number of samples
m = the acceptable level of the test organism
c = the number of samples allowed to exceed m
RU = reporting units
 3. Sampling plan
Type of sampling plan
2. “Two-class” attributes plan
 3. Sampling plan
Choice of sampling plant
 In general, a two-class attributes plan is preferred when the
organism of concern is not permitted in food sample. If the
number of microbes in a unit-volume is allowable, a three-class
attributes plan is usually adopted.
 By changing the value(s) of c and/or n, the stringency of
sampling plan can also be adjusted.
 To enhance food safety and improve food quality, more
stringent microbiological limits (by decreasing values of m
and/or M) should be adopted.
 4. Sampling preparation

1. A known weight of food sample (10 or 25 g) is taken except where

swabs or rinses have been used.
2. The food is added to a suitable sterilized diluent such ¼ strength
Ringer’s solution or 0.1% peptone water.
3. The food in a known volume of diluent is then mechanically treated
by blending or stomaching.
4. Suitable dilution can be prepared (10-1, 10-2,10-3, ect) depending
upon the microbiological quality of the food or surface under test.
4. Sampling preparation

An example of dilution
 4. Sampling preparation

The test portion

 Laboratory sample preparation => “test portion” or “analytical unit”
 Definition: the part of the sample that is actually tested by the
laboratory.
 Test portion determines the theoretical (i.e., best possible) sensitivity
of the test i.e.,1 cell/test portion
+ 25-gram-detecting 0.04 cells/gram is possible
+ 325-gram-detecting 0.003 cells/gram is possible
4. Sampling preparation

Frozen products:
 Products stored frozen should be brought to a consistency that
allows sampling.
I.e: storing at 18°C to 27°C (laboratory temperature) for a
maximum of 3 h, or 2 °C ± 2°C for a maximum of 24 h. Samples
should be tested as quickly as possible after that.
 If the product is still frozen when portioning, some diluent at

laboratory temperature may be used to facilitate defrosting.

 Powders should be well mixed in their containers before

sampling.
4. Sampling preparation

 Hard and dry products: do not homogenize in a rotary

homogenizer for more than 2,5 min at a time.
 Liquid and non-viscous products: Before analyzing, the test
sample should be taken after having shaken the laboratory
sample by hand (e.g. 25 times through an arc of 25 cm) or by
mechanical means in order to ensure that the microorganisms
are uniformly distributed
 Heterogeneous products: to avoid an excessive rise in
temperature, do not mince or grind for more than 1 min.
 4. Sampling preparation

Very hard products:

- Take a larger amount of the test sample than is required for analysis
and aseptically grate into small pieces or break into small pieces in a
sterile plastic bag using a hammer.
- Add 1 part test sample to 9 parts of peptone salt solution and mix.
- Before homogenization, leave to stand for 20 - 30 mins at 18 °C to
27 °C (laboratory temperature).
- Blend in a rotary blender.
 4. Sampling preparation

Specific procedure
Flours, cereal grains, cereal by-products, animal feeds and cattle
cake:
 Mix according to the product with either a peristaltic homogenizer

for 1 min, or in a rotary blender

 A test portion of 50 g is recommended when analyzing cereals and
other heterogeneous products.
 Add 1 part test sample to 9 parts of peptone salt solution and mix.

 NOTE Hard materials (e.g. grains and bone meal) will puncture bags

for a peristaltic homogenizer; double bagging may prevent this.

5. Enrichment

 Test portion is incubated 8-48 hours in a culture broth

 Must grow to high levels so very small volumes have
enough for later detection steps.
 Different pathogens require a different broths

 One vs. two-stage

 Enrichment resuscitation vs. selective growth.

Example: High salt concentration will select for halophiles.

High temperatures will select for thermophiles
5. Enrichment

Considerations for proper enrichment

 Resuscitation (lag phase) can require 2-3 hours before log-
phase growth begins. Some samples support slower growth

 Has enrichment broth been tempered to warm temperature

prior to incubation? Particularly critical for large test portions
or shorter incubation periods.
 6. Selective and Differential Media

By inhibiting unwanted organisms with salts, dyes, or other chemicals,

selective media allow growth of only the desired microbes.
•Bismuth sulfite agar inhibits gram-positive and most gram-negative
bacteria, used to isolate Salmonella typhi.
•Brilliant green agar inhibits gram-positive and most gram-negative
bacteria and is used to isolate Salmonella species.
•Sabouraud glucose agar has a pH of 5.6; inhibits most bacteria and is
used to isolate fungi.
Differential media are used to distinguish among different organisms.
6. Selective and Differential Media
6. Selective and Differential Media
[Link] Testing
 Non-culture confirmation (e.g., PCR).
 Culture confirmation (e.g., FSIS confirmation): Plating the
enrichment on selective and differential agar media.
Immune magnetic separation (IMS) necessary prior to
plating for E. coli O157:H7

 Suspect colonies = “presumptive positive”

Purification and confirmatory identification tests including:
Biochemical (e.g., identifies “E. coli”).
Serological (e.g., identifies “O157” and “H7”).
Genetic (e.g., identifies “stx” = Shiga toxin genes)
[Link] Testing

Biochemical test
 Identification of microbial isolate usually follows from a
sequence of morphological, biochemical, immunological and
genetic techniques.
 Biochemical reactions, for the purpose of identification, often
utilize a limited combination of metabolic and enzymatic
activities prominent and pertinent to a specific group of
microorganisms that share these common activities.
 Among the most commonly utilized microbial biochemical
activities are fermentation of sugars (carbohydrates),
utilization of certain carbon sources, production of certain
unique fermentation products, possession of specific enzymes.
 Biochemical test performed in test headed towards
miniaturization
Biochemical test

Indole production
 How to perform test: inoculate Tryptone broth with inoculating
loop.
 Property it tests for: this test is performed to help differentiate
species of the family Enterobacteriaceae. It tests for the bacteria
species’ ability to produce indole.
 Bacteria use an enzyme, tryptophanase to break down the amino
acid, tryptophan, which makes by-products, of which, indole is
one.

tryptophan + water = indole + pyruvic acid + ammonia

Biochemical test

Indole production

 Media and Reagents used: SIM media (sulfide-indole-motility

media) or Tryptone broth contains tryptophan.
a. Inoculate the tryptophan broth with broth culture or emulsify
isolated colony of the test organism in tryptophan broth.
b. Incubate at 37°C for 24-28 hours in ambient air.
c. Add 0.5 ml of Kovac’s reagent to the broth culture.
 Reading results: Kovac’s reagent (p-dimethylaminobenzaldehyde)
reacts with indole and creates a red color (red dye rosindole) at the top
part of the test tube.
Biochemical test

Indole production

Indole test using Tryptone broth

Possitive: Edwardsiella, Enterobacter, Proteus vulgaris, P. multocidea, [Link].,

Citrobacter Koseri , Klebs‪iella oxytoca
Negative: Salmonella, Pasteurella haemolytica, Proteus mirabilis, Citrobacter
freundii , Klebsiella pneumoniae
Biochemical test
Hydrogen Sulfide (H2S) Production in SIM
(Sulfide-Indole-Motility medium )
 Test performing: to inoculate, use a needle to stab agar (SIM
media) and use a loop to streak the top slated region.
 Test properties: used to help differentiate species of the
Enterocateriacea. The test is used to determine the bacteria
species’ ability to reduce sulfur into H2S from some amino
acids (cystine, methionine) or reducing inorganic sulfur-
containing compounds (such as sulfite, sulfate or thiosulfate).

 Media and Reagents used: SIM media contains the sulfur

containing amino acid, cystein, sodium thiosulfate and
peptonized iron or ferrous sulfate.
Biochemical test
Hydrogen Sulfide (H2S) Production in
SIM (Sulfide-Indole-Motility medium)
Biochemical test
Hydrogen Sulfide (H2S) Production in SIM
(Sulfide-Indole-Motility medium)
Reading results:
- pH indicator will change the color of
the media in response to fermentation
where that colour change occurs in the
tube will indicate what sugar or sugars
were fermented.
- The presence of a black color
indicates that H2 S was produces. H2S
will react with the iron or ferrous
sulfate and produce a black precipitate.
- A positive result has a black
precipitate present and a negative result Positive: Staphylococus gallinarum,
has no black precipitate. Proteus vulgaris
Negative: [Link]
Biochemical test

Sulfide-Indole-Motility medium

Indole and Hydrogen Sulfide (H2S) production tests using SIM media
(A) uninoculated tube
(B) contains the non-motile and indole-negative bacterium Klebsiella pneumoniae
(C) contains the motile and indole-positive bacterium Escherichia coli
(D) contains the motile, indole-negative, and H2S-producing bacterium Proteus mirabilis

[Link]
Biochemical test
Methyl Red/Voges proskauer
(MR/VP)
 Preparing: inoculate 2 glucose broths with inoculating loop.
After 48 hours of incubation, add a few drops of MR to one tube
and VP reagents to other tube.
 Properties of the test: both test are used to help differentiate
species of the family Enterobacteriaceae:
 MR: test for acid and products like lactate, acetate and formate
from glucose fermentation.
 VP: test for the present of acetylmethylcarbinol (acetoin, an
intermediate of the 2,3-butanediol fermentation pathway) from
glucose fermentation
Biochemical test
Methyl Red/Voges proskauer
(MR/VP)
 Media and reagents used: Glucose broth
[Link] two tubes containing MR-VP Broth with a pure culture of the
microorganisms under investigation.
2. Incubate at 35 °C for up to 4 days.
3. Add about 5 drops of the methyl red indicator solution to the first tube
(for VP test, Barrit’s reagent is added to another tube).
4. A positive reaction is indicated, if the colour of the medium changes to
red within a few minutes.
Biochemical test
Methyl Red/ Voges proskauer
(MR/VP)
 Reading results
- Methyl Red
+ a red color indicates (pH = 4.2 < 6) a positive result (glucose
can be converted into acidic and products such as lactate, acetate and
formate).
+ A yellow colour indicates (pH > 6.2, no acid production) a
negative result, glucose is converted into neutral end products.
- Voges Proskauer: the culture should be allowed to sit for about 15
minutes for color development to occur.
+ If acetoin was produced then the culture turns a red color
(positive result).
+ If acetoin was not produced then the culture appears yellowish
to copper in color (a negative result).
Biochemical test
Methyl Red/ Voges proskauer
(MR/VP)

[Link] (+), Enterobacter aerogenes (-), E.

cloacae (-), L. monocytogenes (+)
Biochemical test

Citrate Utilization

 Test performing: inoculate slant with inoculating loop.

 Properties of the test: the test is used to help differentiate species of
the family Enterocateriaceae. It is selective for bacteria that has
ability to consume citrate (an intermediate Kreb’s cycle) using enzyme
citritase. In this media, citrate is the only carbon source available to
the bacteria and ammonium as sole nitrogen source.

 Media and Reagents used: Simmon’s Citrate agar contains sodium

citrate (carbon source). pH indicator: bromothymol blue.
Biochemical test

Citrate Utilization
Procedure of citrate utilization test:
 Inoculate Simmons Citrate Agar lightly on the slant by touching
the tip of a needle to a colony that is 18 to 24 hours old.
 Incubate at 35oC to 37oC for 18 to 24 hours.

 Observe the development of blue color; denoting alkalinization.

Example:
Production of sodium bicarbonate (NaHCO3) as well as
ammonia (NH3) from the use of sodium citrate and ammonium
salts results in alkaline pH. This results in a change of the
medium’s color from green to blue
Biochemical test

Citrate Utilization
Reading results:
+ If bacteria can not use citrate then it will not grow.
+ If it can use citrate, then the bacteria will grow and the media
will turn a bright blues from green as a result of an increase in the pH of
the media.
Note: Bromothymol blue is yellow at acidic
pH's (around 6), and gradually changes to
blue at more alkaline pH's (around 7.6)

Example:
 Positive: Klebsiella pneumoniae and
Enterocateriacea aerogenes, Citrobacter
spp, Klebsiella pneumoniae
 Negative: Escherichia coli, Proteus
mirabilis
Biochemical test

Urea Hydrolysis test

 Test performing: inoculate liquid Urea broth with inoculating loop.
 Test’s properties: to determine a bacteria’s ability to hydrolyze urea
to make ammonia using the enzyme urease.
 Media and reagents: urea broth contains a yeast extract,

momopotassium phosphate, disodium phosphate, urea and phenol red

indicator.
 Reading results: urea broth is a yellow-orange color. The enzyme

urease will be used to hydrolyse urea to make ammonia.

+ If ammonia is made, the broth turns to bright pinkish-red
colour. It is positive.
+ If the test is negative, ammonia is not made and therefore Broth
has no color change.
Biochemical test

Urea Hydrolysis test

Biochemical test

Carbohydrate Utilization
 Bacteria produce acidic products when they ferment certain
carbohydrates
 The carbohydrate utilization tests are designed to detect the
change in pH which would occur if fermentation of the given
carbohydrate occurred.
 The carbohydrate tests are performed:

1) Glucose (dextrose) test

2) Lactose test
3) Sucrose test
Note: the test is commonly used to identify Gram-negative enteric
bacteria, all of which are glucose fermenters but only some of
which produce gas.
Biochemical test
pH indicators for carbohydrate
fermentation media
pH Uninoculated Acid Alkaline
indicator media (fermentation) (negative)

pH Color pH Color pH Color

Yellow,
Andrade’s 7.1-7.2 Light pink 5.0 Pink-red 12.0-14.0
colorless
Bromocresol Deep
7.4 5.2 Yellow 6.8 Purple
purple purple
Deep
Bromothymol
7.0 Green 6.0 Yellow 7.6 Prussian
blue
blue
Reddish-
Phenol red 7.4 6.8 Yellow 8.4 Pink-red
orange
Biochemical test

Carbohydrate Utilization

 Results of carbohydrate fermentation test

Peptone media with phenol red indicator. Peptone media with bromocresol purple
From left to right: uninoculated tube; indicator. From left to right: uninoculated
Escherichia coli, a glucose fermenter with tube; E. coli, glucose fermenter with gas
gas production (visible air bubble in the production (visible air bubble in the inverted
inverted Durham tube); Shigella sonnei, a Durham tube); S. sonnei, glucose fermenter
glucose fermenter without gas production without gas production (no visible air bubble
(no visible air bubble in the inverted Durham in the inverted Durham tube); P. aeruginosa,
tube); Pseudomonas aeruginosa, nonfermenter.
nonfermenter.
Biochemical test

Carbohydrate Utilization

 Tube 1: No fermentation. The pH indicator remains purple.

 Tubes 2A and 2B: Fermentation with the production of acid (yellow color) but no
gas.
 Tubes 3A and 3B: Fermentation with the production of acid (yellow color) and
insoluble gas (bubble in Durham tube).
Biochemical test Carbohydrate Utilization

Glucose fermentation and gas production

 Test performing: inoculate broth with inoculating loop.

 Test properties: This test for the bacteria’s ability to ferment glucose
and produce gas and an acid end-product.
 Media and reagent use: glucose broth contains beef extract, gelatine
peptone and glucose.
+A phenol red indicator is added to indicate an acid end-product.
+ A Durham tube is added to indicate gas production.
 Results reading:
- A positive results for acid is yellow after indicator is added (indicating
glucose fermentation)
- A positive result for gas is a bubble in the Durham tube.
- A completely negative result has no color change or reddish color and
no bubble.
Biochemical test Carbohydrate Utilization

Glucose fermentation and gas production

Biochemical test Carbohydrate Utilization

Lactose fermentation
Biochemical test Carbohydrate Utilization

Sucrose fermentation
Biochemical test Carbohydrate Utilization

Carbohydrate Utilization
Positive results Negative results
Glucose Staphylococcus aureus: yellow, no gas Pseudomonas aeruginosa:
fermentation Proteus vulgaris and Escherichia coli: no change in color
yellow+ gas
Lactose Staphylococcus aureus: yellow, no gas Proteus vulgaris and
fermentation Escherichia coli: yellow + gas Pseudomonas aeruginosa:
no change in color

Sucrose Staphylococcus aureus: yellow, no gas Escherichia coli and

fermentation Proteus vulgaris : yellow + gas Pseudomonas aeruginosa:
no change in color
Biochemical test Carbohydrate Utilization

Carbohydrate Utilization

E. coli fermentation test for Glucose on the left, Sucrose center,

and Lactose on the right
Biochemical test

Coagulase test
 Test performing: inoculate rabbit plasma with one single
colony. Break up colony and stir until blended in plasma.
Incubate at 370 C for 24 hours.
 Test’s property: this test for the bacteria’s ability to clot blood
plasma using the enzyme coagulase. If the organism has
coagulase it will clump rabbit plasma.
 Reading results:
 If the organism is has coagulase, it will clump the plasma. If
the organism does not have coagulase, it will not clump the
plasma.
Biochemical test

Coagulase test

[Link]
manual/atlas/biol2420photoatlas_037.htm
Biochemical test

Catalase test
 Test performing: smear a small amount of test organism onto
the lid of a Petri plate/culture dish. Add a drop of hydrogen
peroxide (H2O2) to the smear.
 Test properties: the test can be used to detect enzyme catalase
responsible for protecting bacteria from H2O2 accumulation
occurring during aerobic metabolism.

 Media and reagents: solid or liquid broth

 Results reading: if bubbles become visible, the test is
positive. A lack of bubbles indicates the absence of catalase
Biochemical test

Catalase test
Biochemical test

Oxidase test
 Test performing: swab some of test culture into one of the
boxes on an oxidase dry slice. Incubate at 370 C for 1-5 days.
Add oxidase reagent on the colonies.
 Test’s properties: test is based on detecting the production of
the enzyme cytochrome oxidase by gram-negative bacteria. It
is a hallmark test for the Neiserria. It is also used to
discriminate between aerobic gram-negative organism like
Pseudomonas aeruginosa and other Enterobacteriaciae.
 Media and Reagents: solid or liquid broth in petri dish or
tube. Oxidase reagent is added contains 0.5% tetrametil-p-
fenilendiamine
Biochemical test

Oxidase test
 Result reading: if color change
to purple or blue at 30 second – 1
minutes: positive
 Positive: Pseudomonas
aeruginosa strain.
 Negative: [Link] strain
Biochemical test

Motility test
 Test performing: SIM or stab motility media with inoculating
needle. Stab the media in as straight a line as possible and
withdraw the needle very carefully to avoid destroying the straight
line. Observations can be made after incubating the sample for 24 –
48 h.
 Test properties: to differentiate species of bacteria that are motile.
Also this test can be used to check for the ability of bacteria to
migrate away from a line of inoculation due to physical feature like
flagella.
 Media and reagents: motility media contains tryptose, sodium
chloride, agar and a color indicator.
 Reading results
Biochemical test

Motility test
Motility agar
Biochemical test

Multiple tests
 Multiple tests for organism identification can be performed in
a simple manner by using commercially prepared kits.
 These kits combine several biochemical tests into miniaturized
formats which use a computer to analyze the test results and
provide specific identifications.
 Examples:
- The Enterotube
- The API strip
- Vitek card
Screening tests

 Usually commercial testing products.

 Most validated screening tests are: Immunoassays (ELISA,

ELFA, immuno chromatographic devices, etc.).

 Polymerase Chain Reaction (PCR) assays.

 Must be validated for performance with a specific broth and

incubation period.
 Screening tests
Incubation period
 PCR screens may require less growth than immunoassays.

 Shorter incubation periods (<15 hours) may warrant additional

scrutiny of laboratory compliance to the validated protocol.
 Has enrichment/screening combination been validated for a
larger test portion? Particular concern for large test portions
incubated for shorter periods.

For example: 375-gram test portion incubated for 8 hours

 Proposed incubations < 8 hours may warrant OPHS review.

Considerations for testing methods

 Is the method fit for the intended purpose of the analysis?

 Has the method been optimized and experimentally validated

for sensitive detection of pathogens?

 Is the laboratory complying to the validated method protocol?

 Is the method intended for detecting the lowest possible levels

of potentially injured pathogen cells in meat/poultry products
like the corresponding FSIS method?

 Was this demonstrated by the validation study?

 Choice of tests
Some of the factors influencing the choice of test
include:
• Type of ingredients in the finished product
• Status of ingredients (i.e. cooked vs. raw)
• Type of cooking/processing undertaken
• Level of handling after cooking or processing
• Presence of preservatives, including acids and salt
• Presence and type of packaging
• Distribution and storage of finished product
Choice of tests
 Indicator and spoilage organisms
 Pathogens
 Accuracy of the result
 Limit of detection of the method
 Recovery of target organisms
 Comparability of results between different methods
 Inhibition of growth of microorganisms
 Competitive growth
 Test methods
 Microbial Testing
Microbiological testing can be grouped into the following
categories:
1- Testing for the presence of specific organisms (Listeria
monocytogenes, Salmonella, etc)
2- Enumerating specific organisms (Staphylococcus aureus,
etc.)
3- Enumerating groups of organisms (coliforms, fecal
coliforms)
Microbial Testing

 4- Enumerating all organisms which will grow

under certain defined conditions (aerobic plate
count)

 5- Testing for the presence of microbial toxins

(Clostridium botulinum)

 6- Testing for the presence of microbial

metabolites (histamine)
Value of validation

 Determines performance characteristics of the method in

comparison to a “gold standard” method (i.e. ,usually is Food
Safety and Inspection Service (FSIS) or FDA method).

 Independent evaluation provides credibility

 Still must consider fitness for purpose and how the method is
applied. e.g., some AOAC-validated methods are not
consistent with FSIS goals or Compliance Guidelines.
 Method validation

 Recognized independent method validation organizations:

 Government-FSIS and FDA
 AOAC International (U.S.A.) AOAC Official Method (OM)
validations
 AOAC-RI “Performance Tested Method” validations
 AFNOR (France)-e.g., bioMerieux-Vitektests
 Others (ISO, NMKL, etc.)
 Testing method specifications

Common specifications determined through experimental

validation studies:
 How well does the method work for low levels of
contamination? e.g., sensitivity, false negative rate, limit
of detection (LOD)
 How specific is the test for the target pathogen? e.g.,
inclusivity, exclusivity
 How reliable is the method in different hands? e.g.,
repeatability, reproducibility
Specific NOTES

 Bacteria can mutate and evolve into forms that defy the
traditional rules. As a result, much diversity within a pathogen
species
 False negative potential-Does the test miss some subgroup of
the target pathogen?
 False positive potential-Is an unconfirmed result a potential
problem?

 Depends on context (industry vs. FSIS testing)

 Method application

 Can the method accommodate the necessary test portion?

 Does the lab specifically comply to the validated method

instructions, or have they altered the method in some
way?

 AOAC/AFNOR validations typically apply only to

commercial screening methods without regard to any
necessary follow-up tests.
References
1. [Link]
standards/microbiological-standards-and-guidelines
2.[Link]
on/publications/[Link]
3. ISO 6885-2012
4. ISO 17604
5. ISO 6887-4
6. ISO 6887-3
7. ISO 7218