INTRINSIC AND EXTRINSIC PARAMETERS OF

FOODS
AFFECT MICROBIAL GROWTH

INTRINSIC PARAMETERS: The parameters of plant

and animal that are inherent part of the tissues are
referred as intrinsic parameters. The intrinsic
parameters associated with food are
1. pH
2. Moisture content
3. Oxidation – reduction potential (Eh)
4. Nutrient content
5. Anti microbial constituents
6. Biological structures

pH
Most microorganisms grow best at pH 7.0
while few microorganisms grow best at pH 4.0. In
food, pH values are dependent on other growth
parameters of microorganisms. For Example
Lactobacilli dependent on the type of acid used.
The Citric acid; Hydrochloric acid; Phosphoric acid
permits the growth at lower pH than acetic acid
and Lactic acid. Alcaligenes faecaleis has shown
to grow at wide pH in presence of Fruits, Soft
Drinks, Vinegar & Wine normally fall below the

1
point at which bacteria normally grow. Fruits
undergo spoilage by the Molds & Yeasts, which
have the capacity to grow at a pH range of 3.5
Minimum pH for the most food spoilage &
all food poisoning bacteria

Fruits & Vegetables pH range

Asparagus 5.7 – 6.1
Beans (String) 4.6
Lima Beans 6.5
Sugar beet 4.2 – 4.4
Cabbage 5.4 – 6.0
Carrot 4.9 – 5.2
Cauliflower 5.6
Egg plant 4.5
Lettuce 6.0
Onions 5.3 – 5.8
Potato 5.3 – 5.6
Tomato 4.2 – 4.3
Turnip 5.2 – 5.5
Apple 2.9 – 3.3
Fig 4.6
Banana 4.5 – 4.7
Lime 1.8 – 2.0
Water Melons 5.2 – 5.6
Orange (juice) 3.6 – 4.3
Plum 2.8 – 4.6
Grapes 3.4 – 4.5
Dairy products
Butter 6.1 – 6.4
Butter milk 4.5
Milk 6.3 – 6.5

2
 Cream 6.5
Cheese (American) 1.9
Cheddar Cheese 5.9
Meat & Poultry
Beef 5.1 – 6.2
Chicken 6.2 – 6.4
Fish
Fish 6.6 – 6.8
Crabs 7.0
Oysters 4.8 – 6.3
Shrimp 6.8 – 7.0
Salmon 6.1 – 6.3
Vegetables have higher pH values than
fruits and consequently vegetables are subjected
more to bacterial than fungal spoilage.
Meat: Fastigiated animal meat spoils faster
than rested animal. Well-rested animal causes
depression in pH values from 7.4 – 5.6 (depends
upon type of animal). During rigor mortis, 1%
glycogen is converted in to lactic acid, which
leads to depression in pH
Beef: 5.6 – 6.2
Lamb: 5.4 – 6.7
Pork: 5.3 – 6.9
The source of acidity in foods may be due to
Natural or inherent acidity or may be due to
biological acidity. Natural or inherent acidity is
the nature’s way of protecting the respective plant
and animal tissue. The biological acidity is due to

3
the action of certain microorganisms. For example
fermented milk, Sauerkraut and pickles.
Regardless of source of acidity, the keeping
quality of food appears to be the same. Some foods
are able to resist changes in pH. The foods that
resist changes in pH are called as buffered foods.
Meats are highly buffered than vegetables. The
buffering capacity of meats is due to various
proteins when compared to vegetables, which are
generally low in protein and lack the buffering
capacity to resist the changes in pH by growth of
microorganisms.
Effect of pH in foods
Adverse effect of pH on a respiring microbial
cell is by two ways:
(i) Enzyme activity
(ii) Transport of nutrients into the cell
Enzyme Activity:
Cytoplasmic membrane is impermeable to
H+ and OH- ions. The internal pH of the almost all
cells shows neutral. When microorganisms are
placed in environments below or above neutrality,
their ability to proliferate depends on their ability
to bring the environmental pH to a more optimum
value or range. When placed in acid environments,
4
the cells must keep either keep H+ ions very
rapidly. When microorganisms grow in acidic
media, their metabolic activity results in the
medium or substrate become lens acidic, and
those that grow in high pH environments tend to
effect a lowering of pH. In Clostridium
acetobutylicum raise the substrate pH by reducing
butyric acid to butanol. In Enterobacter aerogenes
produces acetonin from pyruvic acid to raise the
pH of the environment. When amino acid is
decarboxylated, increase in pH occurs due to the
formation of amines. The amino acid
decarboxylases have the optimum activity at pH
4.0, and no activity was observed at pH 5.5. At
alkaline range (pH 8) the enzyme amino acid
deaminases brings the pH to neutral as a result of
organic acid accumulation.
Transport of Nutrients:
In General, the bacterial cells have negative
charge. So non-ionized particles can enter into the
cell whereas ionized cannot enter into the cell. At
Neutral / alkaline pH the organic acids don’t enter
because organic acids are in ionized state
whereas at acid pH the organic acids are non-
ionized and can enter into the cell. In particular the
5
morphology of the microorganisms are affected by
pH. For ex. – In Pencillium chrysogenum, the
length of hyphae gets reduced at pH 6.0 whereas
at pH 6.7, Pellets of hyphae are formed instead of
+
free mycelium. Presence of extra cellular K and
H+ ions plays a major role while K + stimulates
fermentation and H+ ions represses it. The
metabolism of Glucose in yeast cell is stimulated
by presence of K+ ions, in an acid medium. In
presence of K+ ions is more consumption of
glucose up to 83% in anaerobic condition whereas
69% more consumption under aerobic condition.
Environmental Factors:
Temperature and salt concentration
The pH of the substrate becomes more acid
as the temperature increases. Concentration of
salt has a definite effect on pH. Addition of 0.2
NaCl increases he pH growth range of Alcaligenes
faecalis and E.coli when salt concentration
exceeds the optimal level, the pH growth range is
narrowed. Adverse pH makes the microbial cell,
more sensitive to toxic agents and young cells
more susceptible to pH changes than older or
resting cells.

6
 Microorganisms when grown on either side
of optimum pH range have increased lag phase.
The increased lag phase occurs if the substrate is
a highly buffered one when compared to poor
buffering capacity. The length of the lag phase
depends on the time necessary for the organisms
to bring the pH of the external environment within
their optimum pH growth range.
For example Salmonellae, initiate growth at
pH 4.5 when hydrochloric and citric acids were
used. Whereas the same organisms initiate growth
at pH 5.4 and 5.5 when acetic and prop ionic acids
were used.
2. MOISTURE CONTENT
Preservation of foods by drying or
desiccation is to remove or binding of moisture
without which microorganisms don’t grow.
Water requirement of microorganism is
defined in terms of water activity in the
environment. Water activity is defined by the ratio
of the water vapour pressure of food substrate to
the vapour pressure of pure water at the same
temperature.

aw = P/Po
P – vapour pressure of solution
7
 Po – vapour pressure of water

R.H. = 100 x aw
Pure water a w - 1.00

22% Nacl - 0.86

Saturated solution - 0.75
Water activity of most fresh foods: is above
0.99. Minimum aw values for growth of
microorganisms in food is given in Table.2

8
 Table.2 Minimum aw values for the growth of
microorganisms in food
Organism aw

Spoilage bacteria 0.90

Yeast 0.88
Molds 0.80
Halophilic bacteria 0.75
Xerophilic molds 0.61
Osmophilic yeast 0.61
E.coli 0.96
Aspergillus glacuas 0.70
Aerobacter 0.95
aerogenes 0.70
A.Conicus 0.97
Pseudomona 0.96
Acinetobacter 0.95
Bacillus subtilis 0.84
Pencillium patulum 0.97
Clostridium
botuliuum

Relationship found to exist among water

activity, temperature and nutrition. At any
temperature, if the water activity is lowered the
growth of microorganisms will be reduced. The
9
range of a w over which the growth occurs is

greatest at the optimum temperature for growth.

The presence of nutrients increases water activity
over which the organism can survive.

Effect of low water activity (a w )

The effect of lowering of a w below optimum is

(i) Increase the length of lag phase growth of

microorganisms
(ii) To decrease the growth rate
(iii) Reduction in size of the population
Due to low water activity, all metabolic
activities get lowered, since all chemical reactions
of the cell require an aqueous environment.
Osmotic stress results in accumulation of
compatible solutes like potassium ions, glutamate,
glutamine, proline, alanine and Glycerol. Gram-
negative bacteria accumulate proline by the
mechanisms of enhanced transport. Halotolerant
and xerotolerant fungi produce polyhydric
alcohols, such as glycerol, Erythritol and arairol.
The germination of Bacillus and Clostridium
spores shows strong inhibition when water activity
(aw) is controlled by NaCl and CaCl2 but glucose
and sorbital shows less inhibition and very little

10
inhibition when glycerol, ethylene glycol,
acetamide or urea used. The overall effect of
lowered aw on the nutrition of microorganisms
appears to be of a general nature where cell
requirements must be mediated through an
aqueous medium. Also lowered aw has adverse
effect on functioning of cell membrane which
should be kept in a fluid state.
In microorganisms, due to reduction in
water activity, K+ ions accumulate inside the cells
and catalyze the formation of proline precursors. If
more amount of proline is accumulated in
microorganisms and when the water activity is
lowered, some of M.O. shows proline-stimulated
respiration.
3.OXIDATION – REDUCTION POTENTIAL
Microorganisms display varying degrees of
sensitivity to the oxidation / reduction potential
(O/R, Eh) of their growth medium. When an
element / substrate loose electrons substrate is
said to be oxidized while the substrate that gains
electrons becomes reduced.
Oxidation

CU CU + e
Reduction
11
Oxidation is also achieved by the addition of
oxygen
2Cu + O2 2 CuO
A substance that gives up electrons / is a
good reducing agent and one that takes up
electrons is a good oxidizing agent. When electrons
are transferred from one compound to another;
potential difference is created; between two
compounds. The difference is measured by an
appropriate instrument, and expressed as millivotts
(mv)
The more highly oxidized substance is more
+ive will be its electrical potential where as highly
reduced substance have negative electrical
potential. When Oxidant / reductant potential is
equal; the potential difference (PD) is zero. O/R of
the system is expressed by Eh
Aerobic organisms require positive Eh
values for growth, while anaerobic organisms
require negative Eh values (reduced). The
substances in food that help to maintain reducing
conditions are SH groups in meats and ascorbic
acid and reducing sugars in fruits and vegetables.

12
 In general, oxidation / reduction is
determined by
1. The characteristic oxidation / reduction
potential of the original food
2. The poising capacity, that is resistance
to change in potential of the food
3. Oxygen tension of the atmosphere about
the food.
4. The access that the atmosphere has to
the food
Some bacteria require reduced conditions
for growth initiation (Eh – 200 mv) Ex. Clostridium,
while others require a positive Eh for growth, Ex.
Bacillus. Some aerobic bacteria grow better under
slightly reduced conditions and these organisms
are referred as microaerophilic. Ex . Lactobacilli
and Streptococci.
Eh effect
The aerobic organisms lower the Eh of the
environment. Due to the growth of these
organisms, the oxygen in the medium is depleted
which results in the lowering the Eh.
Also the Eh, can be reduced by the
microorganisms by their production of certain

13
metabolic byproducts such as hydrogen sulphide
which has the capacity to lower the Eh to –300mv.
4. Nutrient Content:
In general the microorganisms require
water, source of energy, source of nitrogen,
vitamins and related growth factors, and minerals
for their growth and function. The fungi requires
lowest nutrient requirement followed by yeast,
gram – ive bacteria and Gram +ive bacteria.
The sugars, alcohols and amino acids acts
as a source of energy and some of the
microorganisms utilize complex carbohydrates
such as starch and cellulose as source of energy
by degrading these compounds to simple sugars.
Fats are also used as a source of energy for small
number of microorganisms in food. In general
almost all organisms will utilize amino acids for
growth and nucleotides, peptides and proteins
acts as a source of nitrogen.
The microorganisms require B vitamins in
low quanities and almost all natural foods tend to
have an abundant quantity of vitamins for those
organisms that are unable to synthesize their
essential requirements. Gram –ive bacteria and
molds can synthesize most or all of their nutrient
14
requirements and these organisms even grow in
foods with low vitamin B content
5. ANTIMICROBIAL CONSTITUENTS
The stability of foods against attack by
microorganisms is due to the presence of naturally
occurring substances which posse’s antimicrobial
activity. For example spices that contain essential
oil have antimicrobial activity
Eugenol from cloves; allicin from garlic;
Cinnamic acid and Eugenol from cinnaman; allyl
isothiocynate from mustard ; lactoferrin and
congltinum from cows milk; Lysozyme from eggs;
P- coumaric , Ferrulic / Caffeic and Chlorogenic
acids from fruits, vegetables, tea, and molasses.
6.BIOLOGIACL STRUCTURES
Natural covering offers protection against
entry and subsequent spoilage of microorganisms.
For example Testa of seeds, Outer covering of
fruits, shell of nuts, hide of animals and shell of
eggs.
The skin covering of fish, meats, beef, pork
and eggshell prevents the entry of microorganisms

EXTRINSIC PARAMETERS

15
 The extrinsic parameters of foods are
those properties of the storage environment that
affect the food and microorganisms.

1. Temperature of storage
2. Relative humidity of the environment
3. Presence and concentration of gases in the
environment
1. TEMPERATURE OF STORAGE
Microorganisms grow at a wider range of
temperature. The lowest temperature at which
microorganisms can grow is - 34 C and the highest
temperature is somewhere in excess of 90C. The
minimum, maximum, and optimum temperature
required for the growth of microorganisms in food
is given below

16
 Type of Temperature C
microorgani
sms
Minimum, Optimum Maximum
,
Thermophile 40-45 55-75 60-90
s
Mesophiles 5-15 30-40 40-47
Psychrophile -5 to +5 12-15 15-20
s
(Obligate)
Psychrotrop -5 to +5 25-30 30-35
hs
(Facultative)

17
 Some of the psychrotropic organisms like
Alcaligenes, Corneybacterium,Psedomonas,
Shewanella, Flavobacterium, enterococcus ,
Brochothrix, Lactobacillus grow at refrigerated
temperature and cause spoilage of meats, fish,
poultry and eggs. Among these microorganisms
Pseudomonas and Enterococcus are commonly
found. Some of the organisms can grow at a wide
range, Enterococcus faecalis can grow from 0 C
to 30C. The optimum temperature for the growth
of thermophilic bacteria Bacillus and Clostridium
was 37C and this two-organism cause major
spoilage in Canning.

The molds are able to grow over wider

ranges of pH, osmotic pressure, nutrient content
and also wider ranges of temperature than
bacteria. Some strains of Aspergillus,
Cladosporium and Thamnidium are able to grow at
refrigerated temperature and cause spoilage in
eggs, sides of beef and fruits
The yeasts grow at psychrotrophic and
mesophilic temperature but not at thermophilic
range.

18
2. RELATIVE HUMIDITY OF ENVIRONMENT
Relative humidity of the storage
environment is important both from the standpoint
of aw within foods / and growth of microorganisms
at the surfaces.
Foods with low water activity when stored
in high RH environments will take the moisture.
Foods with high water activity when stored in low
RH environment lose moisture. Improperly packed
Meat while chickens / beef cuts due to high RH of
the refrigerator suffer surface spoilage before deep
spoilage occurs.
The storage of fresh fruit and vegetable
requires very careful control of relative humidity. If
the RH is lowthen many vegetables will lose water
and become flaccid. If relative humidity is too high
then water condensation may occur on the
surface, which leads to surface spoilage by
microorganisms.

3. PRESENCE AND CONCENTRATION OF GASES IN

THE ENVIRONMENT

Storage of foods in the atmosphere upto

10% CO2 is called CA or MA storage. MA of plant
foods known from 1917 and the Commercial
19
application is known from 1928.MA storage of
fruits is done no. of countries with apples and
pears.
The concentration of gases doesn’t exceed
more than 10 % CO2 level. Application of CO2 is by
mechanical sources or by uses of dry ice (solid
CO2). CO2 also acts as a competitive inhibitor of
ethylene action. Ethylene acts as senescence
factor in fruits and its inhibition have the effect of
maintaining a fruit is better state of natural
resistance.
Application of ozone to the food storage
environments has preservative effect on certain
foods. Ozone at high ppm was found to be effective
against the spoilage microorganisms. Ozone is a
strong oxidizing agent It should not be used on
high-lipid – content of foods, since it causes
rancidity.
Both CO2 and O3 are commonly used in
retarding surface spoilage of beef quarters for long
period. Use of CO2 atmosphere and now vacuum
packaging of meats is applied for extending the
shelf life of meats
Effect of CO2 / O3

20
 1. Inhibitory effect of CO2 increase with
decreasing temperature is due to the
solubility of CO2 at lower temperature
2. pH of meats stored in high CO2
environments is slightly lower than that of
air stored controls due to carbonic acid
formation.
3. Gram-ive bacteria are more sensitive to CO2
than Gram +ive. Pseudomonas is more
sensitive than lactic acid bacteria and
anaerobes are more resistant steaks stored
in 100% CO2 recorded lower counts on 16-27
days after slaughtering compared to steaks
stored in 100% N2 or 100% O2.
4. Effect of high concentration of CO 2 in meat
packs is to shift the flora from nierogenous
one consisting of Gram –negative to one
consisting primarily or Lactobacillus / and
other lactic acid bacteria.
Smoked pork lions

0 Day Vacuum CO2 N2

48 days 48 days 48
days
APC/g 2.5 7.6 6.9 7.2
pH 5.8 5.8 5.9 5.9

21
 Lactos Lactos Lacto
(52) (74) s
(67)
Dominant Flora
Flavobacterium
(20)
Arthrobacter
(20)
Yeast(20)
Pseudomonas (11)
Corynebacterium
(10)

22
 INCIDENCE AND TYPES OF MICROORGANISMS IN
FOODS:

The number and type of microorganisms

present in a finished food product are influenced
by

1. General environment from which the food

and originally obtained.
2. Microbiological quality of food in its raw or
unprocessed state.
3. Sanitary condition under which the product
is handled / processed.
4. The adequacy of subsequent packaging,
handling and storage conditions in
maintaining the flora at low level.
To produce good quality market foods, it is
important to keep microorganisms at low levels for
reasons of aesthetics, public health and product
shelf life. All foods should be expected to contain
a certain number of microorganisms of one type or
another, unless the food materials are made
sterile.
High number of microorganisms in fresh
foods causes alarm on the quality. The inner part
of plant / animal tissues are generally sterile, and
it is theoretically possible to produce many foods
free of microorganisms.

23
 The objective become impractical when,
mass production and other economic
considerations are realized. The number of
microorganisms in fresh food product is taken to
reflect the overall conditions of raw product
quality, processing, handling, storage and soforth.

24
 No. of Microbial %
samples group/Target Samples
meeting
torque

Raw 735 APC, Log 76

meat 10.6.00/g
parties

Coliforms 2/g 84

E.Coli 2.00/g 92

S.aureus 2.00 85

Presence of 0.4
Salmonella
Fresh 1830 APC,.6.70 89
ground orles/g
beef

S.aureus 92
3000/g

E.coli 2.00/g 84

Salmonella 2

Clostridium 20
keefugus

(i) Comminuted or ground beef generally

have high number of microorganisms

25
 than no comminuted meats such as
steaks.
(ii) Commercial ground meats generally have
more timing from various cuts. These
pieces have been handled excessively
which leads to more number of
microorganisms that meat cuts such as
steaks.
(iii) Ground meat provides more surface area,
which itself accounts for increased flora.
In this particle size reduced, surface
area increases with a consequent
increase in surface energy. The greater
surface area of ground meat favors the
growth of aerobic bacteria, the usual low
temperature spoilage flora.
(iv) In some commercial establishment, the
meat grinders, cutting curves, and
storage utensils are rarely cleaned,
which leads to buildup of microbial
numbers.
2
Mean log/inch

Saw blade Cutting block

Total count 5.28 5.69

26
 Coliforms 2.30 2.04

Enterococci 3.64 3.77

Staphyloccoc 1.60 1.00

us
Micrococcus 3.69 3.79

Bacillus / Clostridia are found in meats of all

types.The incidence of Clostridium perfringenes
in a variety of meats is given below
16.4% - raw meats, poultry / fish;
5%in spices;
3.8% in fruits / vegetables;
2.7% in Commercially prepared frozen foods
and
1.8% in home prepared foods.

Most commonly found microorganisms in fresh and

frozen beef, pork, and related meats.
E. coli (29%);
Serratia liguefaciens (17%);
Pantoea agglomerous (12%).
Citrobacter freundii
Klebesiella pneumoniae 32%

27
Enterobacter cloacea
E.hafniae

SOY EXTENDED GROUND MEATS

Addition of soy protein (Soybean flour) soy

flakes texturized soy protein) at levels of 10 – 30%
to ground meat patties. The no of microbial
population per gram of the Soy product is given
below

Fungi /gColiform E.coli / S.

/g g aureus /g
25 3 3 10
The bacteria grow faster in the meat soy
blends than in non-soy controls.
Generally, addition of soy protein increases
the surface area of soy-meat mixtures and also
increases the pH of the soy extended products
which leads to flourish of aerobic bacteria even at
refrigerated condition
Mechanically Deboned meat, poultry and fish
Meat animals when slaughtered, meat from
the carcasses is removed by meat cutters. The
most economical way to salvage the small bits and
pieces of lean meat left on carcasses bones is by
mechanical means (Mechanical deboning) The
mechanically deboned meats is removed from
28
bones by machines. During deboning, the small
quantities of calcium / bone peeler become a part
of the finished product. In Mechanical deboned
meat, amount of bone should not exceed 0.75%
with a minimum 14% protein; Not more than 30%
fat and Increased pH due to the addition of Ca and
pH 6- 7.0
High counts of microorganisms are found in
MD meat. In Mechanical deboned poultry coliform
counts by MPN method shows that 460 – 1100 / g
 Presence of Salmonellae and Clostridium
perferigens. The APC of hand boned lamb is
6,80,000 / g whereas
 Mechanical deboned lamb allowed for 1 week –
6,50,000 / g. Mechanically deboned fish were
found to contain ten fold higher number of
organisms than conventionally processed fish.
 MDM support the more rapid growth of
psychrotrophic bacteria than lean ground beef.
 Absence of S.aureus in MDM because these
products are less handled by meat cutters.
HOT BONED MEATS
In conventional processing of meats,the
carcasses are chilled after slaughter for 24 h and

29
processed under chilled condition. This process is
known as Cold boning.
Hot boning involves the processing of meats
generally within 1 – 2 h after slaughter while the
carcasses is still hot. In, Hams the cold boned
hams have less number of microorganisms when
compared to hot boned hams. In general, the hot
boned hams contain 67% more Staphyloccoci

whereas cold boned ham contains 47% only.

Generally hot boned meat / pork has more

microorganisms than cold boned meat. Mostly
mesophiles are dominant.
Hot boning accompanied by pre rigor
pressurization consisting of the application of
around 15,000 PSI for 2 minutes improves muscle
colour and overall shelf appearance and increases
tenderization.
Effect of electrical stimulation
Before hot boning, carcasses are
electrically stimulated to speed the conversion of
glycogen to lactic acid. In this method, an electric
stunner is attached to a repeated pulses of 0.5 to
1.0 or more seconds are administered to the
product at 400 + volt potential difference between

30
the electrodes. This process has no effect on
microflora.
Vacuum packed meats
Vacuum packaging is achieved by placing
meats into plastic bags or pouches followed by the
removal of air using a vacuum packaging machine
and the closure of the bag is often by a heat
sealer.
From the microbiological point of view, the
most detrimental point is the change in the
gaseous environment of the product. In this
method, O2 is not removed fully, some amount of O 2
is present which favors the growth of aerobic flora
and meat itself results in an increased level of CO 2,
which is inhibitory to the flora.
O2 – permeable packaging is used –
refrigerated fresh meats under go spoilage by gram
negative organisms by increased pH and foul odors
– Pseudomonas sp. Is being predominant
organisms.
If O2 impermeable packaging is used the
growth of lactic acid bacteria is favored and
Brochothrix thermosphacta is favored because of
increased levels of CO2 and lowered oxidation-
reduction potential.
31
 In vacuum packaged lamb the flora consists
of Streptococci, Micrococci, Staphylococci,
Moraxella, Aceinetobacter and Pseudomonas. In
vacuum packaged lamb in pre film 86% flora
consists of Pseudomonas. O2 barrier film 80 – 90%
flora consists of Lactobacillus . L. cellobiosur. In
Vacuum packaged Beef
Initial surface count was log 2.48 /cm2
Initial flora – 85% consisted of Aeromonas sp.
- 15% Gram positive, caloretibacteria
- After 8 weeks – B.thermosphacta – 39%
- Lactobacilli – 22%
- Psychrotrophic Enterobacteria – 39%
When nitrites are present in the meat; domination
of lactic and bacteria is predominant, since these
bacteria are insensitive to nitrite and
microaerophilic environment is more favorable for
Gram –ve.
B.thermosphacta and Enterobacter are inhibited by
nitrites. In Raw beef, stored under vacuum-packed
condition, Cl.botulinum was present and the toxin
was observed after 6 days. Changes in
organoleptic characters occur.
Chicken:
Initial count – log 3.30 /cm2
32
After chickens were cut – log 3.80 /cm2
During packaging – 4.08 /cm2
Cutting block – 4.68 /cm2
Chilling studies
1 day – E.coli – 85% Enterobacter – 6%
10 days - 4C – E.coli – 1.4%
- Encrobacter – 88%
- Salmonella infantis Turkey
- S.reading are more
common in poultry
- S.blocklary
- Clostridium perfringenes
Sea food
The incidence of microorganisms in sea food
such as shrimp oysters, depends on the quality of
water from which it is harvested. Most of the
organisms get into the fish during processing. In
haddock fillets, most microbial contamination was
found to occur during filleting, and subsequent
handling prior to packaging.
For Ex. Pacific shrimp – Moraxella – 30 – 60%
Pseudomonas – 8 – 22%
Acinetobacter – 4 – 24
Listeria monocytogenes – low levels

33
 The retail samples of fresh fish and shellfish
found to contain cl.perfringens. In general frozen
seafoods have lower microbial count than the
fresh products.
Studies on microflora of fish at retail outlets
shows that the coliforms are present @ 1 to 7.77
cells / g in frozen and 7.9 to 4800 / g of fresh. Plate
count are generally higher an sea food when
incubated at 30C than at 35C.
Vegetables
The incidence of microorganisms in
vegetables depends on
(i) Sanitary quality of processing steps
(ii) Microbial condition of the raw product at
the time of processing.
In fresh style beans, the number of
microorganisms buildup immediately
after slicing. Processing operation often
leads to more number of
microorganisms.
Total count ranged from log 5.60 to 6.00
After blanching log 3.00 to 3.60
Processing, stages / packaging log 4.72
to 5.94

34
 Nearly, 40 – 75% of bacterial flora found to consist
of Leuconostoc, Streptococci – Fresh peas, snap
beans and corn. Initially 41 – 75% of samples
found to contain lactic acid cocci and after
processing, the number of staphylococci will build
up.
The presence of Staphylococci was observed;
these organisms were unable to proliferate due to
the presence of more number of lactic acid
bacteria.
In vacuum pouch – pack vegetables,
C.botulinum is present mostly in spinach and in
Cauliflower, Coliform < 20 / g and in Corn, Coliform
was present
Dairy products
The microflora of raw milk consists of those
organisms that may present in cow’s odder and
hide and on milking utensils or lines. Under proper
handling and storage conditions, the predominant
flora is gram positive. The yeast, molds and gram-
negative bacteria are found along with lactic acid
bacteria, most or all these type are heat sensitive
than gram +ive and are destroyed during
pasteurization.

35
 Psychrotophic spore formers and
mycobacteria were present in raw milk. The
psychrotropic Bacillus, Clostridia, can survive
pasteurization temperature and cause problems in
the refrigerated products. In cheese, Salmonella is
present. Ice cream often contaminated by egg –
“salmonella”
DELICATESSEN AND RELATED FOODS
Delicatessen foods such as salads,
sandwich are involved in food poisoning outbreaks.
These foods are often prepared by hand and this
direct contact may leads to an increased incidence
of food poisoning agents such as S.aureus.
S.aureus was present in 60% sandwiches; 39%
salads.
Retail trade salads – coliforms, S.aureus.
In frozen meat pees, the addition of ingredients
increase the number of organisms and the total
count of the finished product reflect the overall
quality of ingredients / handling and storage.
In general, these products should not exceed log
5.00 /g. In dehydrated space food,
E.coli – absent
S.aureus – negative in 5 g
Salmonellae – negative in 10 g
36
Steptococci – 1.30 /g

ENTERAL NUTRIENT SOLUTION (ENTERAL FOODS)

These are liquid foods administered by tube. They
are available as powdered products requiring
reconstitution or as liquids. The microbiology of
ENS found to contain Staphylococus epidermidis,
Corneybacterium and Citrobacter.

37
 Microbial examinations of foods
The examination of foods for the presence,
types and number of microorganisms / or their
products is basic to food microbiology. The four
basic methods employed for total numbers are
(i) Standard plate count for viable
cells (SPC)
(ii) Most probable number (MPN)
method as a statistical
determination of viable cell.
(iii) Dye-reduction techniques to
estimate the numbers of viable
cells that possess reducing
capacities
(iv) Direct microscopic count
(DMC) for both viable and
nonviable cells.

Standard plate count method

In this method, the food samples are
blended or homogenized, serially diluted in an
appropriate diluents and plated in or on to a
suitable agar medium at an appropriate
temperature for a given time, after which colonies
are counted by use of a Quebec or electronic
counter.
SPC – is the most widely used method for
determining the number of viable cells or colony

38
forming units (Cfu) in a food product. When the total
viable counts are reported for a product, the counts
should be viewed as function of at least some of
the following factors.
1. Sampling methods employed
2. Distribution of organisms in the food
sample
3. Nature of food flora
4. Nature of food material
5. Pre examination history of food products
6. Nutritional adequacy of the plating
medium employed.
7. Incubation temperature / time used
8. pH, aw, and oxidation – reduction
potential of the plating medium
9. Type of diluents used
10. Relative number of organisms in food
sample
11. Existence of other competing or
antagonistic organisms.
In addition, the plating procedures for
selected groups are further limited by the degree
of inhibition and effectiveness of the selective /
differential agents involved. SPC is more often

39
determined by pour plating, comparable research
can be obtained by surface plating.
Surface plating offers advantages in
determining the number of beat sensitive
psychrotorophs in a food product because these
organisms don’t come in contact with melted agar.
Strict aerobes are favored by surface plating but
microaerophilic organisms tend to grow slower.
The disadvantage of surface plating was spreading
in the agar plates. Also crowing of colonies, which
make enumeration more difficulties.
Homogenization of food samples
Microorganism is extracted from food
specimens for plating almost universally by use of
Mechanical blenders. In 1971, Sharpe and
Jackson, developed colwell stomacher, and this
device is used for homogenizing the foods.
It is a simple device, for homogenizing the
specimen in a special plastic bag by vigorous
pounding of two paddles. The pounding effects
the shearing of food specimens and
microorganisms are released in to the diluent.
Three models are available and the model 400 is
commonly employed in the homogenization of food
samples. Stomacher is similar to that of blenders
40
but the stomacher is preferred over blending for
the following reasons.
1. The need to clean and store blender
containers is obviated
2. Heat buildup doesn’t occur during normal
operational time
3. The homogenates can be stored in frozen
in the stomacher bags
4. The noise level is not as unpleasant as
that of mechanical blenders.
Standard plate count method (SPC)
SPC is determined by pour plating and
similar results can be obtained by surface plating
method. In the surface plating method, prepared
and hardened agar plates with dry surfaces are
employed.
The diluted specimens 0.1 ml inoculum /
plate is placed on the surface hard agar plates,
and with the aid of bent glass rods the inoculum is
carefully and evenly distributed over the entire
surface. Surface plating offers advantages in
determining the numbers of heat sensitive
psychographs in a food product, because these
organisms don’t come in contact with melted
agar. Strict aerobic organisms are favored by
41
surface plating but micro aerophilic organisms
tend to grow slower.
The main disadvantages of surface plating
are the problem of spreaders, (i.e. when the agar
surface is not adequately dry prior to plating) and
the crowding of colonies, which makes
enumeration more difficult.

SPIRAL PLATER: is a mechanical device that

distributes the liquid inoculums on the surface of
the rotating plate containing a suitable poured
and hardened agar plates. The dispensing arm
moves from near center of the plate towards
outside, depositing the sample in Archimedes
spiral. The attached special syringe dispenses
continuously decreasing volume of sample so that
the concentration range of upto 10.,000:1 is
effected on a single plate. Incubation of plates at
an appropriate temperature, colony development
reveals a higher density of deposited cells near
the center of the plate with progressively fewer
towards the edge.
The enumeration of colonies on plates
prepared with a spiral plater is achieved by use of
special counting grid. The advantage of spiral
42
plater over standard plating was less quantity of
agar is used and 3 to 4 times more samples / hour
can be examined.
Among the disadvantages, the food particles
may cause in blocking the dispensing styles. It is
more suited for use with liquid foods such as milk.
The spiral plating disease is developed by Giletin’s
et al.
The efficiency of spiral plating is similar to
that of standard plating.
Membrane filters
Membranes with a pore size of 0.45 m will
retain bacteria but allow water or diluent to pass
are used. The known volume of samples is
allowed to pass through the membrane and the
bacteria are collected on the membrane after
filtering. Then the membrane is placed on an agar
plate, or an absorbent pad saturated with the
culture medium of choice and incubated
appropriately. After incubation the colonies are
enumerated and also direct microscopic count
can be made.
This method is suitable for samples that
contain low numbers of bacteria and only small

43
samples of dilute homogenates from certain foods
can be used for single membrane.
The efficiency of membrane filter method for
determining microbial number by direct
microscopic count has been improved by
introduction of fluorescent dyes. The use of
fluorescent dyes and epifluorescent microscopes
to enumerate bacteria in water has been
employed widely. At early stages, cellulose filters
were used and now polycarbonate nucleopore
filters after the advantage of retaining all bacteria
on the top of the filter.
DIRECT EPIFLUORESCENT FILTER TECHNIQUE
DEFT – employs fluorescent dyes and
fluorescent microscopy. It is a rapid method for
enumeration of microorganisms in food. A diluted
food homogenate is filtered through a 5 m nylon
filter and the filtrate is collected and treated with
2 ml of Triton x-100 and 0.5 ml trypsin. Trypsin is
used to lyse somatic cells and to prevent clogging
of filters. After incubation, the treated filtrate is
allowed to pass through 0.6 m nucleopore
polycarbonate membrane and the filter is stained
with acridine orange.

44
 After drying, the stained cells are
enumerated by epifluorescent microscopy and the
number of cells / g is calculated by multiplying the
aerage / field try the microscopic factor. DEFT
has been employed successfully to estimate
number of microorganisms in meat and poultry
and on food contact surfaces.
MICROCOLONY – DEFT
In DEFT allows direct microscopic
determination of cells whereas micro colony –
DEFT allows to determine only viable cells. The
food homogenates are allowed to pass through
DEFT membranes. The membrane is then placed
on the surface of appropriate culture media and
incubated for micro colony development.
Three-hour incubation period is
sufficient for Gram-negative bacteria and 6 hr for
gram positive. The micro colonies that develop
must be viewed with a microscopic.
Coliforms, Pseudomonas and
Staphylococci could be detected within 8 h.
HYDROPHOBIC GRID MEMBRANE FILTER (HGMF)
This method was developed by Sharpe and
Michaud to enumerate microorganisms in variety
of food products. This method employs a
45
specially constructed filter that consists of 1600
wax grids on a single membrane filter that
restricts the growth and colony size to individual
grids.
On one filter, 10 to 9 x 10 4 cells can be
enumerated by an MPN procedure.
It is widely used to enumerate all cfu or
specific groups such as indicator organisms,
fungi, Salmonellae and Pseudomonas.
For ex. 1 ml of 1:10 homogenate is filtered
through a filter membrane, and followed by
placing the membrane on a suitable medium for
incubation overnight to allow colonies to develop.
The grids that contain colonies are counted and
MPN is calculated.
MICROSCOPIC COLONY COUNTS
It involves counting of micro colonies that
develop in agar layered over microscopic slides.
The method was devised by frost. In this
method, 0.1 ml of milk-agar mixture was spread
over a 4 cm2 area on a glass slide. Following
incubation, drying and staining, micro colonies are
counted with the help of microscope.
2 ml of melted agar are mixed with 2 m of
warmed milk and after mixing 0.1 ml of inoculated
46
agar is spread over a 4 cm2 area. The slide is
stained with Thionin blue and observed in a
microscope.
AGAR DROPLETS
Developed by Sharpe and Kilsby. The food
homogenate is diluted in tubes of method agar.
For each food sample, three tubes of agar are
used. The first tube being inoculated with 1 mo of
food homogenate. Mixing it a sterile capillary
pipette is used to transfer a line of 5 x 0.1 ml
droplets to the bottom of an empty petridish. With
the same capillary tube, three drops (0.1 ml) from
the first 9 ml tube are transferred to the second
tube and mixed and similarly another line of 5 x
0.1 ml droplets is placed next to the first.This step
is repeated for the third tube of agar petriplates
containing the agar droplets are incubated for 24
h and colonies are enumerated with aid of 10 x
viewer. This method is three times faster and 24 h
incubation gave counts equal to those obtained
after 48 h by conventional plate count. Dilution
blanks are not required and only one petridish /
sample is needed.
DRY REDUCTION METHOD

47
 Two days are commonly employed to
estimate the number of viable organism in
suitable products viz., Methylene blue and
resazirin. To conduct a dye reduction test,
properly prepared supernatants of foods are added
to standard solutions of dye for reduction from
blue to white for methylene blue and from slate
blue to pink or white for resazurin.
The time for dye reduction is inversely
proportional to the number of organisms in the
sample. Resazurin reduction method is a rapid
procedure for assessing the ground beef spoilage.
One of the problems of using dye reduction for
some foods is the existence of inherent reductive
substances. Ex. Raw meat.
Methylene blue reduction test has a long
history of use in the dairy industry for assessing
the microbial quality of raw milk. Dye reduction
method has advantages like, simple rapid, and
inexpensive and only viable cells actively reduce
the dyes and disadvantages are
(i) Not all organisms reduce the dye
equally
(ii) Not applicable to food specimens that
contain reductive enzyme
48
 (iii) Cell morphology cannot be assessed.

Direct microscopic count

49