Chapter 7

Microbial Analysis of foods

Microbiological Standards
Found in the Food Regulations Act 1984
Cheese
The cheese shall contain not more than:
100 Escherichia coli per gram.
100 Staphylococcus aureus per gram.
A 50 g sample shall be free from Salmonella.
Ice cream, frozen confections
When subjected to the test described in the Fourth Schedule to the Food Regulations AAct
1984, 3 out of 5 replicate portions of 0.1 millilitre shall not give evidence of acid formation and
gas formation as described in that schedule. Tubes showing evidence of acid and gas formation
shall be subcultured to an additional tube of test media and incubated according to the test
prescribed in the Fourth Schedule as a confirmatory test.
Milk
(a) Raw milk. raw cream
Standard plate count at 30°C <150,000 colonies/ml.

(b) Pasteurized milk and pasteurized milk products

Includes standardized milk, recombined milk, flavored milk, skim or non-fat milk, reduced-fat
milk, cream, whipping cream or whipped cream, reduced cream or pouring eream, light cream,
sour cream and recombined cream and any other milk product

Standard plate count at 35°C <50,000 colonies/m

(c)UHT (Ultra heat treated) milk and UHT milk products

UHT products shall be sterile when subjected to the test in the Sixth Schedule to the Food
Regulations Act 1984.

Pulped egg
When subjected to the resazurin test described in the Seventh Schedule to the Food
Regulations Act 1984 the reduction time shall be not less than 7 hours.

Yoghurt
pH<4.5
Lactic acid producing bacteria 2 106 per ml

Food containers
The Food Hygiene Regulations Act 1974, contains
requirements governing the "Microbiological
dlandards" for bottles, jars, or jugs, in that residual bacterial plate counts may not exceed:

() More than 1/millilitre of

containing capacity, or

More than
1/square centimetre of surface area.
 analysis
Microbiological

of
i n t e r p r e t a t i o n

1.
The Action
Table

Interpretation

Result None

expected
category
within type
Results
are
levels
for this
Good microbiological and
range)
(lower c o n c e r n .

product
of food
safety
no
present None
expected
within
type
for this
ae
Results
levels
Acceptable microbiological and
range)
(upper
o f product concer
no
food safety are taken for
present
Further samples
return good or
outside the expected
type
testing. If
these
Results are this results no action is
Unsatistactory levels for
microbiological

present no
food safetyY | acceptaDie
taken. If these
return
ofproduct,
but might
indicate poor
unacceptable
results the business
to determine if food
concern,
food handling practices. is inspected
controls and hygiene
handling
are adequate. A product
practices
withdrawal may be Considered
occurs.
while further testing
determine if
outside
Inspect supplier to
of the
Results are controls and
Potentially
microbiological levels for food handling
expected àre adequate;
a |hygiene practices
hazardous
this type of product and present Consider a product lecall.
potential food safety
concern.

MICROBIOLOGICAL EXAMINATION
OF MILK

Methylene blue reductase test

Principle:
This reductase test is based on the oxidation-reduction activities of the bacteria present in the milk
sample. The indicator used in the reaction is methylene blue which is color sensitive to oxygen
concentration. The indicator is blue in the oxidized state and leuco or white in the reduced
conditions. The speed of color disappearance of methylene blue is proportional to the microbial
load in the milk sample. The more the bacteria present the faster will be the reduction.

The classification of milk as per methylene blue reductase test is as follows.

Class I- Excellent, not decolorized in 8 hours.

. Class II-Good, decolorized in less than 8 hours but not less than 6 hours.
2 Class II- Fair, decolorized in less than 6 hours but not
less than 2 hours.
3. Class IV Poor, decolorized in less than 2 hours.

Requirements:
Milk sample, methylene blue solution, Mc
Cartney bottles, pipettes,
distilled water, Bunsen burner. water bath set at 5
before
Note: All the glass wares were sterilized use.
 Praedure

ution was
Methylene blue soluti
prepared by
of distilled water.
25 ml dissolving mg methylene blue ppowder aseptically
inred 10 ml of milk sample into sterile
Transferm

Me
Added methylene blue solution to the milkCartney bottle using sterile pippet.
bottle was closed with the sample using a separate sterile
tents of the tube were
stopper pipette.
The conte mixed by
Mc Cart
gently inverting it 2-3 times.
Incubate
te the
the Mc Cartney bottle in a water
bath
conrntrolled tubes containing 10 ml
at 37"C for 6 hours.
boiled milk and 1 ml of
incubated.
methylene blue was also
Recorded the time for discoloration

aaRIAL ANALYSIS OF ICE CREAM AND SOFT

DRINK
Principle

hotled beverages including non-pasteurized

in imum requirement for microbiological criterianon-carbonated
of the WHO
soft drink should conform to the
used in the manutacture of ice cream. standard. Only pasteurized milk
re reiected.
The frozen samples were Samples were received in the frozen state, melted one
melted immediately before examination.
lates ensure an aerobic environment for The
microorganisms present in the food sample. spread
Reguirements
le cream sample, soft drink sample, nutrient agar, pipettes,
petriplates, test tubes, L rod, alcohol.
Procedure

lce cream sample:

1. 1gof ice cream sample was weighed and added to 10 ml sterilized distilled
water blanks and
was labeled as 10 dilution.
Mixed gently by
inverting the test tubes for several times.
.
Prepared serial dilutions of ice cream sample by
distilled water and mixed well. transferring I ml from first dilution to sterile

4. This test tube was labeled as 10.

he procedure was repeated up to 7th test tube with respective dilution 10, 10,
10 10, 10,
using different sterile pipetes.
Discarded I ml of the sample from 10 dilution.
ml of the diluted sample from particular dilution were pipetted out into nutrient agar
plates and was spread
uniformly using a sterilized L-rod.
.
Aept the plates in an upright position for few minutes.

Cubated the plates in an inverted

position at 37 Cfor 24 hours.
Examined the plates for bacterial colonies.
b) Soft drink sample:
water blank
and was labeled as 1
sterilized distilled
added to 9 ml
m l of soft drink was

dilution.
several times.
MIx gently by inverting the test tubes for
transferringI ml
from first dilution to
drink sample by
SPrepared serial dilution ofthe soft
sterile distilled water blank and mixed well.

4. This test tube was labeled as 10 dilution.

5. to 7th test tube with respective dilution 10, 10", 10, 1o

he procedure was repeated up
10 using different sterile pipettes.
6. Discarded I ml of the sample from 10 dilution.
7. 0.1 ml of the diluted sample from particular dilution were pipetted out into nutrient agar

plates and was spread uniformly using a sterilized L-rod.

8. Kept the plates in an upright position for few minutes.

9. Incubated the plates in an inverted position at 37C for 24 hours.

10. Examined the plates for bacterial colonies.

Observation
The number of colonies formed in each counted for both ice
plates were cream and soft drink
samples. Plates with fewer than 30 colonies were designated as "too few to count" (TFTC) and
plates with more than 300 colonies as "too numerous to count" (TNTC).
MICROBIOLOGICAL ANALYSIS OF FRUITS AND VEGETABLES
Principle
Fruits and vegetables are readily susceptible to microbial decomposition, and hence considered as
perishables. The total number of bacteria present in the sample of fruits and
frozen food can be enumerated by traditional vegetables and other
pour plate or spread plate technique. A sterile food
blender is essential to obtain the
microorganism in suspension.
Requirements
Nutrient agar, fruit sample, vegetable sample, petriplates, pipettes, L-rod,
blender), alcohol. Homogenizer (food
Procedure

a) Fruit sample

. Using aseptic technique, I g of fruit was

weighed.
2 Weighed food sample was transterred into 10 ml sterile
distilled water blank and was labeled
as 10 dilution.
3. Mix gently by inverting the test tubes for several times.
 Ising a sterile pipette, I ml
of sample was transferred from 10 diution sterile distilled
water blank and was labcled as 10 dilution. to

Mixed gently.

The procedure was

repeated up to th
7 test tube wv ith respective diution 10. 10. 10. 10
10 using ditferent sterile
pipettes.
Discarded 1 ml of the sample from 10 dilution

0.1 ml of the diluted sample trom particular diution were pipetted out into nutrient agar
nlates and was spread uniformly using a sterilized L-rod.

Kept the plates in an upright position for few minutes.

Incubated the plates in an inverted position at 37C for 24 hours.

10.
Examined the plates for bacterial colonies.

b)Vegetablesample
Using aseptic technique, I g of vegetable was weighed.

transferred into 10 ml sterile distilled water blank and was labeled as

Weighed sample was

10 dilution.
tubes for several times.
Mix gently by inverting the test

transferred from 10 dilution to sterile distilled

Using a sterile pipette, I ml of sample was
4
10 dilution.
water blank and was labeled as
5. Mixed gently.

repeated up to 7th
was test tube with respective dilution 10", 10*, 10, 10°
6 The procedure
10 using different sterile pipettes.
dilution.
7. Discarded 1 ml of the sample from 10
out into nutrient agar
sample particular dilution were pipetted
from
8. 0.1 ml of the diluted
a sterilized L-rod.
plates and was spreaded uniformly using
for few minutes.
9. Kept the plates in an upright position
24 hours.
the plates in an inverted position at 37 C for
10. Incubated

bacterial colonies.
11. Examined the plates for

Observation vegetable
fruit and
counted for both
colonies formed in each plate were
count" (TFTC) and
The number of were designated as "too
few to
colonies
fewer than 30
samples. Plates with numerous to count" (TNTC).
colonies as "too
plates with more than 300