Here we discused about the various microbial testing of food product and the industrial aspects of the test

1. Microbial testing of Food
Products
Mujeeb rehman M.K-
19msfood06
R.Vidyaeswari- 19msfood12
Aswin kumar. K- 19msfood15
Anirban Das – 19msfood19

2. Why microbial testing?
It is done to identify and restrict harmful microorganisms,
which can spoil food products and to protect from food
borne diseases.

3. AEROBIC MESOPHILIC PLATE COUNT
It indicates microbial counts for quality assessment of foods.
Procedure:
1. A food homogenate of 1:10 ratio is prepared.
2. A pour plate is prepared where agar is used as a medium.
3. The petri dishes are incubated at the desired temperature
and for 48 hours or more (as per the food regulation).
4. Following incubation the colonies are counted and
calculated and are recorded per dilution form.
5. It is expressed as 1CFU/g.

4. ACIDURIC FLAT SOUR SPORE-FORMERS.
To determine and confirm Aciduric Flat sour spore formers in foods:
The organism of this group is Bacillus coagulans. It is responsible for
spoilage of canned products.
Procedure:
1. the dilution of the sample are taken in test tube and heat shocked at
88 degree Celsius for 5 mins.
2. the test tubes are immediately cooled and one ml of the heat
shocked sample is transferred into petri plate.
3. 18 to 20 ml of melted bromocresol purple agar is added and then
the plates are incubated at 55 degree for 48 hours.
4. it is calculated by multiplying average plate count with dilution
factor (X).
5. and it is expressed as X/g.

5. Bacillus cereus
Detection and determination of Bacillus cereus in foods, and in beverages:
*Most probable number method:
This is suitable for food samples containing less than 1000 B.cereus per gram.
Procedure:
1. The food homogenate is prepared.
2. the decimal dilution is prepared by pouring 1ml in 9ml of dilution water.
3. it is inoculated in three test tubes of prypticase-soy-polymyxin broth with 1
ml food homogenate and its dilutions. Incubate at 30 degrees for 48 hours.
4. examine for dense growth and transfer one loopful from each growth
positive tube to dried MYP medium plates. Streak to obtain isolated colonies.
5. incubate at 30 degrees for 48 hours. Transfer to nutrient agar which has
colonies surrounded by precipitate zone.

6. *Plate count techniques:
This is suitable for examination of foods expected to contain more
than 1000 B.cereus per gram.
Inoculate duplicate MYP agar plates with the homogenate and each
dilution of homogenate by spreading 0.1 ml evenly on to each plate in
duplicate with sterile bent glass streaking rods.
Incubate plates 24 hours at 30 degrees Celsius.
The number of eosin pink colonies surrounded by lecithinase zone are
counted. Plates must have 15-150 colonies.
It is expressed as following:
Bacillus cereus = present/absent

7. Detection, determination and conformation of coliforms, faecal coliforms
and e.coli in foods and beverages.
* Solid medium method
Procedure:
1. pipette 1ml of food homogenate and of each dilution into each of the
appropriately marked duplicate petri dishes.
2. Pour into each petri-dish 10-12 ml of VRBA and swirl plates to mix and
allow to solidify. Overlay with 3-5ml VRBA and allow to solidify.
Incubate the dishes, inverted at 35 degrees for 18-24 hours.
3. Count all the colonies that are purple red in colour and are surrounded
by a zone precipitated bile acids. Each plate approx. has 30-100 colonies.
4. It is calculated by multiplying the total number of colonies per plate
with the reciprocal of the dilution used and reported as coliforms per gram
or ml.

8. * Most probable method:
This method is valuable in those samples where coliform density
is low because higher quantity of sample can be used for
examination.
Procedure:
1. Inoculate each of 3 tubes of LST broth with 1ml of food
homogenate.(1:10)
2. Do the dilution process for every test tubes (1:100);(1:1000)
3. Incubate the LST tubes at 35 degree Celsius for 24-48 hours.
4. It is calculated as coliform = present/absent per gram.

9. Methods
There are two types of method qualitative and quantitative.
Quantitative methods are designed to estimate directly or
indirectly the microbial load in a test material.
Qualitative methods are used to detect the possible presence
of certain foodborne pathogens, especially those capable of
causing high fatality rates among consumers.

10. Methods & techniques
Traditional method
• Plate counts
• Membrane filtration
• Most probable
number
• Direct microscopic
count
• Dye reduction tests
• Indicator
Rapid Method
• Direct epifluorescent
filter technique
(DEFT)
• Electrical impedance
Enzyme-linked
immunosorbent
assay(ELISA)

11. Plate count method
• Diluent
• 0.85%NaCl
• 0.1% peptone
• Phosphate buffer
• Medium
• Elective medium
• Selective medium
• General
• Petri dish plate
• Replication
•Pour plate
•Spread plate
•Drop plate

12. Plate count depends on
Diluent
Food homogenate
Dilution series
Medium
Plating method
Incubate conditions

13. Application of plate count
Check quality of RM & final products
Check condition hygiene
Estimate storage life of products
Determine
Production
Transport
Storage
Determine pathogens

14. Dye Reduction Test
Some dyes such as methylene blue and resazurin are colored
in oxidized states but colorless under reduced conditions.
This change can occur because of microbial metabolism and
growth.
It is assumed that the rate of reduction during incubation of a
specific concentration of methylene blue added to a food is
directly proportional to the initial microbial load in the food.
 This method is generally used to determine the
microbiological quality of raw milk.

15. Membrane epiflurescent

16. Bioluminescence
 The bioluminescence method measures the ATP content in a
sample as an indirect measurement of microbial load.
 As only viable cells retain ATP, the amount of ATP is
regarded as directly related to the microbial load in the
sample.
 Using the luciferin-luciferase(from firefly) system in the
presence of Mg, the ATP concentration in the lysed cells in a
sample is measured.
 The method is very rapid.

17. Detection and Determination of Anaerobic Mesophilic spore
formers (Clostridium perfringens)
procedure:
1. Inoculate 2g of food sample into 15-20 ml of cooked meat
medium in duplicates. Incubate for 24 hours at 35 degree Celsius.
2. positive tubes showing turbidity and gas are streaked on to TSC
agar plates. Plates are incubated anaerobically for 18 to 20 hours
at 35 degree Celsius.
3. the colonies that are black in colour surrounded by a zone of
precipitate are counted.
4. Clostridium perfringens = present/absent

18. Detection and confirmation of sulfide spoilage spore formers in
processed foods
Used in canned foods
The culture media is sulfite agar solution
Procedure
•for sample preparation and heat treatment follow the anaerobic
thermophilic spore formers.
•Inoculations of the prepared sample are placed into the sulfite agar
medium with a nail. Incubate at 55 degree Celsius for 24-48h in
anaerobic jar.
•D. nigrificans will appear as jet black spherical areas,
•The color due to the formation of iron sulfide.
•No gas production is occur.
•Colonies can be counted and spore/g of sample

19. DISULPHOTOMACULUM NIGRIFICANS

20. Detection and determination of thermophilic flat sour sporeformers
The culture media is Dextrose tryptone agar
Procedure
• weighed samples are heat treated at 100 degree Celsius for 30
min. Then allow rapid cooling.
• Aliquotes of these solutions are then transferred to petri plates.
• Media is added and swirled gently to distribute the inoculum.
allow to solidify
• Starch sample are added to the sterile dextrose tryptone agar in
100 ml flask.
• Autoclaved flasks are used[51 b for 10 min]
• After the cooling mixture is equally distributed in to 5 plates and
allow harden.

21. • Then plates are incubated at 50-55 degree Celsius for 48-
72 hr.
• Flat sour colonies are round [2-5mm in diameter]
• And dark opaque center, surrounded by a yellow halo in a
field of purple.
• The colonies are counted and expressed in terms of
number of spores per g of the sample.

23. Direct Microscopic count in tomato puree, sauce, paste, chutney
PROCEDURE
1.mould count
2. bacterial count
3. Yeast and bacterial spore count
PREPARATION OF SAMPLE
Tomato juice: Use juice as it comes from container

24. Ketchup or sauce: Place 50 ml stabilizer solution in 100 ml graduated cylinder, add 50 ml
well mixed sample by displacement and mix thoroughly.
Stabilizer solution: 0.5% sodium carboxyl methyl cellulose (NaCMC)-place 500 ml
boiling water in high speed blender. With blender running add 2.5 gms NaCMC and 10 ml
formalin and blend for 1 minute. Keep in a stoppered bottle.
Puree and paste: Dilute the sample stabilizer solution and mix thoroughly so that the
refractive index of 1.3448 to 1.3454 at 20℃ is obtained.

25. Mould count
1. Prepare two mounts and count only 25 field from each, observing in such a manner as to be
representative of all sections of mount.
2. Observe each field noting presence or absence of mould filament and recording results as positive when
aggregate length of not less than 3 filaments present exceeds 1∕6 of diameter of field .
3. In case a single filament is traversing several fields of microscope it is counted as one positive field.
4. Calculate proportions of positive fields from results of examinations of all observed field and report as
percent fields containing mould filaments.
5. Percent positive fields =
𝑛𝑜.𝑜𝑓 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑓𝑖𝑒𝑙𝑑𝑠
𝑛𝑜.𝑜𝑓 𝑓𝑖𝑒𝑙𝑑𝑠 𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑
×100

26. BACTERIAL COUNT
Place the haemocytometer slide under microscope and using 400 to 500 magnification count four small
size squares from each corner of ruled chamber and the central medium square.
No. of bacteria in 20 small squares = B
No. of bacteria in 400 small square= (B x 400) / 20
That is - 20 B bacteria
1.0 mm3 contains - 20 x 10B
1.0 cc contains - 20 x 10 x 103 B or 2 x 105 B
If the material is diluted five times then the number of bacteria per ml
of sample is =5 x 2 x 105 B or 10x105 or 106B or B million/cc.
e.g. If B = 20 then the count will be 20 million per cc

27. Yeast and Bacterial spore count
The same slide prepared for bacterial counts is used and a total of 200 squares comprising of 0, 40 and
80 from the top, middle and base of ruled chamber respectively is counted.
Calculate number of yeasts/bacterial spores per 1/60 mm3 as follows:
No. of yeasts/spores in 200 small squares = Y
No. of yeast/spores in 400 small squares = (400 x Y)/200 or = 2Y
Or 0.1 mm3 contains = 2Y yeast
1.0 mm3 contains = 2 x 10 Y yeasts
 1/60 mm3 = (2 x 10 x 1 Y yeast)/60
 Or 1/3 Y yeasts
If diluted 5 times then 5 x 1/3Y; or
5/3 Y yeasts/bacterial spores per 1/60 mm3 of the sample.

28. ROPE PRODUCING SPORES IN FLOURS
PROCEDURE:
1. 50 g of sample is taken along with 450mk of sterile 0.1% peptone water in a blender for mixing.
2. ten and one ml volumes of peptone water suspension are pipetted into separate 100 ml portions of
melted dextrose tryptone agar contained in 250ml flasks and held at 45 degree Celsius.
3. the flasks and a control flask is submerged in a boiling water bath for 15mins.
4. after heating, the flask contents are cooled to about 45 degree Celsius and contents of each flask
is poured into 5 sterile plates in equal volumes. After agar solidifies, it is incubated inverted at 35
degree Celsius for 48 hours.
5. the total colonies on the set of 5 plates from the flask that received 10ml suspension are
considered as rope spores per gram of sample.
6. rope spore is expressed as ____/g.