AN IN PLANT TRAINING REPORT

VERKA MILK PLANT, AMRITSAR, PUNJAB 

(AN ISO 9002 CERTIFIED)

SUBMITTED TO: SUBMITTED BY:

GENERAL MANAGER Simranjeet kaur
Harminder singh Sandhu MSc. microbiology
GNDU,Amritsar
 Acknowledgement
I would like to show my gratitude and sincere regards
towards the GENERAL MANAGER(Harminder singh
Sandhu) giving me this great opportunity to widen my
horizon of knowledge. I would like to thank(QUALITY
MANAGER) Gurdevpal singh & Mr.Amardeep (
DEPUTY MANAGER OF QUALITY ASSURANCE
LAB) for his immense support through the training.It is
not just acknowledgement rather, Its my deepest
gratitude toward my mentor Miss
Sonia(Microbiologist) . They all were very cooperative
& provided me all the facilities & required information.
Above all, I thank GOD for being with me all time,
supporting me & protecting me from all the unseen
dangers.

Yours sincerely
Simranjeet kaur MSc.microbiology
Guru Nanak Dev
University Amritsar
 COMPANY PROFILE

Verka  Milk  Plant  ,  Amritsar  was  the  first  milk  plant established in Northern 

region  .  The  foundation  stone  of  the  plant  was  laid  by  the  then  Hon’L  Chief 
Minister  of  Punjab  ,  S  .  Partap  Singh  Kairon  on  20  th  August  ,  1959.  The 
plant  was  inaugurated  by  Dr  A.M.  Thomas,  then  Deputy  Minister  of  Food  & 
Agriculture,Govt  of  India  on  23rd  March  ,1963.  The  equipment  of  the  plant 

 
was  gifted  by  the  United  States  of   America  ,  thus  assisting  and  providing  the 
Indian people with a more nutritious diet containing nature’s most perfect food .
 
In  the  beginning  ,  the  processing  capacity  of  the  plant  was  60,000  LPD  ,  with 
5.0  MT  per  day  drying  capacity  &  10,000  litres  Liquid  Milk  Supply under the 
control  of  Punjab  Government  .  This  was  the  first  Composite  Milk  Plant  in 
Northern  India  and  was  established  at  the  Verka  Village  on  the  outskirts  of 
Amritsar city . Therefore the brand  name  of  all  the  products  was  named  as 
“VERKA” which is internationally famous brand name for Milk Products.  
 1.MILK

Milk processing and production

Milk is the fluid secreted by the female of all the mammalian species , primarily to meet
the nutritional requirements of the new born such as energy , essential amino acids ,
vitamins , minerals & water. Milk is an effective and balanced source of lipids and
proteins ( caseins and whey proteins ), carbohydrates (mainly lactose ), minerals
(calcium phosphate ), enzymes , vitamins and trace elements .

Different types of milk

Types of milk Fat% SNF% Package name Packaging color Market price

Standardised
milk 4.55 8.50 Shakti Green 24
Full cream milk
1.5 9.0 Gold Red 27
Double toned
milk 6.0 9.0 Slimmers Yellow 19
Toned milk
3.0 8.5 Tazza Blue 21
Cow milk
4.0 8.3 Cow milk Pink 22
 Details of raw material processing
Milk received from various cooperative societies is collected at dock site of the plant . It includes
weighing of milk and preparation of record sheet . The reception of milk has been illustrated by
the following points:
1. Unloading of the cans
2. Placing the cans on the conveyor belt
3. Dumping the cans of a particular society in weighing bowl
4. Drawing the sample of milk of a particular society
5. Passing the cans to the cans washer
6. Trafficking of the milk to the processing section with a suction motor
7. Washing the cans with steam and water

The place where raw milk is received from vehicles is known as

dock . various chemical and organoleptic tests are conducted at this place
for checking the quality of the milk and termed as dock test or platform
test. The various test carried out for the first hand quality checking of milk
are fat , SNF, acidity , sediments , CLR , and specific gravity.

Filtration of milk
Filtration is done to remove the visible foreign matter. If any foreign particles are present in the
milk then complaints from consumer will come . Filtration of milk is done at two stages :
1.Filtration of raw milk
2.Filtration during pasteurization of milk in the regeneration section

Cooling and storage of milk

The raw milk received at milk plant is usually between temperature 20-30℃. This temperature is
quite suitable for the growth of mesophiles . Further cooling and storage of raw milk is done at
suitable temperature ranges.

Pasteurization of milk
Pasteurization is process that kills pathogenic microbes (mainly bacteria ) in food and drink ,
such as milk, juice, canned food and others .
It is the process of heating milk at least at 63℃ for 30mins or at 72℃ for 15 seconds .It is done
in approved and properly operated equipment .At this temperature phosphatase enzyme is
destroyed to give the negative phosphatase test. After pasteurization milk is immediately cooled
to less than 5℃.

High temperature short time (HTST)pasteurized milk typically has a shelf life of 2-3 weeks ,
whereas ultra pasteurized milk can last much longer , sometimes 2-3 months.

Process of cooling
The milk is cooled in plate type chillers which are made up of stainless steel . One site of the
plate is exposed to the milk and other to the chilled water. The water is supplied at the
temperature of 11.5℃ and milk is cooled upto 5℃.

Homogenization of milk
In this fat globules are broken down to 2 microns or less under high pressure.

Packaging of milk
Paneer
Paneer is prepared by adding food acid , such as lemon juice , vinegar , citric acid or yoghurt to
hot milk to separate the curds from whey. The curds are drained in muslin or cheese cloth and
the excess water is pressed out.

Verka paneer is made from fresh verka milk and is packed in advanced modern packaging
facilities at verka milk dairies , thus meeting the requisite FSSAI standards . it is ideal for dishes
consisting of paneer as the main ingredient. It is rich in calcium which is good for strong bones
and teeth. It also provides protein for active life and growth.

Packaging
Poly packs : 100gm , 200gm
Bulk packs : 1kg , 2kg & 5kg
Vacuum packs : 200gm

Nutritional composition of paneer per 100gm

packaging
Energy value 300kcal
Carbohydrates 3gm
Minerals 0.09gm
Protein 18gm
Fat 23gm

Procedure for the preparation of paneer

Receiving pasteurized milk
Preheating at 85℃
Cool down at 75℃
Coagulation of milk is done by adding citric acid
Collecting the coagulated mass in muslin cloth
Placing the paneer mass into stainless steel hoops
Pressing of paneer is done with the help of pneumatic press for 15-20 mins
Then placing the paneer into chilled and barine water for few hrs and at last cutting is done into
small blocks
At last packaging is done and the paneer is stored at 7℃.

Curd
Curd is obtained by the curdling of milk. Coagulation is done by adding rennet or by any edible
acidic substance such as lemon juice or vinegar and then allowing it to set.

Packaging
400gm
200gm
150gm
100gm

Nutritional composition information of curd per 150gm packaging.

Nutritional information per 100g*
Energy value(kcal) 65.0
Total carbohydrates(g) 5.9
Added sugar(g) 0.0
Protein (g) 3.7
Total fat(g) 3.0
Saturated fat(g) 1.9
Trans fat(g) 0.0
cholesterol(mg) 7.2
Minerals (g) 0.9
calcium(mg) 150

Procedure for making curd

1. Receiving milk
2. Standardization (fat 3% & SNF10.0)
3. Homogenization
4. Pasteurization (above 90℃ for 5mins)
5. Storage of milk below 7℃
6. Preheat at 40℃
7. Transfer of milk to milk balance tanks
8. Addition of starter culture
9. Machine filling & incubation at 42-43℃
10. After 3-4 hrs, it is shifted to cold room at the temperature of 5℃ for storage.

Quality control test

Main objective of the quality control test

1. Testing of the shelf life of the milk for city supply
2. Sensory evaluation of milk and milk products
 3. Checking the quality of reagents, packaging material , glassware etc
4. Hardness and chlorine content of water
5. Controlling the cleanliness of dairy equipments and overall cleanliness of the plant
6. Checking the efficiency of waste treatment of plant
7. Checking the weight , thickness, prints and length of pouches for supply in city

Need for quality control of various dairy products

A) To ensure safety , quality and long shelf life of dairy products , temperature must be held
at 4℃ during transport of milk.
B) All dairy products are code dated to ensure they are purchased and consumed at their
highest quality.
C) Dairy products which are not sold by the date are removed from sale.
D) To keep products fresh.

Tests performed in microbiology lab.

1.Pour plate:
Pour plate method is usually the method of choice for counting the number of colony
forming bacteria present in a liquid specimen. In this method, fixed amount of inoculum
(generally 1ml) from a broth or sample is placed in the centre of sterile petri plate using a
sterile pipette . molten cooled agar is then poured into the petri dish containing the
inoculum and mixed well. After the solidification of the agar , the plate is inverted and
incubated at 37℃ for 24-48 hrs.

Microorganisms will grow both on the surface and within the medium . Colonies that
grow within the medium generally are small in size and may be confluent ; the few that
grow on the agar surface are of the same size appearance as those on the streak plates
. Each (both large and small) colony is carefully counted . each colony represent a
“colony forming unit.”(cfu)
The number of microorganisms present in the particular test sample is determined using
the formula :
CFU/ml = CFU * dilution factor * 1/aliquot 
For accurate counts, the optimum count should be within the range of 30-300 colonies
per plate . To ensure a countable plate a series of dilutions should be plated.
 
 
  

The pour plate method of counting bacteria is more precise than the streak plate method , but ,
on the average , it will give a lower count as a heat sensitive microorganisms may die when they
come in contact with hot , molten agar medium.

Uses:
The pour plate technique can be used to determine the number of microbes/ml in a specimen . it
has the advantage of not requiring previously prepared plates , and is often used to aasay
bacterial contamination of food stuffs.

Materials and equipments

1. Test sample
2. Plate count agar or nutrient agar
3. Hot water bath 45℃
4. Sterile petri dishes
5. Flame
6. Colony counter with magnifying glass
7. Sterile capped 16*150mm test tubes
8. Pipettes of various sizes

 
 Procedure for pour plate technique
1. Prepare the dilution of the test sample expected to contain between 30-300 CFU/ml
2. Inoculate labelled empty petri dish with specified ml (0.1 or 1 ml) of diluted sample.

Pouring the molten agar and incubation

1. Collect the sterlized molten agar from the water bath (45℃)
2. Hold it in the right hand & remove the cap with the help of the little finger of the left hand.
3. Flame the neck of the flask.
4. Lift the lid of the petri plate and pour the sterile molten agar into the petri plate and
replace the lid .
5. Gently rotate the dish to mix the culture and the medium thoroughly and to ensure that
the medium covers the plate evenly. Do not slip the agar over the edge of the petri plate
.
6. Allow the agar to completely gel without disturbing it , it will take approximately 10mins .
7. Seal & incubate the plate in an inverted position at 37℃ for 24-48hrs.

 
Disadvantages of pour plate method  
1. Preparation for pour plate method is the time consuming compared with streak plate
technique.
2. Loss of viability of heat sensitive organisms coming into contact with hot agar.
3. Embedded colonies are much smaller than those which happen to be on the surface .
Thus , one must be careful to score these so that none are overlooked.
4. Reduced growth rate of obligate aerobes in the depth of the agar.

Spread plate:
Spread plate technique is a method employed to plate a liquid sample for the purpose of
isolating or counting the bacteria present in the sample . A perfect spread plate technique will
results visible and isolated colonies of bacteria that are evenly distributed in the plate and are
countable. The technique is most commonly applied for microbial testing of foods or any other
samples or to isolate & identify variety of microbial flora present in the environmental samples
e.g.soil.
Requirements :
Glasswares : screw capped test tubes, sterile pipettes, glass spreader.
Medium : plate count agar or nutrient agar.
Procedure for spread plate technique :
a. Serial dilution :
1. Prepare a series of at least 6 test tubes containing 9ml of sterile distilled water.
2. Using a sterile pipette , add 1ml of sample in the first tube of the set. Label it as
10​-1.
3. Mix the contents well by swirling the tube upside down few times .
4. From the first tube , take 1ml of sample and transfer to second tube. Label it as
10​-2​ .
5. Repeat the procedure with all the remaining tubes labelling them until 10​-6 ​.

Plating :
1. Pipette out 0.1ml from the appropriate desired dilution series onto the centre of the
surface of an agar plate.
2. Dip the L-shaped glass spreader into alcohol .
3. Flame the glass spreader over the bunsen burner.
4. Spread the sample evenly over the surface of the agar using the sterile glass spreader ,
carefully rotating the petri dish underneath at an angle of 45℃ at the same time.
5. Incubate the plate at 37℃ for 24 hrs.
6. Calculate the colony forming units(CFU)value of the sample.

Points to be noted:
1. Red violet bile agar is used to determine the bacterial count (E.coli). Pink coloured
colonies appear on the media after incubation.
2. To determine the yeast and mold count PDA(potato dextrose agar) is used.
3. To determine the SPC standard or total plate count agar is used. Results of SPC & PDA
are noted after 5 days .

BOD (biological oxygen demand)

It is defined as the amount of dissolved oxygen needed by aerobic biological organisms to
break down organic material present in a given water sample at certain temperature over a
specific time period .The BOD value is most commonly expressed in milligrams of oxygen
consumed per litre of sample during 5 days of incubation at 20℃ & is often used as a surrogate
of the degree of organic pollution of water.
Procedure
1. Aerated water was prepared by adding ferric chloride, calcium chloride, phosphorous
buffer and magnesium sulphate in 1l of distilled water.
2. After mixing incubated it in BOD incubator for 1 hr .
3. Sucked 1ml of distilled water and sample for blank and sample tube .
4. Added 2ml of alkaline iodine solution , 2ml of magnus sulphate & sulphuric acid & 1ml of
starch indicator .
5. Mixed well by shaking the bottles.
6. Titrated with sodium thiosulphate. Color changes from black to colorless .

Organoleptic tests
1. Judging the quality of the milk by its taste and smell by laboratory person.
2. If any fly or extraneous matter is found in the raw milk , it is regarded as unfit and
rejected.
3. Color of the milk is noted .

Acidity of milk
Procedure : a) Took 10ml of sample of milk.
b) Added few drops of phenolphthalein indicator and mix it well.
c) Titrated the milk against N/9 NaOH.
d) Titrated till the endpoint appears when color turns light pink.

Result : %age acidity is calculated as = (volume of NaOH used )/(volume of milk taken)
Inference : acidity of milk lies between 0.12-0.15
This method can also be used to determine the acidity of curd , lassi , & kheer.

To calculate the % moisture in cheese:

Procedure : 1. Grind cheese with the grinder finely.
2. Weighed about 3gm of cheese sample in moisture analyser by putting plate in it. And tare the
machine while weighing it .
3. Noted the %moisture in machine.
Inference : %moisture in the cheese should be greater than 45% usually to 57%.
Note : This method is also used to calculate the %moisture of atta , besan and panjeeri.
RM value of ghee
RM(reichert - Miessl value) is an indicator of how much volatile fatty acid can be extracted from
fat through saponification . it is equal to the number of millilitre of 0.1 normal hydroxide solution
necessary for neutralization of the water soluble volatile fatty acids & filtered from 5gm of a
given saponified fat.
Procedure:
1. Weighed 5gm ghee sample in flask.
2. Added 20ml glycerol, 2ml 50% NaOH.
3. Heated the flask over naked flame with ghee until ghee was saponified and liquid
becomes clear.covered the flask with watch glass.
4. Measured 93ml of boiled distilled water which was boiled for 15 minutes in 100ml
cylinder. Added to saponified when it is cooled .
5. Added glass beads to the flask & 50ml of dilute sulphuric acid .
6. Heated the contents until the insoluble acid are completely melted and then increased
the flame & distill it for 20 mins .
7. Kept the water flowing through the condenser at sufficient speed . Maintained the
temperature fissuring distillate 18-21℃ . when the distillator reaches 110ml mark
removed the flame , replace the 110ml flask with stopper and without mixing the
contents placed it in the water at 15℃ for 10mins.
8. Removed the flask from the water dry outside , invert flask , filter the contents and
collected them in dry beaker . Titrated it with N/10 NaOH using phenolphthalein indicator
till the end point is reached .
9. Noted the volume v1. Repeated the procedure with blank and noted the volume v2.

Result :
RM value of ghee = volume of NaOH used * weight of the sample .
Usually RM value of ghee ranges from 27-30. If the value is less than 27 then the ghee
is not of good quality.

Free fatty acid of ghee:

Procedure :
a) Took 10 gm of ghee sample in a container .
b) Heated until the fumes started coming out.
c) Then cooled the sample by placing it in the dessicator .
d) After cooling added 50ml of ethyl alcohol . Added 1-2 drops of phenolphthalein indicator.
e) Then boiled the sample for 30 seconds .
f) Titrated it with N/10 NaOH till pink color appears .
g) Noted the volume oF NaOH used.
 Result :
Free fatty acid of ghee = ( 2.82 *volume of NaOH used )/ weight of the sample.
Inference : FFA of ghee should be 0.30 .

COD (chemical oxygen demand)

COD is an indicative measure of the amount of oxygen that can be consumed by reactions in a
measured solution. A COD tests can be used to easily quantify the amount of organics in water.
Procedure :
1. Took 20ml of sample and put it into the beakers namely A,B,C.
2. Poured 20ml of distilled water in each flask . set 1 flask as blank .
3. Added 10ml of potassium dichromate in each flask .
4. Added 0.4g of mercuric sulphate in each flask.
5. Added 30ml concentrated sulphuric acid in each flask.
6. Kept it for digestion for 2 hrs. After digestion cooled it to room temperature.
7. Titrate it with ferrous ammonium sulphate using ferroin indicator.
8. Noted end point as sharp vine red color.

Formula used :
COD(mg/ml)= ( A-B *N(FAS)8000) *(sample in ml *10)
A= amount of FAS required for blank.
B= amount of FAS required for blank.

To check the hardness of water

Hardness of the water is due to calcium and magnesium ions present in water, so their
concentration should be low.
PROCEDURE
1) Took 50ml of water sample in a flask.
2) Added little amount of Eriochrome T-black indicator.
3) Added 0.5ml of ammonia buffer into it.
4) Titrated with EDTA(Ethylenediaminetetraacetic acid)
Formula Used
Concentration = (volume of EDTA used*1000)/Sample weight

Inference
Concentration should be less than 300 ppm for water to be safe for drinking.
Protein Quantification
This is done to estimate the amount of protein in different milk powder sample.

PROCEDURE
1) Added 7g of potassium sulphate, 0.8g of copper sulphate and 0.5g of powder sample in
Foss tube.
2) Added 15ml of sulphuric acid and 5-6 drops of octane.
3) Added 3ml of hydrogen peroxide.
4) Tubes were kept for 1 hour and 30 minutes at 400 ℃ for digestion.Placed the digested
tubes one by one for distillation in Kjeldhal machine.
5) After digestion titrated distillate with N/10 HCl.
6) Color changes from light blue to colorless.
7) Noted the observations

FORMULA
(HCl used - sample blank * 14.007* 100*0.1)/sample weight * 6.38(in case of dairy products)

Adulteration
Urea test :
Reagent : p-dimethylaminobenzaldehyde (1.6 g in 100 ml ethyl alcohol containing 10% HCl)
Procedure :
a) Take 2ml of milk in test tube .
b) Added 2ml of p-dimethylaminobenzaldehyde

Inference: appearance of distinct yellow color indicates positive result.

Sugar test :
Reagent : HCl , resorcinol
Procedure :
1. Took 15ml of milk in test tube .
2. Added 1ml conc. HCl & shaked well.
3. Added 0.1gm resorcinol.
4. Put the test tube in boiling water bath for 15mins.

Conclusion : reddish color indicates presence of sugar.

Salt test : reagent : nitrate , potassium dichromate

Procedure: 1. Took 5ml of silver nitrate in a test tube .
2. Added 2-3 drops of potassium dichromate solution which will give brick red color.
3. Added 1ml of milk
Conclusion : yellow color confirms the presence of salt whereas brown color shows absence of
salt.

MPN (MOST PROBABLE NUMBER)

​MPN test is performed in 3 steps
1. Presumptive test
2. Confirmatory test
3. Completed test

1. Presumptive test:
The presumptive test, is a screening test to sample water for the presence of coliform organisms.
If the presumptive test is negative, no further testing is performed, and the water source is
considered microbiologically safe.
If the presumptive test is negative, no further testing is performed, and the water source is
considered microbiologically safe. If, however, any tube in the series shows acid and gas, the water
is considered unsafe and the confirmed test is performed on the tube displaying a positive reaction.
The method of presumptive test varies for treated and untreated water.
Requirements :
● Medium: Lactose broth or Mac Conkey Broth or Lauryl tryptose (lactose) broth
● Glasswares: Test tubes of various capacities (20ml, 10ml, 5ml), Durham tube
● Others: Sterile pipettes

Preparation of the Medium

● Prepare medium (either mac conkey broth or Lactose broth)in single and double
strength concentration.
● For untreated or polluted water :
○ Dispense the double strength medium in 10 tubes (10ml in each tube)
and single strength medium in 5 tubes (10 ml in each tube)and add an
durham tube in inverted position.
● For treated water:
○ Dispense the double strength medium in 5 tubes (10ml in each tube)
and 50 ml single strength medium in 1 bottle and add an durham tube in
inverted position.
 ● Examine the tubes to make sure that the inner vial is full of liquid with no air bubbles.
● Sterilize by ​autoclaving at 15 lbs pressure (121°C) for 15 minutes​.

Procedure of the test

A. For untreated water

MPN Water Testing

1. Take 5 tubes of double strength and 10 tubes of single strength for each water sample
to be tested.
2. Using a sterile pipette add 10 ml of water to 5 tubes containing 10 ml double strength
medium.
3. Similarly add 1 ml of water to 5 tubes containing 10 ml double strength strength
medium and 0.1 ml water to remaining 5 tubes containing 10 ml double strength
medium.
4. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to
48 hrs.
 5. Compare the number of tubes giving positive reaction to a standard chart and record
the number of bacteria present in it.
For example: a water sample tested shows a result of 3–2–1 (3 × 10 ml positive, 2 × 1
ml positive, 1 × 0.1 ml positive) gives an MPN value of 17, i.e. the water sample
contains an estimated 17 coliforms per 100 ml

To view full table download the PDF file from the link given in the reference.
B. For untreated water
1. Take 1 tube of single strength (50ml) and 5 tubes of double strength (10ml) for each
water sample to be tested.
2. Using a sterile pipette add 50 ml of water to the tubes containing 50 ml single strength
medium.
3. Similarly add 10 ml of water to 5 tubes containing 10 ml double strength medium
Incubate the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48
hrs.
4. Compare the number of tube giving positive reaction to a standard chart and record
the number of bacteria present in it.
For example: a water sample tested shows a result of 1-4 (1 × 50 ml positive, 4 × 10
 ml positive) gives an MPN value of 16, i.e. the water sample contains an estimated 16
coliforms per 100 ml

2. Confirmed test:
Some microrganisms other than coliforms also produce acid and gas from lactose fermentation. In
order to confirm the presence of coliform, confirmatory test is done.
From each of the fermentation tubes with positive results transfer one loopful of medium to:
1. 3 ml lactose-broth or brilliant green lactose fermentation tube,
2. to an agar slant and
3. 3 ml tryptone water.

Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas formation after 24
± 2 hours. If no gas production is seen,further incubate up to maximum of 48 ±3 hours to check gas
production.
The agar slants should be incubated at 37°C for 24± 2 hours and ​Gram-stained preparations​ made
from the slants should be examined microscopically.
The formation of gas in lactose broth and the demonstration of Gram negative, non-spore-forming
bacilli in the corresponding agar indicates the presence of ​a member of the coliform group​ in the
sample examined.
The absence of gas formation in lactose broth or the failure to demonstrate Gram-negative,
non-spore-forming bacilli in the corresponding agar slant constitutes a negative test ​(absence of
coliforms in the tested sample).​
Tryptone water Test
1. Incubate the tryptone water at (44.5 ±0.2°C) for 18-24 hours
2. Following incubation, add approximately 0.1ml of Kovacs reagent and mix gently.
3. The ​presence of indole ​is indicated by a red colour in the Kovacs reagent, forming a
film over the aqueous phase of the medium.

a. Confirmatory tests positive for indole, growth, and gas production show the presence of
thermotolerant ​E. coli.
b. Growth and gas production in the absence of indole confirm thermotolerant coliforms.
3. Completed test:
Since some of the positive results from the confirmatory test may be false, it is desirable to do
completed tests. For this inoculum from each positive tube of the confirmatory test is streaked on a
plate of EMB or Endo agar.
In this process, a loopful of sample from each positive BGLB tubes is streaked onto selective
medium like ​Eosin Methylene Blue agar​ or Endo’s medium. One plate each is incubated at 37°C
and another at 44.5± 0.2°C for 24 hours.
High temperature incubation (44.5 ±0.2) is for detection of thermotolerant E.coli.
Following incubation,all plates are examined for presence of typical colonies.
● Coliforms produce colonies with greenish metallic sheen which differentiates it from
non-coliform colonies (show no sheen). Presence of typical colonies on high
temperature (44.5 ±0.2) indicate presence of thermotolerant ​E.coli.

Advantages of MPN :
● Ease of interpretation, either by observation or gas emission
● Sample toxins are diluted
● Effective method of analyzing highly turbid samples such as sediments, sludge, mud,
etc.
● that cannot be analysed by membrane filtration.

Disadvantages of MPN:
● It takes a long time to get the results
● Results are not very accurate
● Requires more hardware (glassware) and media
● Probability of false positives.