Mother dairy training report

PROJECT REPORT
INTERNSHIP
Topic- MICROBIOLOGICAL ANALYSIS OF MILK AND MILK PRODUCTS
Submitted by –
KOPAL MITTAL

AKNOWLEDGEMENT
I feel pleasure in expressing my profound sense of gratitude and
indebtedness to Mother Dairy Organization who has me the authority to get
training under them.
I am highly oblige to Dr. TSR Murli Chief Research development officer
Of Mother Dairy Food Processing Ltd. Patparganj Delhi, to arrange for my
training in their esteemed organization.
I am grateful to Mrs. Nita Sen, Senior Manager Quality Assurance and
Quality Control of Mother Dairy Test Laboratory, for her constant moral support
during the tenure of training.
I express my heart-left gratitude to my guide and Dr. Vikas Sangwan, Assit.
Manager and Mr.Rahul Verma Research and development Executive whose
unique guidance, all embracing help, valuable suggestions and encouragement have
enabled me to complete this task which would not have been a success without
them.
I also thank all the workers of Innovations Department who really showed
co-operation and support.
Last but not the least I with due respect pay my regards to My Parents and
Almighty God who has been the source of motivation, strength and support, which
led me to attain success.
KOPAL MITTAL

CONTENTS
1) ABOUT MOTHER DAIRY
2) MICROBIOLOGICAL ANALYSIS
3) PRODUCT SPECIFICATION

NATIONAL DAIRY DEVELOPMENT BOARD (NDDB)
The National Dairy Development Board (NDDB) was designed in 1969 on Anand
Dairy Cooperative outlines of creating district cooperative of milk producers at village
level which aim at collection of whatever amount of milk they produce and payment to
them according to the quality of milk they supply. Then this milk is processed and
supplied to the people at metropolitans and other cities at nominal charges.
For about 5 yr., NDDB was dormant till the time they received presents of Whole
Milk Powder and Butter Oil from European Union and came into action with the help of
Indian Dairy cooperation.
Operation flood was started in 1970 under the guidance of Dr. Kurein, Chairperson
NDDB, Anand and Mr. Jamnadas Patel in Khera, Gujarat. The objectives of Operation
Flood are:
 To buy the milk from the farmers and villagers as much milk as they
could supply at reasonable prices and keep the price of milk stable
whole year.
 To operate, procure, process and market milk and milk products.
 Production of good milk after its qualitative and quantitative
estimation.
 System to eliminate middleman.
 Providing technical inputs and services to yield of milk animals.
 Organizing an efficient transport system to collect milk from
villagers.
 Set up of balancing dairies and storage system to convert excess milk
to milk powder and butter oil.
 Train personnel to plan and manage the system as well as operate the
services described.
 To meet out these objectives, NDDB started dairy plants all over
India and Mother Dairy was one of them. It was set up in 4 major
cities at Delhi, Mumbai, Chennai and Kolkata.
Operation flood was a major dairy development program. Funds were generated by
the sale of dairy commodities donated by the World Food Program (FAO-UN) which
assisted project to carry out its mission.
This included establishing dairy cooperatives in rural milk sheds, setting of animal
husbandry units, constructing modern dairies in cities, organizing storage and long distance
transportation and project planning with manpower development. The first phase of
operation flood was completed in 1979, followed by second phase (1979-1988), assisted by
European Economic Community. Its third phase ended in 1996.

MOTHER DAIRY PROFILE
Mother Dairy was set up under the operation flood project on second December 1974. It is
equipped with latest technology. Products are prepared as per the PFA standards.
The dairy was established on behalf of the Govt. of India, Ministry of Agriculture (Dept. of
Animal Husbandry and Daring). Mother Dairy is being managed by NDDB as a subsidiary
unit retaining its independent character.
For the purpose of managing the dairy NDDB appoints managing committee. All the policy
matters decided by the management committee but the execution of all the policy decision
and management of dairy rests with General Manager.

QUALITY ASSURANCE
Mother Dairy follows a strict system of quality assurance to maintain a high quality
of milk provided to the consumer. Every batch of incoming milk is tested for Fat, SNF,
Acidity and bacteriological quality, adulterants and presence of any preservative or
neutralizer which are not permitted by the law. The outgoing milk is also checked for
bacteriological and keeping quality besides Fat and SNF.
QUALITY POLICY
Our commitment is to excellence. The evolving needs of our customers drive our continuous
innovation in product development, application of state-of-the art technology and selection of
appropriate processes to be used by our employees and our vendors. To ensure that we are
second to none:
 We maintain a vibrant work environment that encourages excellence.
 We empower our people that lead to continuous improvements in our
processes and systems.
 We choose such processes that ensure quality at competitive price while
protecting the environment, in which we work and live.
 We apply HACCP (Hazard Analysis Critical Control Points) principles for
safety of our products to customers.
We shall constantly strive to be leaders in the dairy industry in Delhi, in
India and in the World.

ENVIRONMENTAL POLICY
We are committed to be a leader in Dairy Industry in India by being a benchmark
in environment protection.
We shall continue to improve our environmental performance by:-
 Adopting processes, procedures and systems for clean
production, pollution prevention and continual improvements in
all our activities.
 Optimizing resource utilization including energy and water.
 Complying with applicable environmental regulations and
legislation.
 Continuing distribution of milk through environment friendly
vending systems.
 Maintain green environment through horticultural activities.
 We pledge ourselves towards providing quality and safe
products under clean and hygienic environment.
Mother Dairy also has HACCP certification as per ISO 15000: 1998
National Accreditation Board For Testing And Calibration Laboratories (NABL),
MDTL, Delhi has been accredited to Mother Dairy on the basis of its compliance to NABL –
criteria which are based on Chemical Testing Discipline. This accreditation is valid for a
period of 3 years.

PRODUCTS
NDDB has also set up an oil storage unit by the name of edible oil tank farm (EOTF)
in this area which has a capacity to store 50000 MT TONNES of mustard oil. Adjoining this
is also an oil lab notified by the govt. of India that do the job of testing of oil at the time of
purchase and dispatch. It aims at providing the farmers the nominal charges for their oil
produce according to its quality and to prevent hoarding of oil by private people.
Mother Dairy has also set up an ice cream plant (ICP) about 5 years back. Through
ICP Mother Dairy markets the ice cream in and around Delhi at nominal charges and in
varied flavors. Mother Dairy also markets Safal frozen vegetables, Verka, Parag, etc.
Mother Dairy has obtained the International std. Certificate issued by International
Organizations of Standards (ISO) for its 23 years service, it’s the first in Indian food sector to
MOTHER DAIRY VISION
With total commitment and teamwork, we at Mother Dairy shall strive to delight our
customers with quality products and services.
We shall create an environment where employees feel happy to work.
Through innovation, consistent learning and continuous improvement, we shall achieve
sustained growth and excellence and provide remunerative returns to farmers and other
stakeholders.

MOTHER DAIRY MISSION
“We are the market leader in fluid milk in Delhi. Our mission is to retain the leadership and
further serve our customers in and around Delhi by providing quality milk and select dairy
and food products.
We shall strive to delight our customers by providing regular and timely services at
convenient points under clean and hygienic conditions, with a product mix for different
income segment. We shall create an environment of commitment and teamwork where
employees feel happy to work.
By innovation, continuous improvement, training and development and most efficient use of
resources we shall achieve consistent and sustained growth and provide remunerative returns
to farmers and other stakeholders.”
Mother Dairy has received the ISO 9002 certificate in 1994 for the quality of its food
products.

MILK
FUNDAMENTAL KNOWLEDGE OF MILK
Milk is the secretion derived after complete milking of healthy milk animal, excluding
that obtained within 15 days before or after 10 days of calving.
COMPOSITION OF MILK
01. Milk Fat
Milk fat is the mixtures of 19 fatty acids. The bulk of fat in milk exist in the
form of small globules with size approx. 0.1 to 22microns. It is an oil-in-water
type emulsion.
02. Proteins
Protein is the most complex organic substances. They are vital for living
organisms as they constitute an indispensable part of the individual body cell.
The protein of milk consists of Casein, Lacto globulin and Lacto albumin. It is
in colloidal state. Casein is only found in milk. It is easily coagulated by heat
treatment.
03. Water
It provides the medium in which all the milk constitutes are either dissolved or
suspended. Most of it is free and only a small portion is in bound form, being
firmly by protein and phospholipids.
04. Lactose(Milk Sugar)
It is found only in milk. It exist in true solution phase and is fermented by
bacterial to yield lactic acid. It is very important for cultured milk products. It
can also cause souring in milk and its products.
05. Mineral Matter:

Although it is present in small quantity, it is very essential for human body.
These are mostly salts like Mg, Na, P, N etc. It influences physio-chemical
properties of milk and also affects the nutritive value.
06. Phospholipids:
There are three types of phospholipids (Lecithin, Cephalic and Sphingomylein).
Lecithin is important constituent as it helps in the formation of outer membrane
of fat globules.
07. Pigment:
There are two type of pigment present in milk, Fat Soluble and Fat insoluble.
Carotene is fat soluble and is responsible for the yellow color of milk. The other
two are Xanthophylls and riboflavin. Carotene also acts as an anti oxidant and
as a source of Vitamin A.
08. Milk Enzymes
The important milk enzymes and their specific action are as follows:
- Anise (diasterase) Starch
- Lipase Fat splitting, leads to rancid flavor
- Phosphatase It is capable of splitting certain phosphoric
esters.
- Protease It is capable of splitting proteins.
- Catalase decomposed hydrogen peroxides.
09. Vitamins
Vitamins are present in minute quantities in milk or any other food but play a very important
role in vital functioning of human body. There are 25 vitamins present in milk. These are fat
soluble e.g. Vit. A, D, E,K. apart from fat soluble there are water soluble vitamins as B-
complex(B1, riboflavin or B2, pantothenic acid, niacin, pyridoxine or B6.)

Role of milk in human life
Milk is one of the most complete food available in nature for human consumption.
Milk contains all nutrients in balanced proportions to meet the demand of humans. It is not
only necessary for children but it is every essential for adults too. Milk proteins are one of
the most high quality proteins. It is essential proper growth of body. Casein, the milk
protein, is only found in milk. Milk contains lactose (milk sugar) which is only found in
milk and on hydrolysis gives galactose and glucose. Lactose on fermentation gives butter,
cheese and dahi, which have high nutritive value and thus is very important for human life.
 1 Liter of Cow’s milk gives 720 Calorie (3000J) of energy.
 1 Liter of Buffalo’s milk gives 1100 Calorie (4600J) of energy.
 1 gm of milk fat gives 9 Calorie of energy.
 1 gm of Protein gives 4 Calorie of energy
 1 gm of Carbohydrate gives 4 Calorie of energy
Most milk is composed of 80 to 90 percent water. The remaining 10 percent
consists of an abundance of the major nutrients needed by the body for good health,
including fats, carbohydrates, proteins, minerals, and vitamins.
Cow milk typically contains about 3.5 to 5 percent fat, which is dispersed
throughout the milk in globules. In addition to providing milk’s characteristic taste and
texture, fat supplies vitamins A, D, E, and K, as well as certain fatty acids that the body
cannot produce on its own.
Lactose, a kind of sugar found only in milk, gives milk its sweet taste. Making up
about 5 percent of milk’s content, lactose is a carbohydrate that is broken down by the
body to supply energy. Infants digest lactose easily, but many adults, especially those of
Asian and African ancestry, have lost some of their ability to digest this sugar. When these
adults drink milk, they often suffer gastric distress and diarrhea.
The most important protein in milk is casein, accounting for 80 percent of milk
protein. Casein is a complete protein, meaning that it contains all of the essential amino
acids, which the body cannot manufacture on its own. Casein molecules and globules of fat

deflect light rays passing through milk, giving milk its opalescent appearance. Other
proteins present in milk include albumin and globulin.
Milk contains many minerals, the most abundant of which are calcium and phosphorus, as
well as smaller amounts of potassium, sodium, sulfur, aluminum, copper, iodine, manganese,
and zinc. Milk is perhaps the best dietary source of calcium—one liter (about 1 qt) of milk
supplies as much calcium as 21 eggs, 12 kg (26 lb) of lean beef, or 2.2 kg (5 lb) of whole
wheat bread. Milk is an excellent source of vitamins A and B2 (see riboflavin). All other
vitamins are present also, but in lower doses. Vitamin D is typically added to commercially
sold milk. Vitamin A, which is found in the globules of fat, is removed when fat is skimmed
away to make low-fat or skim milk. Generally, vitamin A is replaced during the production of
commercially sold low-fat milk.
Preparation of sample:
1. Mix the sample well before opening
2. Milk samples must be kept at < 8 °C during shipment and in laboratory. The sample
should not be kept for longer in the lab and shall be analyzed within 24 h after
receiving.
3. Clean the exterior of the pouch with a tissue paper to remove any dirty material
adhering to it and wipe it off with a tissue paper /cotton soaked in 70% alcohol to
avoid external contamination.
4.Allow the traces of alcohol to evaporate.
5. Cut the pouch with the help of sterile scissors or cutter (sterilized by dipping in
alcohol followed by flaming to burn off the alcohol and cool).
6. Aseptically transfer sample for further analysis
Parameters to be analysed
1) Aerobic plate count
2) Coliform count
3) Psychrotropic count
4) Thermoduric count
5) Thermophillic count

MILK POWDER
Powdered milk or dried milk is a manufactured dairyproduct made by
evaporating milk to dryness. One purpose of drying milk is to preserve it; milk powder has a
far longer shelf life than liquid milk and does not need to be refrigerated, due to its low
moisture content. Another purpose is to reduce its bulk for economy of transportation.
Powdered milk and dairy products include such items as dry whole milk, nonfat (skimmed)
dry milk, dry buttermilk, dry whey products and dry dairy blends. Many dairy products
exported conform to standards laid out in Codex Alimentarius. Many forms of milk powder
are traded on exchanges
. Preparation of sample:
3.Allow the traces of alcohol to evaporate.
5. Aseptically transfer sample for further analysis in diluent bottles.
2) Coliform count
3) S. aureus count

PANEER
Paneer is prepared by adding food acid, such as lemon juice, vinegar, citric
acid or yogurt to hot milk to separate theCasein protein from the whey. The Casein
protein collectively known as curds are drained in muslin or cheesecloth and the
excess water is pressed out. The resulting paneer is dipped in chilled water for 2–3
hours to improve its texture and appearance. From this point, the preparation of
paneer diverges based on its use and regional tradition.
PREPARATION OF PANEER SAMPLE
1. Paneer samples must be kept at < 8 °C during shipment and in laboratory. The sample
receiving.
2. Record the sample particulars such as; name of the sample, sample collection date,
batch no., mfg. date, expiry date, etc.
adhering to it and wipe it off with a tissue paper /cotton soaked in 70% alcohol to avoid
external contamination.
4. Allow the traces of alcohol to evaporate.
5. Cut the pouch with the help of sterile scissors or cutter (sterilized by dipping in alcohol
followed by flaming to burn off the alcohol and cool).
6. Aseptically cut the paneer block in number of pieces all along the length, width and
depth by using a sterile spatula.
7. Transfer the pieces into a sterile sampling bag to crush and blend the sample either by
using blender/smasher/stomacheror by hand to get prepared sample.
8. Use the prepared paneer sample for preparation of dilutions.
2) Coliform count
3) Yeast and mould count
4) S. aureus count

ICE CREAM
Ice cream (derived from earlier iced cream or cream ice) is a sweetened frozen food
typically eaten as a snack or dessert. It is usually made from dairy products, such as milk and
cream, and often combined with fruits or other ingredients and flavours. It is typically
sweetened with sucrose, corn syrup, cane sugar, beet sugar, and/or othersweeteners.
Typically, flavourings and colourings are added in addition to stabilizers. The mixture is
stirred to incorporate air spaces and cooled below the freezing point of water to prevent
detectable ice crystals from forming. The result is a smooth, semi-solid foam that is solid at
very low temperatures (<35 °F / 2 °C). It becomes more malleable as its temperature
increases.
1. Samples must be kept at < 8 °C during shipment and in laboratory. The sample
receiving.
5. Aseptically transfer 100 gm sample for further analysis in a sterile jar.
6. Allow the sample melt to for 30 mins.
2) Coliform count
4) S. aureus count

BUTTER
Butter is a solid dairy product made by churning fresh or fermented cream or milk, to
separate the butterfat from thebuttermilk. It is generally used as a spread on plain or toasted
bread products and a condiment on cooked vegetables, as well as in cooking, such as baking,
sauce making, and pan frying. Butter consists of butterfat, milk proteins and water.
Most frequently made from cows milk, butter can also be manufactured from the milk of
other mammals, including sheep,goats, buffalo, and yaks. Salt such as dairy
salt, flavorings and preservatives are sometimes added to butter. Rendering butter
produces clarified butter or ghee, which is almost entirely butterfat.Butter is a water-in-
oil emulsionresulting from an inversion of the cream; in a water-in-oil emulsion, the milk
proteins are the emulsifiers. Butter remains a solid when refrigerated, but softens to a
spreadable consistency at room temperature, and melts to a thin liquid consistency at 32–
35 °C (90–95 °F). The density of butter is 911 g/L (0.950 lbs per US pint)It generally has a
pale yellow color, but varies from deep yellow to nearly white. Its unmodified color is
dependent on the animals' feed and is commonly manipulated with food colorings in the
commercial manufacturing process, most commonlyannatto or carotene.
1. Samples must be kept at < 8 °C during shipment and in laboratory. The sample
receiving.
5. Aseptically transfer 100 gm sample for further analysis in a sterile jar.
6. Allow the sample to melt for 30 mins.
2) Coliform count
4) S. aureus count

FERMENTED MILK PRODUCT
Fermented milk products, also known as cultured dairy foods, cultured dairy products,
or cultured milk products, are dairy foods that have been fermented with lactic acid
bacteria such as Lactobacillus, Lactococcus, and Leuconostoc. The fermentation process
increases the shelf-life of the product, while enhancing the taste and improving the
digestibility of milk. There is evidence that fermented milk products have been produced
since around 10,000 BC.[1] A range of different Lactobacilli strains has been grown in
laboratories allowing for a wide range of cultured milk products with different tastes.
CURD
Curds are a dairy product obtained by coagulating milk in a process called curdling. The
coagulation can be caused by adding rennetor any edible acidic substance such as lemon
juice or vinegar, and then allowing it to sit. The increased acidity causes the milk proteins
(casein) to tangle into solid masses, or curds. Milk that has been left to sour (raw milk alone
or pasteurized milk with addedlactic acid bacteria) will also naturally produce curds, and sour
milk cheeses are produced this way. Producing cheese curds is one of the first steps
in cheesemaking; the curds are pressed and drained to varying amounts for different styles of
cheese and different secondary agents (molds for blue cheeses, etc.) are introduced before the
desired aging finishes the cheese. The remaining liquid, which contains only whey proteins,
is the whey. In cow's milk, 80% of the proteins are caseins.
BUTTER MILK
Originally, buttermilk referred to the liquid left over from churning butter from cultured or
fermented cream. Traditionally, before cream could be skimmed from whole milk, the milk
was left to sit for a period of time to allow the cream and milk to separate. During this time,
naturally occurring lactic acid-producing bacteria in the milk fermented it. This facilitates the
butter churning process, since fat from cream with a lower pH coalesces more readily than
that of fresh cream. The acidic environment also helps prevent potentially harmful
microorganisms from growing, increasing shelf-life.[4] However, in establishments that used
cream separators, the cream was hardly acidic at all.
The tartness of buttermilk is due to acid in the milk. The increased acidity is primarily due
to lactic acid produced by lactic acid bacteria while fermenting lactose, the primary sugar in
milk. As the bacteria produce lactic acid, the pH of the milk decreases and casein, the
primary milk protein, precipitates, causing the curdling or clabbering of milk. This process
makes buttermilk thicker than plain milk. While both traditional and cultured buttermilk
contain lactic acid, traditional buttermilk tends to be less viscous, whereas cultured buttermilk
is more viscous.[2]
Buttermilk can be drunk straight, and it can also be used in cooking. Soda bread is a bread in
which the acid in buttermilk reacts with the rising agent, sodium bicarbonate, to
produce carbon dioxide which acts as the leavening agent. Buttermilk is also used

in marination, especially of chicken and pork, whereby the lactic acid helps to tenderize,
retain moisture, and allows added flavors to permeate throughout the meat.
LASSI
Lassi is a popular traditional yogurt-based drink from the Indian Subcontinent and originates
from the Punjab. Lassi is a blend of yogurt, water, spices and sometimes,
fruit. Traditional lassi is a savoury drink, sometimes flavoured with ground and
roasted cumin. Sweet lassi, however, contains sugar or fruits, instead of spices. Salted mint
lassi is highly favoured in Bangladesh.
4. Cut the pouch or remove the seal/ lid with the help of sterile scissors or cutter
(sterilized by dipping in alcohol followed by flaming to burn off the alcohol and
cool).
5. Aseptically transfer sample for further analysis..
1) Coliform count
3) S. aureus count

UHT PRODUCTS
Ultra-high temperature processing (UHT), ultra-heat treatment, or ultra-
pasteurizationsterilizes food by heating it above 135 °C (275 °F) – the temperature required
to kill spores in milk – for 1 to 2 seconds.UHT is most commonly used in milk production,
but the process is also used for fruit juices, cream, soy milk, yogurt, wine, soups, honey, and
stews. UHT milk was first developed in the 1960s and became generally available for
consumption in the 1970s.
The heat used during the UHT process can cause Maillard browning and change the taste and
smell of dairy products.[4] An alternative process is HTST pasteurization (high
temperature/short time), in which the milk is heated to 72 °C (162 °F) for at least 15 seconds.
UHT milk packaged in a sterile container, if not opened, has a typical unrefrigerated shelf
life of six to nine months. HTST pasteurized milk has a shelf life of about two weeks from
processing, or about one week from being put on sale.
4. Cut the pouch or remove the seal/ lid with the help of sterile scissors or cutter
(sterilized by dipping in alcohol followed by flaming to burn off the alcohol and
cool).
5. Aseptically transfer sample for further analysis..
2) Spore count

PREPARATION OF DILUENTS
Diluent for Primary Dilution – Phosphate Buffer Diluent
1. Dissolve 42.5 g of potassium dihydrogen phosphate in 500 mL of water.
2. Adjust the pH using 1N (40 g/ L) NaOH and 1 M (36.5 g/ L) HCL solution, so that after
sterilization it is 7.2 at 25 °C.
3. Dilute it to 1000 mL with distilled water.
4. Distribute in 90 ml screw capped glass bottles.
5. Sterilize at 121 °C /15 psi for 15 minutes.
6. The stock solution can be stored under refrigeration for not more than 6 months.
Discard the solution if precipitate is observed.
Diluent for Primary Dilution – Sodium Citrate Solution in case of high fat containing
products
1. Dissolve 20.0 g of tri-sodium citrate dihydrate in 1000 mL of water.
2. Distribute 90 mL in 250 mL of conical flasks / screw capped media bottles.
3. Plug the conical flasks with non-absorbent cotton or close the media bottles with
autoclavable plastic caps.
Diluent for Further Decimal Dilutions- Phosphate Buffer Diluent
Stock Solution
1. Dissolve 42.5 g of potassium dihydrogen phosphate in 500 mL of water.
2. Adjust the pH using 1N (40 g/ L) NaOH and 1 M (36.5 g/ L) HCL solution, so that
after sterilization it is 7.2 at 25 °C.
3. Dilute it to 1000 mL with distilled water.
4. Distribute in screw capped glass bottles.
Working Solution
1. Add 1 mL of the stock solution (as prepared in section 8.2.1) to 1000 mL of water to be
used as diluent.
2. Dispense 9 mL and 90 mL of working solution into test tubes and media bottles
respectively.

3. Plug the tubes with non-absorbent cotton or autoclavable plastic caps and bottles with
their respective screw caps.
4. Sterilize by autoclave at 121 °C/ 15 psi pressure for 15 minutes.
5. The working solution (diluent) can be stored in a refrigerator (5 ± 3 °C), for not more
than 1 month. Discard the solution, if precipitate is observed.
Selection of dilutions:
For routine testing select dilutions of sample so that colonies on at least one plate will be
between 30 and 300.For unknown sample use two or more dilution

PREPARATION OF MEDIA
Media for Aerobic Plate Count
1. Plate count agar (PCA) from Hi-Media (Code No. M 091) is used for enumeration of
APC.
2. Dissolve completely 23.5 g of dehydrated media in 1 L distilled water by heating.
3. Adjust the pH of the media, so that after sterilization it is 7.0 ± 0.2 at 25 °C.
4. Sterilize by autoclaving at 121 °C / 15 psi for 15 minutes, followed by cooling and
labeling of media with name and date of preparation.
5. Temper the media to 45 ± 1 °C in a water bath prior to pouring.
6. The prepared media can be stored in a refrigerator (5 ± 3 °C) for not more than one
month.
7. Prior to use, melt the media by heating in a microwave oven and cool to 45 ± 1 °C in a
water bath prior to pouring.
8. The left over media of a day’s work shall be discarded i.e. it should not be reused for
next day’s work.
Media for Coliform Counts
1. Violet Red Bile Agar (VRBA) from Hi-Media (Code No. M 049) is used for
enumeration of CC.
2. Dissolve completely 41.5 g of dehydrated media in 1 L distilled water by heating.
3. Adjust the pH of the culture media, so that after boiling it is 7.4 ± 0.2 at 25 °C.
4. Bring to boil. Allow it to boil for 2 minutes, DO NOT AUTOCLAVE.
5. To avoid overheating, do not heat the medium for too long and do not reheat.
6. Immediately cool the culture media to 45 ± 1 °C in a water bath.
7. Use the media within 4 hour of its preparation.
next day’s work.
Media for Yeast and Mould Count
1. Yeast Glucose Chloramphenicol Agar (YGCA) from Hi-Media (Medium Code No. M
1590) is used for enumeration of YMC.
2. Dissolve completely 40.1 g of medium in 1 L distilled water by heating.
3. Adjust the pH of the medium, so that after sterilization it is 6.6 ± 0.2 at 25 °C.
4. Sterilize by autoclaving at 121 °C / 15 psi for 15 minutes, followed by cooling and
5. The prepared media can be stored in a refrigerator (5 ± 3 °C) for not more than one
month.
6. Cool the culture media and temper at 45 ± 0.5 °C in a water bath prior to pouring.

7. Prior to use (in case of stored culture media), melt the media by heating in a microwave
oven and cool to 45 ± 1 °C in a water bath prior to pouring.
next day’s work.
Media for coagulase positive S. aureus Count
1. Baird Parker Agar (BPA) from Hi-Media (Medium Code No. M1736) is used for
enumeration of coagulase positive SAC.
2. Weigh 6.3 g of dehydrated culture media of base medium to a clean and washed media
bottle (capacity 200 mL) and add 90 mL of distilled water, mix well and close the cap.
3. Heat to boiling to dissolve the medium completely.
4. Sterilize by autoclaving at 121 °C / 15 psi for 15 minutes followed by cooling and
5. The prepared culture media can be stored in refrigerator (0-5 °C) for not more than one
month.
6. Cool the culture media and temper at 45 ± 0.5 °C in a water bath prior to pouring.
7. In case of stored culture media prior to use, melt the culture media by heating in a
microwave oven, cool and temper at 45 ± 0.5 °C in a water bath.
8. Rehydrate the content of supplement of Fibrinogen Plasma Trypsin Inhibitor from Hi-
Media (Medium Code No. FD195), using 10 mL of sterilised distilled water 90-120
minute prior to its addition to base medium.
9. Add the rehydrated content of media supplement to tempered base medium, mix well
and use immediately for pouring.
10. The left over culture media of a day’s work shall be discarded i.e. it should not be
reused for next day’s work.

ANALYSIS PROCEEDURE
Aerobic Plate Count Test:
Materials required:
1. Plating chamber
2. Petri plates
3. Plate count agar
4. Auto pipette with tips (1.0)/pipette 1000 l
5. Dilution tubes containing 9 ml of batch phosphate buffer solution.
Procedure:
1. Transfer 1 ml of well mixed sample to 9 ml diluents (phosphate buffer solution).Mix
2. Well and transfer 1 ml of this suspension to second tube to make second dilution.
3. Similarly make third and fourth dilution as per requirement
4. Arrange the Petri plates for each dilution to be tested, mark them with sample no. and
Date.
5. Transfer 1 ml in each plate from respective dilution of sample being tested.
6. Melt the plate count agar, cool it about 45°C and pour 10 ml of this medium into thePetri
dishes. Mix the agar by rotating the plate.
7. Allow the agar to set, invert and incubate the plates at 37°C for 48 hours.
8. At the end of 48 hours remove the plate from the incubator and count the colonies.
9. Also prepare a control plate with 15 ml media for checking its sterility

Coliform Test for Milk:
Materials Required:
1) VRB Agar
2) Dilution Blanks (9 or 99)
3) Auto pipette with tips
4) Petri dishes
Procedure for Solid Media:
1. Transfer 1 ml of well mixed milk sample to 9 ml diluent and mix well to make first
2. dilution. If required make second dilution by transferring 1 ml of first dilution into 9 ml
diluent
3. Arrange two Petri plates for each sample; mark them with sample no. and date.
4. Transfer 1ml in each plate respective dilution of sample to be tested.
5. Add 10 to 15 ml of VRB agar, previously melted and cooled to about 45°C.
6. Mix the contents thoroughly by rotating the plates and allow the agar to solidify. Pour
additional about 5 ml of the over the surface of the solidified medium and allow to
Solidify again.
7. Invert plates and incubates the plates at 37° C for 18-24 hours.

Yeast mould count:
Materials Required:
a) Plating chamber
b) Petri dishes
c) Yeast Extract Chloramphenicol Agar / Potato Dextrose Agar
d) Auto pipette with tips (1 ml) /2.2 ml glass pipettes
e) Dilution tubes containing 9ml diluent and bottles with 99ml
diluents
f) Incubator set at 25±1°C.
Media:
Yeast Extract Chloramphenicol Agar
Ingredients:
Yeast extract : 5.0 gms/litre
Dextrose : 20.0 gms/litre
Chloramphenicol : 0.1gms/litre
Agar : 13.0gms/litre
pH : 6.6±(0.2)
Preparation: Suspend 40 gmsready made Yeast Extract Chloramphenicol Agar in 1000 ml
distilled water. Boil to dissolve the medium completely. Fill in flask or bottles.
Procedure for testing:
1- Select two consecutive dilutions of sample to plate.
2- Arrange two Petri dishes for each dilution, mark them with sample no. ,date and dilution
factor.
3- Transfer 1ml in each plate respective dilution of sample.
4- Melt the Chloramphenicol Agar, cool it to about 45°C and pour 10 to 15 ml into the Petri
dishes. Mix the agar by rotating the plates.
5- Allow theto set, invert and incubate the plates at 25±1°C for 5 days. If moulds grow fast
and develop into large colonies plate may be incubated for three days only.
6- Count the no. of colonies grown in each plate and compute the results. Also prepare a
control plate with 15 ml media for checking it’s sterility.

Spore count:
Materials required:
1. Laminar air flow unit
2. Petri plates
3. Plate count agar
Procedure:
1. Transfer 10ml in a testtube and heat it for 10 mins at 80 0C
well and transfer 1 ml of this suspension to second tube to make second dilution.
Similarly make third and fourth dilution as per requirement
Date.
5. Melt the plate count agar, cool it about 45°C and pour 10 ml of this medium into the petri

Thermoduric count:
Materials required:
1. Laminar air flow unit
2. Petri plates
3. Plate count agar
Procedure:
1. Transfer 10ml in a test tube and heat it for 30 mins at 630C.
Similarly make third and fourth dilution as per requirement
Date.
5. Melt the plate count agar, cool it about 45°C and pour 10 ml of this medium into the petri

Thermophillic count:
Materials required:
1. Plating chamber
2. Petri plates
3. Plate count agar
Procedure:
1. Transfer 10ml in a testtube and heat it for 30 mins at 630C
Date.
6. Melt the plate count agar, cool it about 45°C and pour 10 ml of this medium into the
Petri dishes. Mix the agar by rotating the plate.

Psychotropic count:
Materials required:
1. Plating chamber
2. Petri plates
3. Plate count agar
Procedure:
Date.
5. Melt the plate count agar, cool it about 45°C and pour 10 ml of this medium into the
Petri dishes. Mix the agar by rotating the plate.

Coagulase positive staphylococcus aureus counts:
1. Label Petri plates with sample no., test name (SAC), date, and dilution factor.
2. Aseptically transfer 1 mL of 10-1 dilution to sterile Petri plate in duplicate using a
sterile pipette tip.
3. Repeat the procedure with 10-2 and further dilutions (as per the requirement) of the test
sample, using a sterile pipette tip for each decimal dilution.
4. Pour about 12- 15 mL of the freshly prepared complete Rabbit Plasma Fibrinogen Agar
Medium at 45 ± 0.5 °C, into each Petri plate.
5. Carefully mix the inoculum with the medium by rotating the Petri plate and allow the
mixture to get solidify.
6. Prepare a control plate by plating 1 mL of sterile diluent to check the sterility.
7. After complete solidification, Invert the prepared plates and incubate them at 37 ± 1 °C
for 18 h to 24 h. If necessary, re-incubate for 18 h to 24 h.
8. Select the plates containing not more than 150 colonies, whether characteristic or not,
for counting.
9. Count only the characteristic colonies in each of the selected Petri plate (Characteristic
colonies of S. aureus are black or grey or even white, small colonies surrounded by a
halo of precipitation, indicating coagulase activity).

COUNTING AND EXPRESSION OF RESULTS:
Count the colonies measuring at least 0.5 mm in diameter.
Calculation and expression of results
Case 1: Four Petri plates from two consecutive dilutions
1. If four plates from 2 consecutive dilutions are counted , calculate the number N of
Microorganisms present in the test sample as a weighted mean using the following
equation:
N = ΣC
2.2 X d
Where
ΣC = Total no. of colonies counted on all the 4 plates from 2 consecutive dilutions and
d = Dilution factor corresponding to the first dilution
2. Round off the result calculated to two significant figures.
a. If the third digit is 5 or more, raise the second digit by 1 and use zeroes for successive
digits.
b. If the third digit is less than 5, retain the second digit as such and use zeroes for
successive digits
3. Express the result as a number between 1.0 and 9.9 multiplied by 10x, where x is the
appropriate power of 10 or a whole number with two significant numbers
Example
No. of colonies at the first dilution (10-2): 168 and 215
No. of colonies at the second dilution (10-3): 14 and 25
(168+215+14+25)
N = ----------------------------
2.2 X 10-2

422
= -------------- X 100
2.2
= 19182
= 19000 (rounding the result to two significant digits.
The count also can be expressed as 1.9 x 104 per mL of the sample
Case 2: Two Petri plates from a single dilution
1. If 2 plates from a single dilution are counted, calculate the number N of
microorganisms present in the test sample as an arithmetical mean of the colonies
counted on the two plates using the following equation
N = ΣC
2 X d
Where
ΣC = Total no. of colonies counted on 2 plates
d = dilution factor
Example
Number of colonies at the first dilution (10-3) retained: 12 and 13
(12 +13)
N = ---------------
2 X 10-3
25
= ------ X 103
2
= 12500
= 13000
The count can also be expressed as 1.3 x 104 per mL of the sample

Case 3: No colonies in 1 mL of test sample or 1 mL from the first dilution (10-1)
1. If the two Petri plates from 1 mL of the test sample contain no colonies, report the
result as “less than 1 per mL” or “absent in 1 mL”
2. If the two Petri plate from the first (10-1) dilution contain no colonies, report the results
as “less than 10 per mL” or “absent in 0.1 mL” as required by the specification.

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