INTRODUCTION OF MILK

SCOPE AND OBJECT OF

STUDY
INSTRUMENTATION AND
METHODS
OBSERVATION AND
RESULTS
DISCUSSION
CONCLUSION
BIBLIOGRAPHY
INTRODUCTION
INTRODUCTION OF DAIRY

The dairy industry in India is emerging as a sunrise "industry. India is one of the largest and fast
growing markets for milk and milk products to increase in incomes among 300 millions strong
middle classes. India has become the second largest milk producer in the world,

Next only to U.S. India is on the verge of been the world's number one milk producers
with nearly 75 millionmetric tons of milk per annum. It is estimated that our dairy business is
to be around Rs.80.000Crores and contributes approximately 9%of the total GDP.

The Indian dairy industry profile is projected in table

Dairy industry profile;

Human population : 1 OOmillion (75 million dairy fanners)

Milk production :82.3million tones

Average annual growth rate : 13-14(till 2020)

Milk animals :57million cows and 42 million buffaloes

Milk yield per breed able

Buy in milk : 1250kgs.

VISAKHA DAIRY PROFILE
LOCATION OF THE VISAKHA DAIRY:

Visakha dairy is located on the national highway NH5, opposite to B.H.P.V Ltd
Natayyapalem in Visakhapatanam.

ORIGIN AND ESTABLISHMENT:

The milk shed of Sri Vijayavisakha district co-operative milk producers union limited
(visakha cooperative dairy) comprises of three districts namely Srikakulam,
Vijayanagaram, and Visakhapatnam. These three districts are situated in the north
easternpart of the coastal area of Andhra Pradesh state considered to be backward for agriculture
and industrial development.

The Srikakulam district is declared as the back ward district for the industrial
dev-elopment and the government has sanctioned subsidy and also tax exemption for perennial
sources of water for irrigation through rivers and rivulets.
 The government after considering dairying as one of the best instrument inBringing
socio-economic development in the rural area has started a dairy with a handling Capacity of
10,000 liters per day in 1966, dairying not only Creates subsidy occupations to the rural farmers
by creating responsible market price for his produce athis door steps but also meets the demand of
the urban consumer for the supply of hygienic quality will at responsible price.

After observing the success of the small dairy, the present new dairy was constructed
with an initial capacity of 50,000 liters per day taken a loan of 98.50 lakhs from NDDB. The dairy of
register under cooperative societies act in9037 and established in 1977, at the initial stage, the area
operation was limited to Visakhapatnam district only. Th e f ar m e rs t o l ot i nt er e s t i n
dai r yi n g after realizing it as Subsidiary occupation as it is given them regular income for
theirlive hoods, with the above result, more and more small, marginal fanners and agricultural
labors joined in this stream for increasing their economy at village level utilizing the
infrastructure available at their door steps. When the Andhra Pradesh state has adopted
"Anandpattern" for dairy development through operation flood programmer level.

At Srikakulam, Visakhapatnam, Vizianagaram was formed under the name of Sri

VijayaVisakha district cooperative milk producers union limited during the year 1981-82.

As the production and procurement started increasing, year by year with more
participation of rural farmers with more enthusiasm, the handling capacity of Visakha
cooperative dairy also increased from 50,000liters-to 1,00,000 liters per day which came into
operation during the year 1986-87.

Again the necessity to increase the capacity from 1, 00,000 to 1, 50,000 liters per day
since the installed capacity was reached in short time.

Now the dairy procuring the milk from 2744 villages in coastal Andhra areas by serving 2,
07,925 milk producers.

a) The dairy was converted to mutually Aided cooperative Act-1995 in the year 1999Sri Vijay
Visakha milk producers mutually Aided cooperative union and the daiiy was converted to
company Act-1956 with effect from 06.01.2006 as "Sri VijayaVisakha milk
ProducersCompany limited". • The procurement and production graph growth is increasing
year with more participation of the rural farmers.

Now the Visakha dairy is one of the fastest growing milk and milk products.
 INTRODUCTION OF THE MILK

Milk happens to be the "Natures Most Blessed Food" bestowed with almost every
nutrient needed for our survival and growth-)and the to in almost perfectly balanced quantities.
(Milk is the white, fresh, cream, lacteal secretion obtained through the udder (mammary
glands) immediately after calving to feed the young one.. Milk is liquid food for human
infants and adults.

Besides fat, carbohydrates and proteins milk contains almost all essential amino acids,
vitamins and minerals required for growth and disease resistance. Milk, whether it is human or
bovine is exceptionally complex. It includes more than 200 recognized components. The
composition of milk of the various mammal species differ considerably in quality and quantity.
Particularly a comparison of the average composition of human and cow milk shows
substantial differences, however, similar of energy supplied by co and human milk.

The Chemical composition of freshly drawn milk differs widely.

* The Main Bulk of Milk Water is forms 87.3%

* Lactose constitutes 5%
* Fat is 3.8%
* Case in is 2.5%
* The pH of milk varies from 6.7-6.9
* The Specific gravity is 1.035

Milk contains vitamins such as A, Bl, B2, C, and D.It contains body building proteins. Bon

forming minerals, energy giving lactose, milk fat and fatty acids.

All nutrients present in the milk are easily digestible and affirmable. Thus milk becomes a

balanced diet to infants, convalescents and patients.

 COMPOSITION OF MILK

Composition Percentage

Water 87.3%

Lactose 5%

Protein 3.5%

Casein 2.5%

Albumin & Globulin 0.7%

Butter fat 3.8%

Fat 5%

Minerals 0.7%

Vitamins 0.07%

PH 6.7-6.9

Specific gravity 1.035

Dairy about 4,50,000 liters of milk is received. Among that about 4, 25,000 Its of milk is packed in

the form of different milk packets like toned milk, skimmed milk, fatless milk, etc., The remaining

amount of milk products section and which is usedfor the preparation of several milk products

like Pannier, Badam milk, Butter, Ghee, DoodhPeda, Butter Milk, Curd, Burfi, Ice Cream, Lassi,

Milk Cake, Cheese, Cream.

 RAW MILK RECEPTION DOCK

INTRODUCTION:

Raw milk reception dock is the platform where dairy receive milk from chilling in
road tankers, bulk cooling centers and by other ways.

To protect the milk from morning sunshine, the receiving dock wasconstructe d facing,
southern direction. The height of the dock is constructed so that unloading of cans from
the tucks is facilitated easily. 85% of buffalo milk andaround 20% cow milk is received
every day from variousbulkcooling centers and milk chilling centers.

UNLOADLMG FROM MILK TANKERS:

The major quantity of the milk is received from milk chilling centers. The

unloading as follows:

1. Milk tankers are of different capacities. Generally they are of two types
basing on the volume i.e. 15000 liters& 90001 (rs.

♦ Time taken for unloading 15000 liters capacity is 40 mints.

♦ Time taken for unloading 9000 liters capacity is 20 mints.

2. Most of milk tankers consist of two cells front cell and back cell. These
cells are separated by partition.
3. While unloading milk. The milk samples are taken after well mixing with
Plunger and they are taken in two sampling bottles and that instant
organoleptic tests are also evaluated.
4. The milk tankers are unloaded and stored in silos (6, 7, 8&9) by passing
through chiller.
5. At a time tankers can be unloaded. One is through chiller and another
which is already chilled in chilling center.
6. On average 20- tankers per shift are unloaded. If milk is sour then fat is
separated and skim milk is drained out.
 Flow of milk:
Bulk cooling centers and units

Receiving milk through road tankers

Chilling to < 5° C

Storing in silos (6, 7, 8, 9)

Pasteurization

Separation

Homogenization

Standardization

Toned milk

Homogenized double Toned Milk

Standardized Whole milk

Low fat milk

Full fat milk

a Temperature of milk received is taken at 4oc

b. Temperature of milk received from bulk coolers is 6-7oc

 Cleaning of tankers:

1. Immediately after unloading, the milk tankers are flushed with water
2. After flushing through water, then it is cleaned with hot water to remove
residual milk
3. The water is removed from tanker

CIP for Road Tankers:

1. Rinse with tap water
2. Scrub the inner surface with liquid soap manually
3. Rinse with hot water at 70-75 oc
4. Sanitize by spraying with Idophore solution

Milk as a culture medium:

Milk is an excellent Culture medium for many species of microorganisms.

Milk has the necessary moisture, proteins, fats and mineral salts. Lactose can be used as a
nutrient by a considerable species of microorganisms; some organisms can break down
proteins and few can utilize fats. The temperature is generally favourable for b acterial
growth from the time when the milk is drawn until it is cooled and held at
temperature below 4.4° C. By actual test, milk has been found to be a suitable medium
for the development of hundreds of bacterial species, both saprophytic and pathogenic
forms.

Microorganisms are generally of five groups namely

> Bacteria
> Virus
> Yeast and moulds
> Algae
> Protozoa

Milk and its types:

Milk is a lacrial secretion of buffalo which consists of carbohydrates' fats'

Proteins, minerals, vitamins and water.

Composition of milk (%)
Constituent's %
Water 87.0
Casein 2.5
Lacto albumin
and other protein 0.5

Lactose 5.0
Lipid 4.0

Sterols, Vit A, D, E 0.05

The major part of the milk is water. The specific protein, casein is in fact a mixture of three
long Chains of amino acids suspended in fluid. Lacto albumin another protein forms the
surface skin of milk when heated to boiling. The major carbohydrate is lactose.

Most commercially available milk is buffalo's milk however; milk from other mammals,
such as cow's, goats and sheep is also sold.

Raw milk:

Raw milk is unpasteurized milk; drinking it is no longer recommended. The risk of

bacteria such as listeria and salmonella in raw milk has prompted the U.S Government to
require pasteurization, which also extends the milk's shelf life.

Pasteurized milk:

The term "pasteurization" means heating at different temperature heat treatment. The
basic processes are currently in use:

(a) Holding method

(b) Flash method
(c) UHT method.
 Sterilized milk:

It is prepared by subjecting the milk continuously at above EiP of H2O for required time
(from few sec to few min) after heat treatment preserved at R.T not less than 15 days from the date
of manufacture.

Evaporated (or) condensed milk:

To make this concentrated product, half of the milk's moisture is removed by evaporation

Flavoured milk:

It may contain nuts chocolate, coffee, or any other edible flavours, edible food colours
and cane sugar. These are also pasteurized, sterilized (or) boiled.

Homogenized milk:

In the homogenization process, milk fat is broken into small particles that it is emulsified
and held within the milk, instead of floating at the surface. In the United States today, almost
all milk is sold homogenized form.

Toned milk:

It is a product prepared by that mixture of cow (or) buffalo milk (or) both with fresh
skimmed milk that has been standardized to fat and SFN percentage.

Types of bacteria present in milk: Acid

forming bacteria:

The most commonest are lactic streptococci including streptococcuslactis,

streptococcus fecalisand Lactobacilli are also found. They ferment lactose in milk, pro ducing
acid, mainly lactic acids which lead to the formation of a smooth gelatinous curd.

Alkali forming bacteria: these consist of alkali genes species some aerobic spore bearers and
Achromobacter. These render the milk alkaline.

Inert Bacteria: bacteria which produce in visible change is milk are called inert. These include
some cocci of the under, members of the Achromobacter group and most of the pathogenic
organisms in the milk.
Human milk: Breast milk contains small numbers of staphylococcus
epidermidls, streptococcus mitis: Gaffkya tetragena and staphylococcus aureus.

Milk borne diseases:

Infections of animals that can be transmitted to human beings :

The most important are Tuberculosis,Brucellosis yStreptococcualand

Staphylococcal infections, salmonellosis and Q-fever. Diseases of l e s s importance

include cowpox and milker'snodes which are usually transmitted during milking rather than
through ingestion of milk. Foot and mouth disease: anthrax and leptospirosis have been
transmitted on rare occasion .tickborneencepalities vims may be transmitted through goat
milk. Milk borne infections hepatitis has been reported.

Occasionally, milk may be contaminated with Streptobacillusmoniliformsixom nasal

secretions of rats and with camphlyobacterjejunifromanimal feces. Yersinia
enterobacilliticaisnot uncommon in milk and may give rise to gastroenteritis if present n. large
numbers.

The organisms that cause all the diseases mentioned above are destroyed by adequate
pasteurization.

Infections primary to Humans that can be transmitted through milk .Enteric

infections: these caused by consummation of milk which has been mixed with water
contaminated by human excreta. A less common source are the human carriers of enteric
infections employed in the dairies. The disease caused by typhoid and paratyphoid fevers,
shigellosis; cholera and diarrhea are due to E.coli forms.

Streptococcal infections : Buffaloes may have under loss teat infections and the
organisms into the milk.

Staphylococcal food poisoning: milk from buffaloes suffering from Staphylococcal

mastitis is contaminated with the organisms. If the milk is consumed after being allowed to
remain at temperature favour abke fir its multiplication the entero toxin is produced, which
causes food poisoning. Diphtheria: milk contaminated either from human carrier or more
usually through diaphoretic lesions on the teats, when consumed unpasteurized milk causes
disease.
Tuberculosis: milk contaminated by excretions from persons suffering from tuberculosis, when
consumed leads to the disease.

Some of the disease like brucellosis in animals can be detected also by demonstration the
antibodies in the milk,by the milk-ring or the whey agglutination.

Sources of contamination of milk:

> Many microorganisms attacks milk and changes the quality and shelf life of the milk,
Contamination of milk is of various sources are as follows.
> Milk is contaminated by the infected milk animals milk contains relatively few bacteria when it
leaves the udder of infected buffaloes
> Utensils used by the milk man for milking purpose,
> Milk man should maintain hygienic conditions, Otherwise milk taken by such person may be
infected
> In case of dairy products milk may be contaminated by dairy utensils milk
Contact surfaces, including the large round open vessel for carrying liquids milking
machines, as the case may bestainers, milk cans or pipelines and the
bulk milk cooler.

Spoilage of milk:

The number of bacteria in milk range from several hundred to several thousands per milliliter. From the

time the milk leaves the udder of the animal until it is dispensed into containers everything with which it

comes into contact becomes a potential source of contamination. Among Bacterial spsofBacillus,

Escherichia, Lactobadllus, Leuconastoc, Clostridium, MicrococcuSyMycohacteriiim, Propionibacterium,

Proteus, Pseudomonas, Streptococcus are common.

SPOILAGE:

Decay or decomposition of an undesirable nature usually called as spoilage. It is applied to food,

which producing undesirable nature by the action of microorganisms
 TYPES OF SPOILAGE OF MILK
Spoilage occurs when "microorganisms" degrade the carbohydrates, proteins, fats of m
produce toxins end product.

Common types of spoilage is dairy products are.

Spoilage type organisms involved Signs of spoilage

Souring Lactobacillus Sps. Sour milk

Streptococcus Sps Curd formation

Sweet curdling Bacillus Sps Alkaline PH

Proteus Sps formation
Micrococcus Sps

Gas production Clostridium Sps Explosion of curd

Coli form bacteria -

Ropiness AlcaligensSps Stringy (or)

KlebsiellaSps Slimy milk
EnterobacterSps
Red rot Serrratiamarcescens red coloration
Gray rot Clostridium Sps Gray coloration
Foul smell
Dariymould penicilliumSps
Geotrichum SPs Mouldvaocearani

Spoilage mainly occurs due to sealing weakness. This can be verified by dye testing. When
package get Spoiled with any visible appearance there is no need to test But sometimes they may
get spoiled without any visible porosity then we have to investigate thoroughly.- For this purpose
we have a keen observation of milk, package air, and water by utilizing various techniques
This spoilage mainly occurs due to presence of Micrococci and Bacillus Sps. Etc. the

defectives in packaging mainly occurs by following reasons :-

 Fault in package
 Mechanical damage (faults in machinery)
 Improper handling while packing
 SOURCES OF MICROBIAL CONTAMINATION

Milk and milk product constitute highly perishable Commodity because they are extremely
susceptible to microbial contamination. The contaminating Microorganism gain entry into milk
and milk product through different source. The air e borne microorganism entering the dairy
products may cause then spoilage or may be Responsible for the spread of infection, disease and
intoxications.,

 Environment (air)
 Water
 Packaging materials
 Equipment
 Personnel

ENVIRONMENT (AIR)
Certain forms are quite uniformly present Molds and Yeasts are quite commonly found in
the air in some cases out number the bacteria. Spores of yeasts and molds are quite resistant of
light which is lethal to bacterial spores

As per a survey conducted on the dairy environment by "Cannon" 1966, the air-borne

microorganism consisted of

0.4% yeast

249% Gram-Negative rods

22.2% Spore forming bacteria

33.3% Gram » positive Cocci

19.1% Gram positive, non-spore forming rods

Recent problem of Staphylococcus and Salmonella in dairy milk preparation have raised
doubts about the air as the culprit because of large volumes of air being used for drying,
transporting and cooling of those products.

According to "Walter" 1966, microorganism exits in air in three ways

 Asporogenous on solid particles of dust or skin or hair
 Within droplets formed by the atomization of liquids by sneezing stirring or other
activity.
 As isolated organisms resulting from the evaporation of water from droplets or in the
case of mold spores as a result of their natural method of propagation.
 WATER
The quality of the farm Water supply used in the milking parlor for Cleaning, rinsing etc., will have
some effect on the quality of milk. Fecal Contamination through water.

PACKING MATERIALS

Other source of contamination after the milk leaves the form include the tanker track,
transfer pipes, sampling utensils and the equipment at the market - milk plant, the most are the milk
tanks.

EQUIPMENTS
The most significant source of contamination are the milk Contact Surfaces, pipelines, vats, tanks,
pumps, values separators, clarifiers, Homogenizers, coolers, Strainers, stirrers and tillers may serve as
Possible source of bacteria. The amount or level of contamination from each of these sources depends on
cleaning and sanitizing methods.

Probably the two most Significant sources of contamination arc dairy Utensils and milk Contact
surfaces, including the milk pail or milking Machines, as the case may be strainers, milk cams or
pipelines, and the bulk Milk coolers. If dairy utensils or the milk f contact surfaces are Inadequately
cleaned, sanitized and dried, bacteria may develop in large numbers in the dilute milk like residue and
enter the next milk to touch these surfaces.

PERSONNEL;

Milk contains relatively few bacteria when it leaves the udder of a healthy cow, and
generally these bacteria do not grow in milk under the usual Condition of handling. However,
Micrococci and Streptococci have been recovered from aseptically drawn milk.

During the normal operation, however milk is subject to contamination from the animal,
especially the exterior of the udder & adjacent areas. Bacteria found in manure, soil, and water may
enter from this source such contamination is reduced by clipping the cow, especially the Links and
udder or grooming the cow, and washing like udder with Water (or) a germicidal solution before
milking.

Other possible sources of contamination are the employees, particularly their hands and

arms or dairy the air of the barn of milking Parlor and flies.
 Other possible sources of contamination are the employees, particularly their hands and
arms or daily the air of the barn of milking Parlor and flies.

Factors influencing shelf life of milk:

Shelf life of milk and milk products mainly depends on chemical, physical microbiological and
biochemical changes. To control the length of microbial generations, different methods are used such as:

■ Refrigeration
■ Deep freezing
■ Chemical Preservatives
■ Heat Treatment
Refrigeration

Exposure of Microorganisms present in milk to low temperatures reduced their rate of growth and
reproduction, This principle is used in refrigeration and freezing. Usually temperature in refrigeration
ranges from 0-1 OOC. However some organisms like Salmonella spszndStreptocoocispssurvives in freezing
temperature also,

Deep Freezing;

Storage temperature ranges around or below -18 C. Shelf life around 1 year or more is usually
achieved.

Chemical Preservatives:

By adding chemicals the growth of the microorganisms are Suppressed without damaging the quality
of product.

Heat Treatment

• Pasteurization process
• Sterilization process

Pasteurization Process:
The process was developed by Louis Pasteur in 186O's to eliminate bacterial species. The
primary process of pasteurization is to "eliminatepathogenic organisms in milk". Pasteurization
process involves several typesviz.,
1 Long temp long time method (or) Holding method At 63 °C for 20 to 3 0 minutes

2 High temp short time method (or) flash method At 72 °C for 15 seconds

3 Ultra high temp method At 141 °C for 2 seconds

Sterilization Process:

Sterilization is distinguished into two types one is "in-FlowSterilization" and other one is 'In-Batch
sterilization".

In-Flow sterilization: 135 °C-155 °C for a few Sec

In-Batch sterilization: 120 °C for 22-30 mins

PASTEURIZATION QF MILK

The process of heating the milk to 76 c and immediately cooling it to 4 to 5 c so as to kill the
pathogenic and non-pathogenic organisms is called pasteurization.

Pasteurization are two in number. One with 20,000 litrs/hr capacity and the other 10,000 lit/hi",
these are

1. Alfa -Laval pasteurizer.

2. Tetra packs pasteurizer.

1. Alfa-Laval pasteurizer:
Medium : Milk

Throughout : 10,000 lit/hr

Temperature : from 5-50c-72.5-80c-57.5c-12.5c 4c

Cooing medium : Ice water -30,000 lit/hr at 2b

Heating medium : Hot water

Regeneration :2

Type of flow : counter flow

Steam pressure : 6 bar

Plate material : SA 240;GR 304

Plate pack length : 1837 mm

Max. Temperature : 110c

 2. Tetra pack pasteurizer:
Make : Tetra pack - India
Medium : milk
Throughout: 10,000 lt/hr

Temperature range: from 5-50c-72.5-80c- 57.5c-12.5c 4c Cooing medium

: Ice water -30,000 lit/hr at 2c

Type of flow : counter flow Steam pressure : 6 bar Plate material : SA

240;GR 304 Plate pack length : 1109 mm Max .temperature :130c

Pasteurization of milk:
Chilled milk from raw milk storage tank

Pumping

Regeneration I Filtration

Regeneration II (60 °C)

Heating sectioi! (78+_2°C)

Regeneration III (60°C)

Regeneration I (60 °C)

Chilling (4-5 °C)

Storage Tank
 REVIEW OF LITERATURE

Year Scientists Achievements

1674 Mark the birth of Microbiology, when

Antony Van Leeuwenhoek
looked at a drop of lake water through a
glass lens.

1949,1854 John Snow Showed faecal-oral transmission of

Cholera.

Queen Victoria husband was reported

1861 John Snow to have died of typhoid indicating that
even the royal family apparently drank
fecal polluted water.

Cholera Virus
1883 Robert Koch Connected polluted water with

1885 John Snow human disease.

Sewage treatment technology

John snow stared.

1900
Poliomyelitis is due to fecal

1909 Landsteiner -Popper Contamination

Used the biuret test as a measure

Swiatopelk -Zawadski
1916 of proteolysis.

Precipitate the case in from milk

Zaribnicky
1926
with methyl alcohol and magnesium
sulphate and analyzed the filtrate for
total nitrogen.

Computed the MPN to evailuate

Hoskins
1934
Coli- acrogens test by fermentation
tube method.

1950 Chambers
Showed that 40 to 390 million per
 ml coil forms were required to produce
visible Gas in fermentation broth.
1951 Goetz &Tsuneishi
Described a new method for

enumeration of coliforms in water to

which they named as Millipore Filter
Technique

1960 Taylor
Defined the Water Potential as the
Chemical Potential of water.

1962 Kabler there are three chief methods that are

used for the purification of drinking
water in Municipal supplies i.e.
Sedimentation. Filtration, disinfection.

1968 Singh etal Reported cases of endemic

Fluorosis in human being
Kanwan& Mehta caused by the ingestion
of-high fluoride well water
in Andhra Pradesh.
 SCOPEANDOBJECTOFSTUDY

Milk is an excellent diet for all age groups, especially for infants, and it is an
excellent medium for the growth of different kinds of microorganisms. In good olden days,
people usually depended upon milk yielding animals for their survival. Similarly products
were not stored for longer duration, they were consumed immediately. But Now-a-days
people have lots of prospective for their survival due to developing science and technology. 'So
only certain group of people depends on dairy industries, but they can't produce the milk.
Storage methods have been evolved, among them "Pasteurization" and "Sterilization"
are very important processes to keep the quality of milk and increase the shelf life. In that
Way to prove The importance of sterilization 1 selected this project.

Scope of present study i.e., milk and its products endsupto the
examination of locally available raw milk, collected from buffaloes and
pasteurizes milk of Sangam Diary.

Hypothesis:

Pasteurized milk is Worth of Human consumption than Raw Milk

 MILK SAMPLE COLLECTION

I .Milk samples are collected from the premises.-specified by the client, by a trained person.

2. The bottles chosen to collect the samples should be of enough capacity to carry the
milk in the amounts required for the intended analysis and should have stoppers or
other means to properly seal them.

3. The milk for microbiological analysis will be collected in sterilized glass or plastic bottles.

4. The sterilized bottles will be transported to the site in a closed vessel, which can
effectively carry the bottles in a cooled environment.

5. The box should have a covered top.

6. These bottles should have proper identification stickers attached to them.

7. The box should be carried in such way that the contents of the bottles do not spill out.

8.The surroundings oflhe point from where the sample is to be collected should be Inspected
and necessary step should be inspected and necessary steps should be taken to avoid the
contamination of the sample.

9.The tap should be cleaned with spirit (or)alcohol and left open for at least 15 minutes
before collecting the samples aseptically.

lO.The bottles should be sealed immediately after taking the sample.

I1 .The bottles should be kept upright in the ice cooled container being used to carry the
Samples.

12.Upon arrival in the laboratory the sample should be immediately transferred to

Refrigerator and put for analysis as per specifications
 MATERIALS

Instruments and equipment :-

1) Laminar air flow

2) Hoi air oven

3) Incubator

4) Test tubes

5) Pipettes

6) Petri plates

7) Screw cap test tubes

8) Durhan's tubes

9) Autoclave

10) Colony counter

11) Microscope

MEDIAS USED:

DIFFERENTIAL MEDIA:

MacConkey's medium, Pseudomonas agar, Mannitol salt agar, Sabourauddextrose

agar etc.,

1. Brilliant green agar

2. Eosine Methylene Blue agar
3. MacConkey agar
4. Listeria oxford base agar
5. MacConkey Broth
 6. Mannitol salt agar
7. MR-VP broth medium
8. Starch milk agar
9. Rose Bengal chloramphenicol agar lO.S.S
agar
1 l.Tryptone glucose yeast extract agar 12.
Violet red bile agar

Brilliant green agar:

Composition grams/liter

Lactose 10.00

Sucrose 10.00

Sodium chloride 05.00

Phenol red 0.08

Peptone 10.00

Yeast extract 3.00

Brilliant green 0.0125

Agar 20.00

Use:

For the selective isolation of salmonella

Description:

First introduced by Kristiansen et al in 1925 as a selective medium for the isolation of

salmonella (except salmonellatyphi)

The medium is suitable for subcultures from selective enrichment media.

 Eosin Methylene Blue agar:
Composition grams/liter

Peptone digests of animal tissue - 10.00

Dipotassium phosphate - 2.00

Lactose - 5.00

Sucrose - 5.00

Eosine-y - 0.40

Methylene blue - 0.065

Agar - 13.50

pH - 7.2+0.2

Description:
Escherichia cali can be identified with Eosine Methylene blue (EMB) agar based on the
occurrence of a green metallic sheen that appears on the surface of the bacterial colonies. EMB
agar is a blend of two strains eosin-y & methylene blue in the ratio of 6:1 are pH indicators and
inhibitors of gram- positive bacteria and provides a color indicator distinguishing between those
organisms that Fermentlactose.

ListeriaOxford base agar:

Composition Grams/liter

Peptone - 23 .00

Lithium chloride . - 15.00

Sodium chloride - 5.00

Corn starch - 1.00

Esculin - 1.00

Ferric ammonium citrate - 0.50

Agar - 10.00

PH - 7.0+0.2 at 37 c
Description;

It is used for the isolation and cultivation of Listeriamonocytogenesfrorn pathological

specimens.

Special peptone provides nitrogen, vitamins and minerals. Corn starch is omitted to
reduce opalescence.
Naclensuresosmotic balance. Listeria monocytogens hydrolyses esculin to esculating
and forms a black complex with irons. Therefore Listeria monocytogenesgrows as
brown-green colored colonies with a black halo(esculin splitting).

Violet Red Bile Agar:

Composition Grams/liter

Peptic digest of animal tissue - 7.00

Yeast extract - 3.00

Lactose. - 10.00

Bile salts mixture. - 1.50

Nacl - 5.00

Neutral red - 0.03

Crystal violet - 0.002

. Agar - 15.00

pH - 7.4+0.2

Use:

For the selective isolation, detection and enumeration of coli-aerogens bacteria in water, milk and
other dairy products.
Tryptone Glucose Yeast Extract Agar:
Composition

Grams /liter

Yeast extract - 3.00

Glucose - 11.00

Agar - 15.00

Distilled water - 100ml

pH - 7.0+0.2

USE:

This medium is used for the detection and enumeration of totalbacteria in water, air, rnilk and milk
products.

Mannitol Salt Agar:

grams/liter
Composition

Peptic digest of animal tissue 5.00

Pancreatic digest of casein 5.00

Beef extract 1.00

D-Mannitol 10.00

Sodium chloride 75.00

Phenol red 0.002

Nutrient agar:
Agar
Grams/liter
Ingredients
Nad 15.00
5gm
Agar Use: Selective medium for
15gm staphylococcusaureus.

7.2-7.6

5gm Use: 3gm

Peptone It is the basic culture media.

Beef extract
Mac Conkev Broth:
Ingredients Grams/liter

Peptone 17.0

Protease peptone 3.0

Lactose 10.0

Bile salts mixture 1.5

Sodium chloride 5.0

Neutral red 0.03

0.001
Crystal violet

USE:

It is a specific broth used for presumptive test for detection of specific coli form bacteri a.
 MEDIAN AND REAGENTS USED FOR IMVIC TESTS; SIM Agar

Media:

Ingredients Grams/liter

Peptone 30.00

Beef extract 3.00

Ferrous ammonium sulfate 02

Sodium thiosulfate 0.0025

MR-VP Broth:

Ingredients Grams/liter

Peptone Dextrose' 7gm

glucose 5gm

Potassium phosphate 5gm

KOVAC'S REAGENT:

Ingredients Grams/liter

P-Dimethyl amino Benz aldehyde - 5 gm

Buianol Arm I alcohol - 75 ml

Hydro chloric acid - 25ml

 METHYL RED REAGENT FOR MR-TEST:

Ingredients Grams/liter

Methyl red • 0.1 gm

Ethyl alcohol 300 ml

Distilled water 200 ml

Note : Dissolve the methyl red in 95% ethyl alcohol. Dilute to 500 ml with distilled water.

BARRITS REAGENT FOR VP-TEST:

Solution A:

A - naphtha - 5 gm

Absolute ethanol - 95ml

Solution B:
40 gm
Potassium hydroxide
Creatin 0,5 gm

Distilled water 10 ml
Storage tank

Starting of HTST pasteurizer:

10. Switch on the main switch of the control panel

11. Fill up the hot water tank with water
12. Start the hot water pump
13. Check the water level of the hot water tank
14. Fill the FCBT with water and start the milk pump
15. Open the stream valve slowly
16. Sterilize the forward and diversion lines with hit water for 10 min after required
temp (80c) is reached.
17. Set the temp control for required temperature i.e.. 76_+2° C.
18. Open the chilled water valve..
lO.Flush out the hot water and pump the milk to FCBT the silo.

11 .As soon as pure milk start from the outlet turn the valve to the storage tank.

12.Turn the control panel switch to Auto.

13.Observe and record the heating chilling temperature for every Vi hour .

Shut down:

7. When the last milk is leaving the FCBT open the water to FCBT.
8. Flush out plant with water and stop the tap & chilled water .
9. Cleaning of the plant (CIP) and close the stream valve
10. Circulate the tap water through hot water tank until the plant is cooled
11. Stop the milk and hot water pumps & switch off the main switch
12. Open the filter and clean it.
SIMMON'S CITRATE AGAR:

Ingredients Grams/liter

Ammonium dihydrogen phosphate 1

Dipotassium phosphate 2

Sodium chloride 5

Magnesium sulfate 0.2

Agar 15

Bromothymol blue 0.08

pH 6.8
 METHODS USED IN BACTERIOLOGICAL ANALYSIS

• Raw milk and Pasteurized milk:

Both raw milk and pasteurized milk are used for microbiological analysis. When samples are
drawn from tankers care should be taken and the containers used are rinsed with sufficient quality
before taking the sample.

Diluting the givensample:

Diluting the given milk sample is used to enumerate the microbial count at different
dilutions. Dilution of milk samples should be done only under aseptic conditions. During dilutions
one ml of milk is added to 9ml of distilled water or buffer of 10 "' dilution. Again serial dilution
should be done by taking 1 ml of milk from 10 '" dilution and 9ml of distilled water should be added.
Serial dilutions should be done up to 10"5 dilutions. These give 1:10 and 1:100 dilutions of the milk
sample.

Volume of the sample

Dilution =

Total volume of the sample and diluents

 METHODS USED IN BACTERIOLOGICAL ANALYSIS

• Raw milk and Pasteurized milk:

Both raw milk and pasteurized milk are used for microbiological analysis. When samples are
drawn from tankers care should be taken and the containers used are rinsed with sufficient quality
before taking the sample.

Diluting the givensample:

Diluting the given milk sample is used to enumerate the microbial count at different
dilutions. Dilution of milk samples should be done only under aseptic conditions. During dilutions
one ml of milk is added to 9ml of distilled water or buffer of 10 "' dilution. Again serial dilution
should be done by taking 1 ml of milk from 10 '" dilution and 9ml of distilled water should be added.
Serial dilutions should be done up to 10"5 dilutions. These give 1:10 and 1:100 dilutions of the milk
sample.

Volume of the sample

Dilution =

Total volume of the sample and diluents

 METHYLENE BLUE REDUCTION TEST

Principle:

Take length of time taken by milk to decolorizes Methylene blue is a fairly good measure of its
bacterial content and hence it requires utmost care in testing.

Apparatus:

Sterile test tubes, sterilized rubber stoppers, sterile 1 ml &10 ml pipettes, water bath at 37+1 °c.

Procedure:

a) Thoroughly mix the milk sample transfer 10 ml of each sample into separately
identified sterile test tubes.

b) Add lml of methylene blue solution to the milk in each of the test tubes and
replace the cotton plugs with rubber plugs.

c) Mix the dye and the milk by inverting the twice.

d) Place the tubes in the water bath maintained at.

e) Check the reduction of the color for every half an hour interpretation. Match the
reduction with standard.

Microorganisms present in milk and milk products . .. ...

-

1. Coliform

2 .Staphylococcusaureus

3.Salmonella 4.Shigella

5. LysteriaMonocytogens

MBRT GRADE
Above 5 hours very good
3and4 hours Fair
1 and 2 hours Poor
 MICROBIAL EXAMINATION OF MILK BY PLATING METHODS
STANDARD PLATE COUNT (SPC)
Introduction:
Standard plate count means total bacteria (or) Viable cells, Vegetative cells, Spores and
Fungal Spores these are all comes under standard plate count.

Principle:

This method is used for the detection and enumeration of bacteria in milk under the aseptic
conditions. Under aseptic conditions milk is diluted and transferred into standard agar plates. The
agar is permitted to harden on a level surface. The Petri dishes incubated at 37 °C for 48 hours.

Medium Used: Tryptone glucose yeast extract agar. This media is sterilized by Auto clave 121 °C
15lbs pressure for 15 minutes.

Dilution factor:

For Raw milk required dilution is 3rd dilution. For Pasteurized milk required

dilution is 1st dilution.

Procedure:

> Take 1 ml of the milk sample from the above dilution and aseptically
transferred into sterile Petri plates using a sterile pipette.

> Add 15 to 20 ml of sterile Tryptone glucose yeast extract agar media at

45 °C+0.5 °C was poured into Petri plate.

> Contents were mixed gently clockwise and anti clockwise without splitting.
And allowed to solidify.

> After complete solidification plates were inverted and incubated in the
incubator at 37°C for 48 hours.

Observation:

After 48 hours of incubation the colonies were observed and counted by using colony
counter in each plate.

Result: Calculation

No .of colonies * dilution factor CFU/m!

 ENUMERATION OF COLI FORM
Introduction: This microorganism's habitat on Intestinal of human being (or) Animals. Those
microorganisms are called Coli form. This Coli form bacteria can be-divided into two types.

1. Fecal

2. Non-Fecal.

Fecal: Fecal Coli forms can survive at 45 °C.

Non Fecal: These Coli Forms can survive less than 40 °C.

Principle:

This method is used for the isolation of coli form organisms. It includes all aerobic and
anaerobic gram negative, non-spore forming rods able to ferment lactose with production of acid and
gas at 30°, 35° & 37°c within 48 hours.

Medium Used: Violet Red Bile media is not need of Auto clave. This media is boiled at 80-100 °C
and this media is used for only the growth of Bile salt utilized microbes.

Procedure:

> Take 1 Ml of the milk sample and transfer to 1st dilution blank.
> Vortex for few seconds, take lml of sample and transferred into sterile Petri
plate by using sterile pipette.
> About 15 -20 ml of Violet Red Bile Agar 45° C+0.5° C was poured into each
Petri plate.
> Contents were mixed gently clockwise and anti clock wise and allowed to
solidify.
> After complete solidification plates were inverted and incubated in the
incubator at 37° C for 24 hours.

Observation:

After 24 hours incubation the colonies were Observed and Counted by using colony Counter in each
plate.

Result: Calculation

No .of colonies * dilution factor CFU/m!

 BIO CHEMICAL TESTS

IMViC TEST

Identification of enteric bacteria is of prime importance in controlling intestinal

infections by preventing contamination of food and water supplies. The groups of bacteria that
can be found in the intestinal tract of the humans and lower mammals are classified as members
of the family Enterobacteriaceae. They are shot, gram negative, and non-spore forming
bacteria.

The bacteria included in this family are.

1. Pathogens such as members of the general salmonellaandShigella.

2. Occasional Pathogens such as Proteus and Klebsiella.
3. Normal intestinal flora such as Escherichia coli and Enterobacter.

> Differentiation of the principal groups of Enterobacteriaceaecan be accomplished on

the basis of their Bio-chemical properties and enzymatic reactions in presence of
specific substrates.

> The IMViC series of tests- Indole, Methyl red, VogesProskaeur and Citrate Utilization can
be used.

INDOLE TEST

Aim;

To determine the ability of microorganism to degrade the amino acid tryptophan.

Principle:

Tryptophan is decomposed into its metabolic products like Indole, pyruvic acid and
ammonia by the enzyme tryptophanase. The ability of these organisms to produce indole may be
used as one of the differentiation characteristics for Enterobacteriaceae. Indole is detected by
"adding Kovac's reagent, which produces a cheery red reagent are indole positive and absence of red
color indicates Indolenegative test.
 Procedure:

> Take 5ml of sterile peptone water into a test tube.

> Take a sterile loop and pick up 24 hrs cultured isolated colonies
> The testtubes were incubated for 24-48 hours at 37° C
> After the period of incubation few drops of Kovac's reagent was
added to each test tube.
> The color change in each tube was examined.

Interpretation;

* Indole positive : A red colour ring near the surface of the medium.
* Indole negative : A yellow coloured ring appears the surface of the
medium

METHYL RED TEST

Aim:

To differentiate between all glucose oxidizing enteric organisms particularlyA.coz7and

Enterobacteraerogens.

Principle:

Enteric organisms Oxidize- Glucose and the end products produced vary based on the
specific pathway of metabolism. Although all enteric forms glucose with organic acid as the end
product, the methyl red test is used to differentiate between E. coli and Enterobacteraerogens.

Both these organisms utilize glucose and produce organic acids as the end products during
early incubation periods. The low pH is stabilized ad maintained by E.coiklXlhQ end of incubation.
However Enterobacteraerogens convert these acids enzymatically to ethanol and acetion. The
non-acidic end products, which elevate the pH to 6.

Methyl-Red indicator will turn red in pH range 4.0 indicating a positive test. The pH
indicator turns yellow at pH 6.0 still indicating the presence of acid at low hydrogen ion
concentration and is a negative test.
Procedure:

> Using sterile technique, each experimental organism was inoculated into its
appropriately labeled deep tubes by means of stab inoculation.

> The last test-tube was kept inoculated which serves as a control.

> The test tubes were incubated for 24-48 hours at 370C.

> After the period of incubation few drops of Methyl Red indicator is added to
all the tubes.

The color change in each tube was examined

VOGES-PROSKAUER:

Principle:

This test is to determine the capacity of some organisms to ferment Carbohydrates with
production of non-acidic or neutral end products such as acetyl methylcarbonyl(or) its reduced
product 2.3-butylene glycol. These products are produced from organic acids that result from
glucose metabolism, which is a characteristic of Enterobacieraerogens. The positive tubes give a
crimson color to the media.

Procedure:

> Using sterile technique, each experimental organism was inoculated into its
appropriately labeled deep tubes by means of stab inoculation.

> The last test-tube was kept in inoculated which serves as a control.

> The test tubes were incubated for 24-48 hours at 37C

> After the period of incubation few drops of Barrits reagent was added io all the Tubes.

> The color change in each tube was examined.

 CITRATE UTILIZATION
TEST:

Aim:

To differentiate among enteric organisms on the basis of their ability to ferment citrate as
a sole carbon source.

Principle;

In the absence of glucose or lactose some microorganisms use citrate as the carbon source,
which depends on the presence of citrate permease. Citrate is acted upon by the enzyme citrate,
which produces oxalo acetic acid and acetate. These are then enzymatically converted into pyruvic
acid and carbon dioxide. During this reactionmedium becomes alkaline as carbon dioxide combines
with sodium and

changes the bromothymol from green to Prussian blue. In this test the organism utilize
citrate as a sole carbon and energy source for growth and Ammonium salt as the sole source
of nitrogen. Catalase
H2Q2 * H 2O + Vi O2 (Necent oxygen or singlet oxygen)

Procedure:

> Using sterile technique, each experimental organism was inoculated into its
Appropriately labeled Simmons citrate agar slants by means of stab and streak
inoculation.

> The last test-tube was kept uninoculated which serves as a control.

> The test tubes were incubated for 24-48 hours at 37C

> After incubation all the tubes were examined for the presence or absence of growth
and coloration of the medium.

Interpretation: Organisms that produce efferhances (bubbles) are catalase positive

 KB010 Hi.Coli™ Identification Kit

Introduction:

KB010 is comprehensive test system that can be used for identification and
differentiation of Escherichia Coli are gram negative , lactose fermenting coccid bacillary rods
which are frequently isolated from food, feces, water and other relevant clinical sa mples. Hie
Coli can be used for screening pathogenic organisms and can also be used for validating known
laboratory strains. The complete list of organisms that can be identified with this system is given in
the identification index provided with the kit

Principle:

Each KB010 kit is a standardized colorimetric identification system utilizing eight

conventional biochemical tests and four carbohydrates utilization tests. The tests are based on the
principle of p change and substrate utilization. On incubation E.Coli exhibit metabolic changes
indicate by color change in the media that is either visible spontaneously or after addition of a reagent.

Kit Contents

8. Each kit contains sufficient material to perform 10 tests.

9. 10 kits of KBO10.
lO.Technical product insert.
11 .Result interpretation chart and result entry datasheet.

12.Identification index.

IB.Baritt reagent A(R029) for voges-Proskauer's.

H.Bariit reagent B (R030) for voges-Proskauer's.

15.Methyl Red reagent (L007) for Methyl Red test.

16.Kovac's Reagent (R008) for Indole test.

17.Sulphanilic Acid 0.8% (R015) for Nitrate test.

18.N-N Dimethy-1-Napthylamine reagent (R009) for Nitrate test.

Storage and Shelf-life

Store at 2-8°c Shelf-life is

12 monthss
Instruction for Use

3. Preparation of inoculum

KB010 cannot be used directly on clinical specimens. The organisms

to be identified have to be first isolated and purified. Only pure
cultures should be used.
Isolate the organisms to be identified on a common medium like
Nutrient Agar (M001) or a different medium like MacConeyAgar
(M082).
Pick up a single well isolated colony and inoculate in 5ml Brain Heart
Infusion Broth (M210). Incubate for 4-6 hours till the density of the
inoculum is > 0.1 OD at 620nm or 0.5 Mcfarlandstandard.
Alternatively a homogeneous suspension adjusted to 0.1 OD at 620nm
or 0.5 Mcfarlandstandard can also be used for inoculation.

Note:

• Results are more prominent if enriched culture is used instead of

suspension
• Erroneous false negative results may be obtained if the individual
well with a loopful of inoculum
4. Inoculation of the strip

• Open the kit aseptically. Peel off the sealing foil.

• Inoculate each well with 50jul of the above inoculum by surface
inoculation method.
• Alternatively the strip can also be inoculated by stabbing each
individual well a loopful of inoculum.
5. Incubation

• Temperature of incubation 35°c ± 2°c

• Duration of incubation 18- 24 hours.

Interpretation of Results:

Interpret results as per the standards given in the result interpretation chart. Addition
of reagents in well#l,2 and 4should be done at the end of incubation period that is after 18-24 hours.
Following reagents to be added to the respective well
 Methyl Red Test :WellNo.l

• Add 2-3 drops of Methyl Red reagent (] 007).

• Reagent will be red in color if the test is positive.
• Reagent decolorizes and becomes yellow if the test is negative.

VogesProskaeur'sTest: Well No. 2

• Add 2-3 drops of Bariit reagent A(R02/}) and 1 -2 drops of Bariit reagent B
(R030).
• Pinkish red colour development within 5-10 minutes indicates a positive test.
• No change in colour or slight change in colour denotes a negative reaction.

Important points to be taken into consideration while interpreting the result

5. Allow the reagents to come to room temperature after removal from the
refrigeration.
6. In case of carbohydrate fermentation test some microorganisms show weak reaction.
In this case record the reaction as ± and incubate further up to 48 hours. Orange
color after 48 hours of incubation should be interpreted as a negative reaction.
7. At times organisms give conflicting result because of mutation or the media
used for isolation, cultivation and maintenance.
8. The identification index has been compiled from standard references and results of
test carried out in the laboratory.

Precautions:

• Clinical samples and microbial cultures should be considered potentially

pathogenic and handled accordingly.
• Aseptic conditions should be maintained during inoculation and handling of the strips.
• Reagents should not come in contact with skin, eyes or clothing.

Disposal of used material

• After use, strips and the instruments used for isolation and inoculation must be
disinfected and then discarded by incineration or autoclaving in a disposal bag
 ENUMERATION OF SPECIFIC PATHOGENS

Food borne Pathogens: Staphylococciis aureus, E.coli, Salmonella, Sheghilla, Lysteria

moncytogenes.

Enumeration of Staphylococcus aureus.

INTRODUCTION:

Staphylococcus aureus is Gram Positive, Non motile bacteria.

Principle:

Ma nni t ol S a l t A ga r Me di u m sp e ci f i c al l y s up po rt s t he gr owt h of

Staphylococcus aureus. The presence of golden yellow colonies after 24 hours of incubation

indicates the presence of staphylococcus aureus.

Procedure:

> Take lml of milk sample and transfer to Is1 dilution blank

> Vortex for 5-10 seconds, take lml and transferred into Petri plate

> Pour the media 15-20 ml aseptically

> Stir gently clockwise anti clockwise without splitting

> Allow to solidify for few mines.

> After solidify keep in incubator in invert position at 3 7 °C for 24 hours

Dilution:

Required dilution is 1st dilution.

Interpretation:

After 24 hour incubation Golden yellow color colonies are observed was positive
 Confirmation Test:

GramStaining:

It is a differentiate staining technique to differentiate the bacteria whether is Gram Positive

(or) Gram negative.

Procedure:

> Transfer a drop of water onto the slide.

> Pick out an isolated colony from the culture plate an spread it on a slide by means of a
circulatory motion with the inoculating lop.
> Allow the smear to air dry heat fix the smear.
> Flood it with primary stain (Crystal Violet) for 1 minute: Gently wash extra
stain. With tap water.
> Decolorize with 95% ethyl alcohol (add drop by drop until alcohol mn almost clear
showing only blue tinge). And gently wash with tap water.
> Counter stain with saffarrin tor 45 seconds.
> Gently wash with tap water.
> Blot dry with bibulous paper.
> Examine under oil immersion objective.

Observation:
When observe under oil emulsion if presence of Staphylococcus aureuswas appear like
purple colour grape 1 ike structure. _,-•.•

Biochemical test:

Catalase test:

Pick a Suspected colony and place on glass slide. Adcl3% of H 2O2

Observation:

Appearance of gas bubbles is indication of presence of Staphylococcus aureus.

Enumeration of Escherlchia coli

Introduction: E.coins a rod shaped gram -ve bacteria. It is motile.

 Principle:

EMB Medium specifically supports the growth ofE. Coli in the presence of green metallic

sheen indicates the presence ofE. coli.

Procedure:
 About 15 ml of EMB medium at 45°C + 0.5 °C was poured into each Petri plate.
 A loopful of organism was taken from the sample and streaked on the solidified
EMB agar plate.
 The plates were inverted and incubated in the incubator at 37 ° C for 24 hours.

Dilution:

Required dilution is 1st dilution.

Interpretation:

After 24hrs of incubation period green metallic sheen was positive.

Confirmation Test:

Gram Staining:

It is a differentiate staining technique to differentiate the bacteria whether is Gram Positive (or)

Gram negative.

Procedure:
> Transfer a drop of water onto the slide.
> Pick out an isolated colony from the culture plate an spread it on a slide by
means of a circulatory motion with the inoculating lop.
> Allow the smear to air dry heat fix the smear.
> Flood it with primary stain (Crystal Violet) for 1 minute: Gently wash extra
stain. With tap water.
> Decolorize with 95%-ethyl alcohol (add drop by drop until alcohol run almost

> clear showing only blue tinge). And gently wash with tap water.
> Counter stain with saffarrin tor 45 seconds.
A
Gently wash with tap water.

> Blot dry with bibulous paper.

> Examine under oil immersion objective.

Observation:
When observe under oil emulsion if presence of E.coli was appear pink, roads

Enumeration of salmonella

Introduction: It is a gram -ve rod shaped bacteria. Motile with peritrichate

flagella.

Principle:

Brilliant green agar medium specially supports the growth of Salmonella. The presence of red

color colonies indicates the presence of salmonella.

Procedure:

 lml of milk sample was transferred into a sterile Petri plate using a sterile

pipette.

 About 15 ml of Brilliant green agar medium at 45°C+0.5uC was poured into

each Petri plate.

 Contents were mixed gently and allowed to solidified.

 After complete solidification plates were inverted and incubated in the

incubator at 37°C for 24 hours.

Dilution:

Required dilution is 1st dilution

Interpretation:

After incubation colonies will not change from pink to red colour was Nagative.

Enumeration of LYSTERIAMONOCYTOSENS

Introduction:
It is Gram +ve bacteria, non motile.

Media Used:

Required media is Lysteria Oxford agar media.

Dilution :

Required dilution is 1SI dilution.

Procedure:

> 1 ml of milk sample was transferred into a sterile Petri plate using a sterile

pipette.

> About 15 ml of Brilliant green agar medium at 45°C+0.5°C was poured into

each Petri plate.

> Contents were mixed gently and allowed to solidified,

> After complete solidification plates were inverted and incubated in the incubator at

37°C for 24 hours.

Interpretation:

After 24 hrs incubation not black halo colour colonies was Negitive.
 ENUMERATION OF YEAST AND MOULDS

Required media
Rose bengal chlorophenicol
Dilution factor: 1st dilution

Procedure:
Take lml of sample from above dilution and aseptically transfer into petridish and then
add the media then mix well clock wise to anticlock wise direction then keep it in an
incubatorat250cfor 3-5 days.

Observation:
After 3days if presence of moulds black, white, yellow coloured mycelial growth is
observed yeast are like rice bean shaped when stained with lactophenol cotton blue stain.

OBSERVATION:1
SPC count on TGYA Medium and Coli form Count on VRBA Medium incubated at
37 °C:

SNo Sample Sample Standard for SPC result for Standard Coli form
volume SPC 48 hours for Coli results for 24
Cm/ml forms hours

1 Raw milk lml NMT5 2,90,000. Less than 60

lakhs 100
2 Pasteurized lml NMT30.0O0 16;000. ABSENT Nil
milk
3 Elester milk lml NMT1 Nill ABSENT Nil
4 Out let lml NMT 30,000 4000 ABSENT Nil
5 New silo lml NMT 30,000 13,000 ABSENT Nil
6 Storage tamker lmi NMT 30,000 10,0001 ABSENT Nil
7 Homogenized lml NMT 30,000 8,000 ABSENT Nil
8 Toned mild lml NMT 30,000 7,000 ABSENT Nil
 OBSERVATIONS

SPC count on TGYA Medium and Coli form Count on VRBA Medium incubated at
37 °C:

S.No Sample Sample Standard for SPC Standard Coli form

volume SPC result for 48 for Coli results for
Cm/ml hours forms 24 hours

i Raw milk lml 2,50,000. 45

N>IT5 Less than
lakhs 100
2 Pasteurized lml NMT30,000 10,000. ABSENT Nil
milk
3 Elester milk lml NMT1 Nil ABSENT Nil

4 Out let lml NMT 30,000 3,000 ABSENT Nil

5 New silo lml NMT 30,000 8,000 ABSENT Nil

6 Storage tamker lml NMT 30,000 11,000 ABSENT Nil

7 Homogenized toned 1ml NMT 30,000 7,000 ABSENT Nil

milk
8 Toned mild lml NMT 30,000 10,000 ABSENT Nill

OBSERVATIONS

SPC jount on TGYA Medium and Coli form Count on VRBA Medium incubated at
37 °C:

S.No Sample Sample Standard for SPC Standard Coli form

volume SPC result for 48 for Coli results for 24
CFU/ml hours forms hours

.1 Raw milk lml NMT5 1,50,000. Less than 20

lakhs 100
2 Pasteurized lml NMT30.000 12,000. ABSENT Nil
milk
3 Elester milk lml NMT 1. Nil ABSENT Ml
4 Out let lml NMT 30,000 2,000 ABSENT Ml
5 New silo lml NMT 30,000 9,000 ABSENT Nil
6 Storage tamker lml NMT 30,000 10,000 ABSENT Nil
7 Homogenized lml NMT 30,000 7,000 ABSENT Nil
toned milk
8 Toned mild lml NMT 30,000 8,000 ABSENT Nil

Biochemical Tests:

Observation:

Name of the test

Catalse Indole MR VP Citrate

Organism Gram MORPHO

Mature LOGY

Staphylococcus Positive purple + 1+ +

Aureus colour
bunch
(grape)
like cocci

E.coli Negative Pink Note + +

colour done
Small
Rods

i Salmonella Negative Rods Note + +

done

Lysteria positive Purple Note + + - +

monocytogenes colour done
rods
 OBSERVATION FOR SPECIFIC PATHOGENS

SKo Test Medium Sample Observation

Source

1 Salmonella BGA Raw milk absent

2 Salmonella Test BGA Pasteurized Absent

milk
3 Salmonella Test BGA Pasteurized Absent
homogenized
toned milk
4 Staphylococcus Test MSA Raw milk Golden
Yellow
Colonies
Seen
5 Staphylococcus Test MSA Pasteurized milk Absent
;
6. Staphylococcus Test MSA Pasteurized Absent
homogenized
.oned milk
7. Lystenamonocytogens LysteriaOx Raw milk Absent
brd media

8 Lystenamonocytogens Lysteria Pasteurized Absent _

Oxford milk
media
9 Lysteriamonocytogens LysteriaOx Pasteurized Absent
brd media lomogenizcd
toned milk
10 E.coli EMB law milk Green
metallic
sheen seen
11 E.coli EMB pasteurized Absent
12 E.coli EMB Pasteurized Absent
lomogenized
oned milk
 From the above test for specific pathogens of Raw miik sample was found to be
positive for staphylococcus, E.coli .whereas Pasteurized milk and Pasteurized
homogenized toned milk are negative for test pathogens.

Biochemical Tests:

Observation:

Organism Gram
Mature MORPHO
LOGY Catalse Indole VP Citrate
MR

Positive Cocci + + +
Staphylococcus

aureus
E.coli Negative Rods + +
Note

done
 RESULTS

The morphological characterization was done by colony morphology on nutrient

agar and specific media like Eosine Methylene blue agar and Brilliant green agar and Tryptone
soya yeast extract agar, gram staining indicates Si and S2 were stained pink in color by absorbing
counter stain safronin and are gram negative organisms.

The results were tabulated and biochemical characterization was done .Catalase test
showed positive towards all isolates which implies that these release an enzyme catalase which .
breaks down I-^Ch.to water and oxygen giving rise to bubble formation on the slide.

Methyl red showed positive result towards S1, S2- voges-proskeur test was negative
towards S I, positive S2 isolates, citrate utilization test showed negative results towards S|, S2.

utilize sodium citrate present in the medium as carbon sources. SI shows negative for citrate

utilization which does not changes the colour of indicator from green to blue.

By all these above morphological, staining and biochemical tests the selected organisms

identified as E.coli, S.aureus.

 DISCUSSION

A series of studies were carried out on the various microbiological parameters of a local milk
product collected from Kostha Andhra regions of Visakhapatnam .Viable counting requires
incubation time, it may be necessary to determine the total count to estimate the potential
viable count. The total count also gives an estimate of the total number of microorganisms to
which a substance has been exposed. The highest bacterial count was found in the sample
from West Godavari, East Godavari, Srikakulam, while the lowest was from the
Vizayanagaram, Visakhapatnam.

Total coliform count indictes the hygienic standard and the storage quality of the milk
product, rather than the presence of human pathogens. Special care is required during the
production and handling of the milk-based products in order to lower the coliform count.

SUMMARY AND CONCLUSSION

The Selective Pathogenic microorganisms Staphylococcus aureus, E.Coli, and were

screened in Raw milk. The Selective Pathogenic microorganisms, which were observed in Raw

milk and Pasteurized milk samples, were found to be negative for pasteurized homogenized

toned milk . Though colonies were observed for standard plate count and coli form count the

values were well below the standard permissible values.

The absence of pathogenic microorganisms is treated and processing milk samples reflect
the proper hygienic conditions available in Visakha Dairy, milk treated can be used in the
processing section and the products are safe for consumption.