1

NH No.: 8, Village: Dhamdod, Ta. Mangrol, Near Kosamba, Surat – 394 125. (GUJARAT)

LAB MANUAL

INSTITUTE NAME : SCHOOL OF SCIENCES

DISCIPLINE : MICROBIOLOGY

COURSE : [Link]. (H.)

SUBJECT CODE : SSMB3110

SUBJECT NAME : FOOD & DAIRY MICROBIOLOGY PRACTICAL

2
 NH No.: 8, Village: Dhamdod, Ta. Mangrol, Near Kosamba, Surat – 394 125. (GUJARAT).

Index

S. No. Name of Practical PageNo.

1 MBRT of milk samples and their standard plate count. 3
2 Alkaline phosphatase test to check the efficiency ofpasteurization of milk. 7
3 Isolation of any food borne bacteria from foodproducts. 9
4 Isolation of spoilage microorganisms from spoiled vegetables/fruits. 12
5 Isolation of spoilage microorganisms from bread. 14
6 Preparation of Yogurt/Dahi. 16

3
 Experiment No. 1

OBJECTIVE (AIM):

To perform the MBRT test for Milk samples and StandardPlate Count.

INTRODUCTION:

Milk is a good medium for the growth of microorganism. A variety of microorganism can be
found in both raw milk and pasteurized milk. These actively growing microorganisms reduce the
oxidation reduction potential of the milk medium due to the exhausted oxygen by the
microorganism. Normally the milk is contaminated with microorganisms such
as Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeroginosa, Enterobacter
spp., Bacillus spp., Paenibacillus spp., etc. Contaminated milk is one of the important sources
for transmission of diseases from animals to humans. The main reason for this contamination is
the un-proper handling of milk. Normally milk is contaminated during the milking process by the
microorganisms present in the exterior surface of the animals, pipelines such as udder and
adjacent areas. Unsterilized dairy utensils such as milking machines, milk cans are also a good
source of contamination by the microorganism. The formation of Methylene blue reductase is
thus becoming a popular tool for determining the quality of the milk.

PRINCIPLE

The principle of methylene blue reduction test MBRT depends on the fact that the color
imparted to the milk by adding a dye such as methylene blue will disappear more or less quickly,
which depends on the quality of the milk sample to be examined. Methylene blue is a redox
indicator, that lose its color under the absence of oxygen and is thought to be reduced. The
depletion of oxygen in the milk is due to the production of reducing substances in the milk due
to the enhanced rate of bacterial metabolism. The dye reduction time refers to the microbial load
in the milk and the total metabolic reactions of the [Link] the number of
organisms present in milk and greater their activity, more rapidly the dye is reduced.
4
MATERIAL REQUIRED:
1. 3 sterile screw capped 10 ml tubes
2. Sterile 10 ml and 1 l pipettes.
3. Controlled water bath at 37c and boiling water bath
4. Milk Sample
5. Standard Methylene Blue Solution (1:20,000 dilution)
6. Clock
7. Sterile Distilled water

PROCEDURE
1. Mix the Milk sample thoroughly.
2. Using sterile pipettes prepare and label 3 sterile screw capped tubes as:
(i) Positive control = 10 ml milk + 1 ml methylene blue
(ii) Negative control = 1o ml milk + 1 ml sterile D/W
(iii) Test = 10 ml milk + 1 ml methylene blue
3. Close the screw cap tightly and mix the contents by inverting the tubes once or twice.
4. Place the control tubes in boiling water bath for 3 mins to destroy natural reducing
system of milk.
5. Place all the three tubes in waterbath at 37c and note down the time.
6. Examine tubes at every 30 minutes interval and if no color change has occurred , tubes
were slowly inverted once to mix contents.
7. Decolorization is said to be complete when whole column of milk is decolorised upto 5
mm surface. Decolorised milk in test is to be compared with color of negative control in
case of doubt.
8. Record the time of milk decolorization and conclude:

5
Results and Interpretations

MBRT Time (hrs) Quality of Milk

≥5 Excellent
4-5 Very good
3-4 Good
1-2 Fair
≤1 Poor

Indications of Milk spoilage

The milk contains energy sources such as lactose (sugar), nitrogenous compounds such as
proteins, amino acids, ammonia, urea etc. for the growth of microorganism. Acid fermentation
by the bacteria is common under ordinary conditions. Souring of milk indicates the milk is
spoiled. Acid formation in the milk is indicated by the sour flavor, coagulation of milk to give a
jelly like curd appearance or clear whey nature. Lactic acid fermentation is common in the raw
milk at the room temperature. At temperature from 10 to 37 o C souring is mainly due
to Streptococcus lactis, Enterococci, Lactobacilli and other coliform bacteria. At temperatures
o
from 37 to 50 C the most common contaminants of milk are S. faecalis and S. thermophilus.
Thermophilic bacteria such as L. thermophilus can grow in the milk at higher temperatures.
Pasteurization is an important process to kill most acid producing microorganisms while
permitting the growth of heat resistant microorganisms such as Streptococcus
thermophilus and Lactobacilli. Other acid producing microorganisms are Micrococcus
species, Bacillus species (mainly lactic acid) and Clostridium species (mainly Butyric acid).
Microorganisms such as Clostridium spp. and Bacillus spp. can also produce gases such as
hydrogen and carbon dioxide which can be indicated by the formation of foaming at the top of
the milk suspension. The other way of milk contamination is the hydrolysis of milk proteins by
the growing microorganisms. The release of peptides in the milk leads to a bitter flavor to the
milk. Ropiness, sliminess in the milk, is caused by the release of slimy capsular material from
the cells. Enterobacter aerogenes, E. coli, Micrococcus freudenreichii are examples of
microorganisms that can cause ropiness in the milk. Oxidation of unsaturated fatty acids can also
6
lead to change in odor and taste of milk. Production of alkaline products such as ammonia, urea,
carbonates etc can also produce off flavors to milk.

FIGURE:

RESULT:

7
 EXPERIMENT NO: 2

OBJECTIVE (AIM):

To Study Alkaline Phosphatase test to check efficiency ofPasteurization of Milk.

INTRODUCTION
Pasteurisation is an essential process in the production of milk which is safe and free from
pathogens. Alkaline Phosphatase is an enzyme which is naturally present in milk, but is
destroyed at a temperature just near to the pasteurization temperature. Alkaline Phosphatase test
is used to indicate whether milk has been adequately pasteurised or whether it has been
contaminated with raw milk after pasteurisation. This test is based on the principle that the
alkaline phosphatase enzyme in raw milk liberates phenol from a disodium para-nitro phenyl
phosphate and forms a yellow coloured complex at alkaline pH. The intensity of yellow colour
produced is proportional to the activity of the enzyme. The colour intensity is measured by direct
comparison with standard colour discs in a Lovibond comparator. The test is not applicable to
sour milk and milk preserved with chemical preservatives.

MATERIAL REQUIRED
1. Water-Bath -maintained at 37±l⁰C, thermostatically controlled.
2. Comparator - with special discs of standard colour glasses calibrated in µg p-nitrophenol per
ml milk, and 2 x 25 mm cells.
3. Test Tubes - of size 16 x 1.50 mm and rubber stoppers to fit.
4. Pipettes - 1, 5, and 10 ml.
5. Filter Paper - Whatman No. 2 or equivalent.
6. Litmus Paper

8
REAGENTS
1. Sodium Carbonate-Bicarbonate Buffer - Dissolve 3.5 g of anhydrous sodium carbonate and
1.5 g of sodium bicarbonate in one litre of distilled water.
2. Buffer Substrate - Dissolve 1.5 g of disodium p-nitrophenyl phosphate in one litre of sodium
carbonate-bicarbonate buffer. This solution is stable if stored in a refrigerator at 4°C or less for
one month but a colour control test should be carried out on such stored solutions

PROCEDURE
1. Pipette 5 ml of buffer substrate into a clean, dry test tube followed by 1 ml of the milk to be
tested. Stopper the tube, mix by inversion and place in the water-bath
2. At the same time place in the water-bath a control tube containing 5 ml of the buffer
substrate and 1 ml of boiled milk of the same kind as that under test that is pasteurized
homogenized, low fat.
3. After 2 hours, remove the tubes from the bath, invert each and read the colour developed
using the comparator and special disc, the tube containing the boiled milk control being placed
on the left of the stand and the tube containing the sample under test on the right. Record
readings which lie between two standard colour discs by adding a plus (+) or minus (-) sign to
the figure of the nearest standard.

RESULT:

9
 EXPERIMENT NO: 3

OBJECTIVE (AIM):

To Study and Isolate Food borne bacteria from foodproducts.

INTRODUCTION:

Food Products serve not only as sources of nutrition for humans and other animals but also as
substrate for growth of microorganisms. Many foods serve as an ideal cultural media.
Microorganisms produce both desirable (flavour, aroma, taste, preservation) as well as
undesirable changes (decreased nutritional value, unpleaseant appearance, altered taste, odor,
color, texture). Microorganisms are the primary cause of food spoilage and food borne illness.
Food borne diseases are globally important, as they result in considerable morbidity, mortality,
and economic costs. Many different sources like bacteria, viruses, parasites, chemicals, and
prisons, may be transmitted to humans by contaminated food. Outbreaks and sporadic cases of
food borne disease are regular occurrences in all countries of the world. However, the statistical
data of food borne illness is increased due to the unrecognized or unreported outbreaks
particularly in the developing countries. There has been a continuous increase in several food
borne diseases caused by bacterial pathogens such as Salmonella, Campylobacter, [Link],
Listeria, [Link], Klebsiella and Pseudomonas. These pathogens come into contact with foods
during harvest or slaughtering, processing, storage and packaging. Environmental challenges
have caused food-borne bacterial pathogens to evolve and the susceptibility of human population
to infections. Generally, food borne diseases are associated with acute, mild and self-limiting
gastroenteritis with symptoms such as nausea, vomiting and diarrhea as a consequence of
consumption of microbial contaminated food and number of chronic sequences may result from
food borne.

Moreover, food products are at risk of contamination with mishandling during processing and
preparations where there are favorable conditions for pathogens and also responsible for causing
spoilage to the edible portion of the crop. In most of the cases food like dairy product, fish and
poultry product get contaminated while handling of the product, harvesting and processing
10
equipment and transport. Food products may become contaminated at different stages along the
food chain, from growth or production until the final consumption. In most of the cases
microorganism like [Link] in meat product, drinking water and dairy product; Salmonella,
[Link] in meat, eggs, on vegetables and poultry; Listeria in dairy product and shellfish;
Pseudomonas on fruit, dairy product, poultry and in drinking water.

COLLECTION OF FOOD SAMPLE

Clean and sterile Screw Capped jars or plastic bags are commonly used for food sample
collection. From bulk food material, small sample is collected with help of sterile spatula. Jars
are carried to laboratory. Samples are placed in refrigerators and examined at the earliest.

PREPARATION OF FOOD SAMPLE

1. In case of Solid food 10gm of food is homogenised aseptically in 90 ml of 0.1 % peptone

water with help of blender.
2. Food sample prepared in this manner represents 1 : 100 dilutions of food.
3. 1ml contains 0.1 gm of sample.

REQUIREMENTS
1. 5 MLBB (Macckoney’s Lactose Bile Broth) tubes each having 10 ml double strength 2x
medium.
2. 11 MLBB tubes each having 5ml single strength X medium.
3. Sterile 10 ml and 1 ml pipettes
4. Food Sample to be tested.

PROCEDURE
1. Shake the food sample vigorously to ensure uniform distribution of organisms.
2. Dilute the sample by transferring 10 ml of food sample by sterile pipette to 90 ml sterile
D/w. This makes 10-1 dilution. Make more dilutions if needed.
11
 3. With sterile graduated pipettes inoculate the food sample as follows:

(i) 5 tubes of MLBB having 10 ml 2x medium with 10 ml of food sample.

(ii) 5 tubes of MLBB having 5 ml X medium with 1 ml of food sample.
(iii) 5 tubes of MLBB having 5 ml X medium with 0.1 ml of food sample.
4. One tube of MLBB having 5ml X medium is left uninoculated which serves as control.
5. Incubate all tubes at 37c for 24 hrs.
6. Examine the tubes for acid and gas production after 24 hrs.\
7. If no tube shows acid and gas production, inoculate for other 24 hrs.
8. At the end of incubation period, record the number of positive tubes in each of three sets
and interpret the result as per McCrady table.

RESULT

12
 EXPERIMENT NO: 4

OBJECTIVE (AIM):

To isolate and study spoilage microorganisms from spoiledvegetables/fruits.

INTRODUCTION:

Fruits play a vital role in human nutrition by supplying necessary growth factors such as
vitamins and essential minerals in daily diet which help to live a healthy life. The succulent
nature of fruits and vegetables makes them to be easily invaded by microbes. The high
concentration of various sugars, minerals, vitamins and amino acids also provide a good platform
for the successful growth and survival of various microorganisms. Most microorganisms that are
initially observed on whole fruit or vegetable surfaces are soil inhabitants. Spoilage refers to any
change in the condition of food in which the food becomes undesirable or unacceptable for
human consumption. Bacterial spoilage first causes softening of tissues as pectins are degraded
and the whole fruit may eventually degenerate into a slimy mass. Starch and sugars are
metabolized next and unpleasant odours and flavours develop along with lactic acid and ethanol.
Some spoilage microbes are capable of colonizing and creating lesions on healthy, undamaged
plant tissue. Present investigation was carried out to study the presence of various bacteria
responsible for the post harvest decay and deterioration of economically important fruits.

Requirements
1. Various fruit samples

2. Various Vegetable samples

3. Plastic zip bags

4. Nutrients Agar Plates

5. Potato Dextrose Agar Plates

6. D/W

7. Spatulla
13
 PROCEDURE

1. Various unwashed and unprocessed fruits and vegetables were collected in plastic zip bags.

2. The microorganisms were isolated from fruits and vegetables by using serial dilution
agar platemethod.
3. Spoiled fruits and vegetables were crushed crushed into presterilized mortar and
pestle with distilled water to form suspension, which was serially diluted from 10-1 to 10-
5 dilutions.
4. 100µL of food suspension from each dilution was spreaded over Nutrient Agar Medium
(NAM)plates and Potato Dextrose Agar Plates (PDA).
5. The NAM was supplemented with amphotericin B (10µg/mL) before pouring to
prevent fungal growth.
6. The inoculated petriplates were incubated at 370C for 24 hours for bacterial growth.

7. After incubation the morphologically different colonies of bacteria and fungi were
isolated and subcultured.

RESULT

14
 EXPERIMENT NO: 5

OBJECTIVE (AIM):

To isolate and study spoilage microorganisms from bread

INTRODUCTION:

Bread is a food product that is universally accepted as a very convenient form of food that has
desirability to all population rich—and poor, rural and urban. Its origin dates back to the
Neolithic era and is still one of the most consumed and acceptable staple in all parts of the
world. It is a good source of nutrients, such as macronutrients and micronutrients that are all
essential for human health (Potter and Hotchkiss 2006). Bread and other bakery products are
subjected to various spoilage problems, viz., physical, chemical and microbial; the latter is the
most serious one particularly bacterial (Bacillus sp.) and mold growth. Various molds involved in
spoilage of bread include Rhizopus, Mucor, Penicillium, Eurotium, Aspergillus and Monilia.

REQUIREMENTS

1. Bread Loaves from different shops

2. Polythene bags

3. Potato Dextrose Agar Plate

4. Dilution tubes

5. D/W

15
PROCEDURE

1. The 10 loaves of bread used in this study were purchased from different shops in the local
market.
2. The samples collected were brought in sterile polythene bags to the laboratory for analysis.

3. These were exposed to the laboratory environment for 7 days.

4. Potato Dextrose Agar (PDA) which is a common medium to grow fungi was used in this
study.

5. 1g bread sample was mixed with distilled water and a homogenate was prepared.

6. Dilution plate method was carried out to enumerate the fungi.

7. The working surface was sterilized using ethanol.

8. 1ml was taken from the above homogenate.

9. Serial dilution was done at the recommended dilution rate i.e 1:10 (1+9). Dilution was done
using saline water. 0.1 ml of the inoculum was added on the surface of the PDA medium and
spreaded evenly over the surface using a sterile spreader .
10. The plates were incubated in an upright position at 30˚C for 4-5days.

11. The same procedure was carried out for all the samples.

12. The fungal growth was observed and recoreded.

13. A small portion of each sub-cultured colony was cut using a sterile scalpel.

16
14. It was placed on a sterile glass slide using a sterile forceps. The slide was covered with a
cover slip and placed in a petridish. Similar procedure was carried out for other fungal
colonies as well..These petridishes were left at 30 ˚C for 5days. The cover slips were taken
with forceps and placed on slides containing cotton blue. The excess stain was removed and
observed under the microscope. The morphology ie shape, structure of conidia,
conidiophores, pigmentation, shape of sporangia, sporangiophores were recorded.

RESULT

17
 EXPERIMENT NO: 6

OBJECTIVE (AIM):

To prepare Yogurt/Dahi

INTRODUCTION:

The production of yogurt from the fermentation of milk is an ancient practice that requires a
combination of two or more starter cultures such as Streptococcus thermophilus and
Lactobacillus bulgaricus. These Gram-positive thermophilic organisms ferment the sugar lactose
to lactic acid via glycolysis. The lactic acid makes the yogurt tart, discourages the growth of
pathogens, and prevents spoilage. Lactic acid also causes the major milk protein, casein, to form
a solid curd, which results in a thick texture. Lactose is a disaccharide composed of glucose and
galactose. S. thermophilus does not possess the enzymes needed to metabolize galactose, and L.
bulgaricus preferentially metabolizes glucose. This results in an accumulation of galactose,
which adds sweetness to the yogurt. Other components of milk are converted to products such as
acetaldehyde, diacetyl, and acetate that, together with the lactic acid and galactose, give yogurt
its characteristic flavor and aroma. Some strains of S. thermophilus also produce glucose
polymers, which result in a viscous consistency. Type of Milk You will be assigned one of
several different types of milk. Whole fat cow milk will usually produce a mild tasting and firm
yogurt. Nonfat cow milk will produce a more tart and less firm yogurt. Milk from grass fed cows
has a different composition than that of grain fed cows. Goat milk contains less lactose and more
calcium/potassium than cow milk; it is less allergenic and will produce a lighter yogurt with a
stronger flavor. Plant-based milks (almond, soy, coconut, hemp, rice) lack lactose and have less
protein than cow milk; they may require the addition of thickeners such as guar gum or gelatin to
reach the desired consistency. In this exercise you will make highly edible yogurt, observe the
effect of a starter culture on milk, and compare the quality and flavor of yogurts made with
different types of milk.

18
REQUIREMENTS

1. 2 Quarts of Milk (Whole, reduced fat or nonfat cow, goat, or plant-based milk)

2. 5-10 grams of Yogourmet freeze-dried starter culture containing

a. L. bulgaricus, S. thermophilus, and L. acidophilus

3. Ice water bath and thermometer

4. Vessel, spatula, spoon, small bowl/measuring cup, and whisker

PROCEDURE

1. Pour 2 Quarts (1/2 Gallon) of milk into Vessel.

2. Slowly heat the milk over medium fire (not so hot that it burns on the bottom) to a
temperature of 180-195o F (82-90o C) while stirring constantly.
3. Turn off the heat, place the lid on the Vesse and allow the milk to sit for 10 minutes. Do
not allow the milk to boil over.
4. Carefully Cool the vessel and the contents.

5. Dissolve 5-10 grams (one-two envelope sections) of the Yogourmet freeze-dried starter
culture into a small amount (e.g.: 1 cup) of the cooled milk in a bowl.
6. Once the culture dissolves completely, add to the rest of the milk and mix in thoroughly.

5. Pour the milk into clean glass vessels for incubation period.

6. Incubate the covered glass jars for 6-12 hours at 110-115o F (43-46o C) and allow to set.

7. Disturbing the yogurt during early incubation will interfere with proper setting.

8. After the incubation period, refrigerate it.

RESULT

19