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Jagannath University
Department of Microbiology

Laboratory Note Book

Course title: Practical
Course code: MIB-3607

INDEX
Serial Experiment Name Page
No. Number

1. Microbial Analysis of Soil 2-5

2. Microbial Analysis of Frozen Food (Ice Cream) 6-8

3. Dough Fermentation By Yeast 9-11

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Experiment No: 01 Date: 31-07-18

Name of The Experiment: Microbial Analysis of Soil.

Principle

Soil contains myriads of microorganisms, including bacteria, fungi, protozoa,

algae, and viruses. The most prevalent are bacteria, including the
actinomycetes and fungi. It is essential to bear in mind that the soil
environment differs from one location to another and from one period of time
to another. Therefore, factors such as moisture, pH, temperature, gaseous
oxygen content, and organic and inorganic composition of soil are crucial in
determining the specific microbial flora of a particular sample. Just as the soil
differs, microbiological methods used to analyze soil also vary. A single
technique cannot be used to count all the different types of microorganisms
present in a given soil sample because no one laboratory cultivation procedure
can provide all the physical and nutritional requirements necessary for the
growth of a greatly diverse microbial population. In this experiment, only the
relative numbers of bacteria will be determined through the serial dilution–
agar plate procedure.

Materials

1. Soil Sample Preparation: 1 g sample of finely pulverized, rich garden soil

in a flask containing 99 ml of sterile water; flask labeled 1:100 dilution
(10−2).
2. Equipment: Autoclave, Flasks, Pipette, Petri dish, Incubator, Bunsen
burner, glassware marking pencil.
3. Reagents: Nutrient agar and 99-ml flasks of sterile water.

Procedure
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1. The nutrient agar was liquefied in an autoclave or by boiling and maintain in

a water bath at 45°C.
2. The Petri dishes was labeled using a glassware marking pencil as 10−4, 10−5,
10−6, and 10−7 (to be used for enumeration of bacteria).
3. With a glassware marking pencil, the soil sample was labeled as Flask 1, and
the 99-ml sterile water Flasks 2 and 3.
4. The provided soil sample dilution of 1:100 (10−2) approximately 30 times was
vigorously shaken.
5. With a sterile 1-ml pipette, 1 ml of the provided soil sample dilution was
transferred to Flask 2 and shake vigorously as before. The final dilution is
1:10,000 (10−4).
6. Using another sterile 1-ml pipette, 1 ml of Dilution 2 was transferred to Flask
3 and shaken vigorously as before. The final dilution is 1:1,000,000 (10−6).
7. Using sterile 1-ml pipettes and aseptic technique, the proper amount of each
dilution was added into each Petri dish as indicated.
8. Transfer 1 ml of Dilution 2 into plate to effect a 10−4 dilution.
Transfer 0.1 ml of Dilution 2 into plate to effect a 10−5 dilution.
Transfer 1 ml of Dilution 3 into plate to effect a 10−6 dilution.
8. Using the pour-plate technique, the liquefied agar was poured into the
plates and rotated gently to ensure uniform distribution of the cells in the
medium.
9. The plates were incubated in an inverted position at 37ºC for 24 hours.
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OBSERVATIONS AND RESULT

1. Using an electronic colony counter or by hand, all the colonies were

observed on each nutrient agar plate 2 to 3 days after incubation begins.
Plates with more than 300 colonies cannot be counted and should be
designated as too numerous to count (TNTC); plates with fewer than 30
colonies should be designated as too few to count (TFTC) plates with
between 30 and 300 colonies were only counted.
2. The number of organisms per milliliter of original culture on all plates
other than those designated as TFTC or TNTC by multiplying the number
of colonies counted by the dilution factor.
3. Cell counts per gram of sample were calculated in the following chart:
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Dilution Number of colonies Organisms per gram of soil

Bacteria

Discussion
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Experiment No: 02 Date: 31-07-18

Name of The Experiment: Microbial Analysis of Frozen

Food (Ice Cream).

Principle

Ice cream is a nutritious food which contains significant quantity of milk,

saturated fat, protein and calcium as well as vitamin C. Ice cream is consisting
of milk, sweetening and stabilizing agents together with flavoring and coloring
matter. The ingredients of ice cream may have different combinations of milk,
cream, evaporated or condensed milk, dried milk, coloring materials,
sweetening agents, eggs and egg items, flavors, fruits, nuts, and stabilizers.
However any of those ingredients may contribute microbial development and
adversely affect the microbiological quality of the product which is judged by
its bacterial load. Also the high content of nutrients like lactose, proteins and
its neutral pH make it an excellent growth medium for microorganisms some of
which may cause serious disease outbreaks like Cholera, typhoid, bacillary
dysentery in human beings. Many psychrophiles and psychrotolerant
microorganisms like Listeria monocytogens, Staphylococcus aureus, Bacillus sp.,
Salmonella sp., Shigella species, Streptococus spp., Pseudomonas spp.,
Campylobacter spp., Brucella spp. and coliform bacteria are generally present
in ice cream. The presence of these microorganisms in pasteurized ice cream
indicates their capacity to survive the pasteurization process as in the case with
spore formers and they may persist in ice cream product thereafter. Coliforms
were generally used as indicator microorganisms which demonstrate potential
fecal contamination of food and water. Possible sources of these
microorganisms in ice cream have been documented to include raw materials
used for the preparation of ice cream mix, such as milk and milk powder,
cream, flavoring and coloring substances and stabilizer and from contaminated
air during processing. The consumption rate of ice-cream is higher among
children of vulnerable age groups therefore it is essential to maintain a high
microbiological safe standard.

Materials
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1. Sample: Ice cream samples were purchased from local markets of Dhaka
city. The samples were brought to the laboratory immediately for proper
analysis.
2. Reagents: Nutrient agar and 9 mL distilled water test tubes.
3. Equipments: Autoclave, Flasks, Pipette, Petri dish, Incubator, Bunsen
burner, glassware marking pencil.

Procedure

1. 1ml of each sample transferred into a sterile test tube containing 9 ml of

peptone water to give a dilution of 1: 10, homogenized by vortex and
dilutions up to 10-5 were made.
2. Total viable count (TVC) was determined on nutrient agar by pour plate
method.
3. 1 ml of each of this dilution was inoculated on nutrient media and
incubated at 37⁰C for 24-48 hours. Following incubation, plates
exhibiting 30-300 colonies were counted.
4. The numbers of colony forming unit (cfu/ml) of each ice cream sample
for the observation of presence of pathogenic bacteria was recorded.
The bacteria plate counts per ml were recorded using the following
formula:

Where, n= Number of colonies developed on the media

r = Total dilution
v = Volume of the particular dilution being put on the media.

Observation and Results

1. Using an electronic colony counter or by hand, all the colonies were

observed on each nutrient agar plate 2 to 3 days after incubation begins.
Plates with more than 300 colonies cannot be counted and should be
designated as too numerous to count (TNTC); plates with fewer than 30
colonies should be designated as too few to count (TFTC) plates with
between 30 and 300 colonies were only counted.
2. The number of organisms per milliliter of original culture on all plates
other than those designated as TFTC or TNTC by multiplying the number
of colonies counted by the dilution factor.
3. Cell counts per mL of ice cream sample were calculated in the following
chart:
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Dilution Number of colonies Organisms per mL of

sample

Discussion
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Experiment No: 03 Date: 31-07-18

Name of The Experiment: Dough Fermentation By

Yeast.

Principle
The use of yeast as a leavening agent in baking dough back to very early
histories of the Egyptians, Greeks, Romans. In modern baking practice, pure
culture of selected strains of Saccharomyces cerevisiae mixed with the bread
dough to bring desired change in texture & flavor. Saccharomyces cerevisiae
strains have the ability to ferment sugar in dough fermentation & to grow
rapidly. The CO2 produced during the fermentation is responsible for the
leavening or rising of the dough.
Yeast
C6H12O6 ──────────► C2H5OH + 2 CO2
Enzyme

Requirements
• Flour
• Yeast (Saccharomyces cerevisiae)
• Water
• Test tube
• Glass slide

Procedure

1. Flour and yeasts were mixed with water to prepare dough.

2. Another type of dough was prepared with flour water but without yeast.
3. Then both of the yeast containing (test) dough & without yeast (control)
dough were left for observation.
4. Then observed the physical changes both of the dough.
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Observation and Results

Discussion