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1. GOVT. NAGARJUNA P.G. COLLEGE OF SCIENCE
RAIPUR [C.G.]
M.Sc. MICROBIOLOGY
2023 -2024
SEMESTER – III
LAB COURSE I
MICROBIAL PHYSIOLOGY
&
MICROBIAL GENETICS
SUBMITTED TO SUBMITTED BY
DEPT. OF MICROBIOLOGY SNEHA AGRAWAL

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INDEX
MICROBIAL PHYSIOLOGY
SIGNATURE :-
1. QUALITATIVE ASSAY OF DIFFERENT EXTRA CELLULAR
ENZYMES
 Lipase
 Cellulase
 Amylase
 Gelatinase
2. Antibiotic sensitivity test.

3. INDEX
MICROBIAL GENETICS
SIGNATURE:-
1. DETERMINATION OF MIC FOR DIFFERENT ANTIBIOTICS.
2. ISOLATION OF ANTIBIOTIC RESISTANT BACTERIAL
POPULATION BY GRADIENT PLATE METHOD.
3. THE EFFECT OF ULTRAVIOLET LIGHT ON BACTERIAL
GROWTH.

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PAPER I :- MICROBIAL PHYSIOLOGY
AIM – QUALITATIVE ASSAY OF DIFFERENT EXTRA-CELLULAR ENZYMES.
LIPASE PRODUCTION TEST
REQUIREMENT - Soil sample of oil contaminated areas or pure culture of E-coli
unknown bacterial or fungal culture tributyrin or tween so broth and agar medium, sterile
petri plate, centrifuge, Ammonium sulphate, micropipette.
PRINCIPLE - Lipase production test is based on the principle that lipolytic micro-
organism show a zone of lipolytic i.e. a clear surrounding the growth when inoculated on
nutrient agar medium supplemented with the lipid substrate as triglyceride tributyrin.
1) The loss of opacity (clear area) of the agar medium is due to hydrolytic reaction
yielding soluble glycerol and fatty acid.
2) The ability of M.O. to hydrolyze lipid is accomplished with an extracellular enzyme
lipase.
3) In this reaction triglyceride is spilt by the addition of water to form glycerol and three
fatty acid molecules.
4) The glycerol and fatty acid produced in this reaction are assimilated by the organism
to synthesize fats, other cell components and production of cellular enzyme adenosine
triphosphate (ATP) under aerobic conditions.
PROCEDURE
(A) Primary screening –
 Collect soil sample from various oil dumping areas.
 Make serial dilution of the soil samples such as 10-1
,10-2
, 10-3
,10-4
,10-5
etc.
 Prepare tributyrin agar medium and pour in sterile petri plates.

5.  Pour the 10-4
and 10-5
dilution soil sample on the above solidified medium and
spread it with the help of spreader.
 Incubate the plates at 37°C for 24 hrs in the incubator.
 Observe the zone of clearers around growing colony.
(B) Secondary screening–
 Prepare tributyrin broth or tween 80 broth.
 Transform 25 ml of each broth into conical flask.
 Inoculate the broth I conical flask with the culture sample.
 Incubates the flask in incubator of 37°C for 24 hrs.
 Centrifuge the above turbid culture broth at 1200rpm for 15 min.
 Collect the supernatant & discorded the pellet.
 Prepare the tributyrin agar or tween 80 agars medium and pour it in sterile
petri plates.
 Put the well with the help of sterile cork and pour the above supernatant with
the help ofpipette.
 Incubate the plates at 37°C – 24 hrs.
 Observe for zone of clearer around the well.
OBSERVATION
RESULT
INTERPRETATION

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.
PRECAUTION
1. All inoculation should be done carefully.
2. . Care should be taken while handling the centrifuge.
3. All observation should be recovered carefully.
4. The media used for the experiment must be standard quality.
5. All the glassware must be thoroughly cleaned and dried.

7. CELLULASE PRODUCTION TEST
REQUIREMENTS - Modified Czapek -mineral salt medium, Soil sample(Botanical
garden) Carboxymethyl cellulose (CMC), Hexadecyltrimethyl ammonium bromide (1%
solution), Sterile Petri plates (2), Inoculating loop/needle, Glass rod, Bunsen burner, Wax
marking pencil.
PRINCIPLE - A prominent carbonaceous constituent of higher plants and probably the
most abundant organic compound is cellulose. Because a large part of the vegetation added to
soil is cellulose, the decomposition of the carbohydrate has a special significance in the
biological cycle of carbon. Cellulose is a polysaccharide composed of glucose units in a
long linear chain linked together by B-1, 4 glycosidic bonds. Degradation of cellulose is
brought aboutby fungi and bacteria by the secretion of extracellular enzyme, cellulase. It is a
complex enzyme composed of at least three components viz., endoglucanase, exoglucanase,
glucosidase. The cooperative action of these three enzymes is required for the complete
hydrolysis of cellulose to glucose.
PROCEDURE –
1) Preparation of Czapek-mineral salt agar medium, whose constituents are as follows:
 Sodium nitrate 0.02 g
 Potassium phosphate 0.01g
 Magnesium sulphate 0.05g
 Potassium chloride (KCl) 0.05g
 Carboxymethyl cellulose (CMC) 0.5g
 Peptone 0.2g
 Agar 5 g
 Distilled water 100ml
a) Dissolve the agar in 40 mL of hot distilled water by adding in small amounts
and stirring with a glass rod.
b) Dissolve the magnesium sulphate, potassium chloride, peptone, sodium nitrate
in 20 mLof water.
c) Dissolve potassium phosphate in 10 mL of water.
d) Dissolve CMC in 20 mL of water with heat and mix in a Waring blender
(mixi).

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e) Mix all the solutions and make up to 100 mL volume.
f) Adjust the pH of the medium to 6.5 with the addition of acid or alkali.
g) Dispense the medium in 250 mL conical flasks, plug them and autoclave at 15
1b/in² (121°C) for 15 minutes.
2) Pour the autoclaved medium cooled to 45-50°C into sterile Petri plates. Allow the
medium to solidify.
3) Dissolve the soil in distilled water.
4) Make serial dilution of the soil samples such as 10-1
,10-2
, 10-3
,10-4
,10-5
etc.
5) Pour the 10-4
and 10-5
dilution soil sample on the above solidified medium and spread
it with the help of spreader.
6) Incubate the plates at 37°C for 24 hrs. in the incubator.
7) After incubation 5% congo red solution was flood in the petri plates with the help of
dropper for 15 min and observed the clear zone.
OBSERVATION
RESULT
INTERPRETATION

9. PRECAUTION
1. While preparing Czapek-mineral salt medium, phosphate should always be dissolved
separately and be added to the mixture at the last.
2. Proper measurement should be taken.
3. The media used for the experiment must be standard quality.
4. All the glassware must be thoroughly cleaned and dried.

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AMYLASE PRODUCTION TEST-
REQUIREMENTS - Nutrient agar slant cultures of Bacillus subtilis, Escherichia coli
and Aspergillus niger, Starch agar medium,Gram's iodine solution, Sterile Petri dishes
(3), Dropper, Bunsen burner, Wax marking pencil.
PRINCIPLE - Amylase is an exoenzyme that hydrolyses (cleaves) starch, a
polysaccharide (a molecule which consists of eight or more monosaccharide molecules)
into maltose, a disaccharide (double sugars, i.e. composed of two monosaccharide
molecules) and some monosaccharides such as glucose.
These disaccharides and monosaccharides enter into the cytoplasm of the bacterial cell
through the semipermeable membrane and there by used by the endoenzymes. Starch is a
complex carbohydrate (polysaccharide) composed of two constituents-amylose, a
straight chain polymer of 200-300 glucose units, and amylopectin, a larger branched
polymer with phosphate groups.
The ability to degrade starch is used as a criterion for the determination of amylase
production by a microbe. In the laboratory it is tested by performing the starch test to
determine the absence or presence of starch in the medium by using iodine solution as an
indicator. Starch in the presence of iodine produces a dark-blue colouration of the
medium, and a yellow zone around a colony in an otherwise blue medium indicates
amylolytic activity.
This exercise deals with testing the hydrolysis of starch for the production of extracellular
amylase by three test organisms, Bacillus subtilis, Escherichia coli and Aspergillus niger
by inoculating these on starch agar medium.
PROCEDURE
1. Melt the starch agar medium, cool to 45°C and pour into the sterile Petri dishes.
2. Allow it to solidify.
3. Label each of the starch agar plate with the name of the organism to be inoculated.
4. Using sterile technique, make a single streak inoculation of each organism into the
centre of its appropriately labelled plate.

11. 5. Incubate the bacterial inoculated plates for 48 hours at 37°C and fungal inoculated
plates for 72-96 hours at 25°C in an inverted position.
6. Flood the surface of the plates with iodine solution with a dropper for 30 seconds.
7. Pour off the excess iodine solution.
OBSERVATION
RESULT
INTERPRETATION
PRECAUTION
1. All the glassware should be clean before use.
2. All inoculation should be done carefully.
3. Carefully use the chemicals.
4. The media used for the experiment must be standard quality.

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GELATINASE PRODUCTION TEST
REQUIREMENTS- 24-48 hours nutrient agar slant cultures of Bacillus subtilis,
Escherichia coli and Proteus vulgaris, Gelatin agar medium (60 ml), Nutrient gelatin deep
tubes (4), Mercuric chloride solution in a dropping bottle (20 ml)Sterile Petri dishes
(2),Refrigerator ,Inoculating needle, Bunsen burner,Wax marking pencil.
PRINCIPLE- Proteins are organic molecules composed of amino acids, in other words
proteins contain carbon, hydrogen, oxygen and nitrogen, though some proteins contain
sulphur too. Amino acids are linked together by peptide bonds to form a small chain (a
peptide) or a large molecule (polypeptide) of protein. Gelatin is a protein produced by
hydrolysis of collagen, a major component of connective tissue and tendons in humans and
other animals. It dissolves in warm water (50°C) and exists as a liquid above 25°C, and
solidifies (gels) when cooled below 25°C.Large protein molecules are hydrolyzed by
exoenzymes, and the smaller products of hydrolysis are transported into the cell. Hydrolysis
(liquefaction) of gelatin is brought about by microorganisms capable of producing a
proteolytic exoenzyme known as gelatinase, which acts to hydrolyze this protein to amino
acids. Hydrolysis of gelatin in the laboratory can be demonstrated by growing
microorganisms in nutrient Hydrolysis of gelatin, a protein (production of gelatinase)gelatin.
Once the degradation of gelatin occurs in the medium by an exoenzyme, it can be detected by
observing liquefaction (i.e. even very low temperature 4°C will, not restore the gel
characteristic) or testing with a protein-precipitating material (ie. flooding the gelatin agar
medium with the mercuric chloride solution and observing the plates for clearing around the
line of growth) because gelatin is also precipitated by chemicals that coagulate proteins while
the products of degradation (i.e. amino acids) are not precipitated by the same chemicals.This
exercise deals with testing of gelatinase production by three microorganisms Bacillus subtilis,
Escherichia coli and Proteus vulgaris by two methods: stab inoculation of nutrient gelatin
tubes to see liquifaction of gelatin; and inoculation of gelatin agar plates to see the formation
of clear zones around the line of growth, when flooded with mercuric chloride.
PROCEDURE-
1. Melt the gelatin-agar medium, cool to 45-50°C and pour into four sterile Petri dishes
(approx. 15 ml in each) and allow it to solidify.

13. 2. Label each of the nutrient-gelatin deep tubes and gelatin agar-medium plates with the name
of the bacterial isolate to be inoculated.
3. Using inoculating loop, make a stab inoculation (i.e. puncture of the agar column from top
to bottom with withdrawal of the needle through the same path) from each culture into its
appropriately labelled deep tube of nutrient gelatin. Uninoculated deep tube should be used as
a control.
4. Make a single streak inoculation from each culture into its appropriately labelled Petri
plate across the surface of the medium.
5. Incubate all the inoculated tubes, uninoculated deep tube and plates at 37°C for 4 to 7 days.
6. After incubation, place the tubes into a refrigerator at 4°C for 15 minutes.
7. Flood the incubated agar plates with mercuric chloride solution and allow the plates to
stand for 5 to 10 minutes.
OBSERVATIONS
RESULT
INTERPRETATION

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PRECAUTIONS
1. All the glassware should be clean before use.
2. All inoculation should be done carefully.
3. Carefully use the chemicals.
4. The media used for the experiment must be standard quality.

15. EXPERIMENT NO. 2
AIM :- ANTIBIOTIC SENSITIVITY TEST
REQUIREMENTS :- Petri plates, forceps, spreader, bunsen burner, NAM media,
bacterial culture E.Coli, test tube, inoculation loop, distilled water, antiobiotic disc.
PRINCIPLE :- This method is given by KIRBY BAUER in 1956 also known as KB
method, disc diffusion antibiotic susceptibility test.
Antibiotic sensitivity test is performed by the commonly used agar diffusion method in which
the antibiotic impregnated disc, placed on agar previously inoculated with test bacterium pick
up moisture and antibiotic diffuse radially and a clear zone or ring is formed around an
antibiotic disc after incubation if agent inhibit bacterial growth. The resulting value is called
the minimal inhibitory concentration (MIC) which is determined by measuring the diameter
of growth inhibition (clear) zone surrounding the antibiotic disc.
PROCEDURE
1. Took a sterile cotton swab and dipped it into a culture or cell suspension of E.coli
2. Inoculated the entire agar surface of each plate, first in horizontal direction and then in
vertical direction to ensure the even distribution of the bacteria over the agar surface, using
the swab.
3. Allowed the agar surface to dry for 5 minutes.
4. Picked up a sterile filter paper disc by the outer edge using flamed, sterile, forceps and dip
the opposite edge of the disc in the Penicillium culture filtrate or into a drop of yellow liquid.
5. Placed the disc near the edge of the agar surface of the inoculated plate.
6. Pressed gently with the sterile forceps to ensure firm contact with the agar surface.
7. Placed a second disc impregnated with an antibiotic solution at the opposite side of the
plate.
8. Placed a third disc moistened in sterile medium as a control.
9. Incubated all plates at 37°C for 24 to 48 hours in an inverted position.
OBSERVATION –

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RESULT –
INTERPRETATION –
PRECAUTION
1. All inoculation should be done carefully.
2. Sterile forceps should be used to place the antibiotic disc.
3. Sterile all equipment and material.
4. The media used for the experiment must be standard quality.
5. All the glassware must be thoroughly cleaned and dried.

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PAPER II :- MICROBIAL GENETICS
EXPERIMENT NO. 1
AIM:- DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION
(MIC) OF AN ANTIBIOTIC
REQUIREMENTS :- Nutrient broth, Culture tubes, Inoculation loop, Bacterial
culture
Spectrophotometer, Incubator. Streptomycin, Test tube, Test tube stand, distilled water.
PRINCIPLE :- THE MINIMUM INHIBITORY CONCENTRATION (MIC) OF AN
ANTIBIOTIC which may inhibit the growth of a particular bacterium. There is different
degree of inhibition in different bacteria with reference to a particular antibiotic. Usually, the
cell wall synthesis in bacteria occurs by synthesizing peptidoglycan layer. The synthesis of
peptidoglycan occurs by lengthening long glycan polymer cross linked with amino acid
chains to connect the long parallel glycan chains. Penicillin is effective against Gram-positive
bacteria while streptomycin kills Gram-negative such as E.coli. Penicillin blocks the amino
acid formation and such cells continue to enlarge with weak cell due to missing cross-link
Osmotic pressure exerts on the wall and cell breaks and lyse.
On the other hand, streptomycin is used against Gram-negative bacteria. It
binds the protein of the 30S subunits of ribosome. The bacterial ribosome is inactivated by
streptomycin, blocking protein synthesis in the cell. The cells stop dividing due to check of
new protein synthesis and lose viability. Streptomycin is, therefore, bactericidal. It enters the
Gram-negative cells more easily in comparison Gram-positive cells.
PROCEDURE :-
1. The solution was prepared by measuring 2 mg/ml of streptomycin.
2. 2 ml of the antibiotic solution were mixed with 2 ml of nutrient broth in test tube and
shake well. Subsequently, 2 ml were transferred to tube 2 and then to the rest of the tubes
containing 2 ml of nutrient broth except the last one that does not contain any antibiotic
solution. The concentration of antibiotic in each tube could be calculated.

19. 3. Each tube was inoculated with one drop of culture and incubated at 37°C for 48 hours.
4. The turbidity was masured in terms of optical density (OD) by spectrophotometer and a
table or a graph was prepared between antibiotic concentration and turbidity.
OBSERVATION:-
RESULT :-
INTERPRETATION:-
PRECAUTIONS:-
1. All the glassware should be clean before use
2. Take the appropriate readings
3. All inoculation should be done carefully.
4. The media used for the experiment must be standard quality.

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EXPERIMENT NO. 2
AIM :- THE EFFECT OF ULTRAVIOLET LIGHT ON BACTERIAL
REQUIREMENTS :- Bacterial culture {E.COLI}, Inoculation loop, NAM plates,
Bunsen burner, Test tubes, Distilled water, Laminar air flow, Incubator.
PRINCIPLE :-
Most of the bacteria are damaged by ultraviolet radiation. Although sunrise contains
ultraviolet radiations of short to long wavelengths yet it is the short wavelengths that are
injurious to the bacteria. The rays emitting from UV lamp also cause cataract and other injury
to human eyes. Generally, UV rays fall between 2000 and 4000 A (1 A angstrom = 10-8
cm)
and 10 Angstrom is equal to 1 mm ( nanometer). The maximaan germicidal effect is at 2650
Angstrom. The germicidal effect of UV on the organisms that have been streaked on nutrient
agar will be exposed to determine the minimum amount of exposure required to effect 100%
all.
PROCEDURE:-
(1) The nutrient agar media plate was prepared.
(2) A bacterial suspension of E. coli was prepared, and 0.1 ml of the suspension was
inoculated onto the agar plate using a spreader.
(3) The plate was placed under UV light, and the lid was removed.
(4) The plate was exposed for 5, 10, 15, 20, and 25 minutes, respectively.
(5) After the exposure, the plate was covered with a lid and incubated at 37 degrees for
48 hours.
(6) The growth on the plates was recorded and observed.
OBSERVATION:-
RESULT:-

21. INTERPRETATION:-
PRECAUTIONS:-
1. Always wear protective gear including gloves, masks, and lab coats
2. Ensured that the lab area and equipment were properly sanitized to avoid
contamination
3. Followed safety guidlines when working with UV radiations.
4. All the glassware should be cleaned and dried.

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EXPERIMENT NO. 3
AIM:- ISOLATION OF ANTIBIOTIC RESISTANT BACTERIAL
POPULATION BY GRADIENT-PLATE.
REQUIREMENTS:- 24 hour nutrient broth culture of E. Coli, Two nutrient agar deep
tubes (10 ml per tube/culture) , Streptomycin sulphate solution (1% ie., 10 mg/100mL), A
beaker with 90%.ethanol, Sterile Petri plates Sterile 1-ml pipette, Glass rod spreader, Water
bath, Glass marking pencil.
PRINCIPLE:-
A microorganism that exhibits a natural, nonmutated inheritance is termed as a wild type or
wild strain while a mutant strain refers to the altered version. Mutant strains can show
variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to
chemicals, temperature preference and nearly any type of enzyme function. Applications of
mutant strains are in: to trace genetic events, unravelling genetic organization, and
pinpointing genetic markers.
Phenotype refers to the observable property of the organism and the genotype
refers to the genetic constitution of an organism. Genes are designated by a three letter code
written in italic lower case letters, eg, abc, abc signifies the name of a locus on the bacterial
chromosome.
An excellent way to determine the ability of organisms to produce mutants that
are resistant to antibiotics is to grow them on a gradient-plate of a particular antibiotic. This
technique requires preparation of a double layered agar plate consisting of two different
wedge-like layers of medium: A bottom slanted plain agar medium and a top layer of agar
medium with the antibiotic. Since the antibiotic is only in the top layer; it tends to differ with
the lower layer producing a gradient of antibiotic concentration from low to high. For
isolation of antibiotic-resistant mutants of gram-negative enteric bacteria, the antibiotics
commonly used are rifamycin (100 ug/ml.), streptomycin (100 ug/ml) and nal dixic acid (100
µg/ml.).
This exercise deals with the isolation of streptomycin resistant mutants using a prototrophic
(wild-type, streptomycin-sensitive) Escherichia coli by the use of a simple gradient-plate
technique where streptomycin at the rate of 100 ug/mL is added in the nutrient agar medium.
Following a spread-plate inoculation of E. coli and incubation for 4-7 days. the development
of bacterial colonies in the high streptomycin concentration region will be streptomycin-
resistant mutants.

23. PROCEDURE:-
A. GRADIENT PLATE PREPARATION
(1) Two nutrient agar deep tubes were melted in a water bath maintained at 96°C and cooled
to 50°C.
(2) Poured the contents of one agar tube into a sterile Petri plate and allowed to solidify
medium in a slanted position by placing a pencil under one edge.
(3) After the solidification of the agar medium, remove the pencil from the plate and place it
in a horizontal position.
(4) 0.1 ml. streptomycin solution (100 ug/mL) was pipetted into the second tube of the
melted nutrient agar medium, the tube was rotated between the palms, and the contents were
poured to cover the gradient agar layer and allowing it to solidify on a level table.
(5) The low and high concentration areas on the bottom of the plate were labelled.
B. FIRST INOCULATION
(6) 0.2 ml. of the E. coli suspension was pipette onto the gradient-plate after 24 hours of its
preparation.
(7) With an alcohol-dipped, flamed and cooled bent glass rod, spread the inoculum evenly
over the agar surface by rotating the plate.
(8) The inoculated plate was incubated in an inverted position at 37°C for 48 to 72 hours.
OBSERVATIONS
RESULTS
C. SECOND INOCULATION
(9) The selected colony was picked with a sterile inoculating loop, and streaked onto a
second gradient-plate toward the high concentration portion.
(10) This step was repeated with two or three colonies of streptomycin-resistant mutants
from the HSC area.
(11) The inoculated plates were incubateds in an inverted position at 37°C for 24 to 72 hours.

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OBSERVATIONS:-
RESULT:-
INTERPRETATION:-
PRECAUTIONS:-
1. Always label the bottom of the plate to show the low and high streptomycin
concentration areas.
2. The gradient-plate must be prepared at least 24 hrs before inoculation to allow the
establishment of antibiotic gradient in the plate.
3. Always grow the organism overnight in a rich broth.
4. Don't disturb the nutrient agar plate when kept in a slanted position, until complete
solidification has occurred.
5. Glassware should be clean before use.
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