GOVT. V.Y.T.

PG AUTONOMOUS COLLEGE, DURG

Practical Examination
Session: 2020-21

“Lab Course-2”

Submitted By:-
Ananya Singh
Roll No. 90702
M.Sc 3rd semester
Department of Microbiology
 Experiment No. 1
• Aim:- Effect of detergents and soaps on the Normal flora of skin.
• Principle:- The skin serves as a natural protective barrier against invasion by most microorganisms
because of the acidity of the skin, the presence of indigenous flora, the temperature of the skin, sebum
secreted by oil glands and creation of hypertonic environment by the salts in perspiration.
However, perspiration and sebum serve as a source of nutrition for certain microorganisms and help in
their establishment as part of the normal flora of the skin. Most frequently encountered bacteria on the
skin are gram +ve cocci including micrococcus spp. and Staphylococcus epidermidis.
Staphylococcus aureus is the part of the normal flora of the skin and is also considered a pathogen.
Sebum secreted from oil glands helps the survival of Propionibacterium acne, an anaerobic gram
positive rod in hair follicles.
The so-called normal flora of the skin varies according to the skin region and the environmental factors.
Most pathogenic organisms do not penetrate the unbroken skin but as soon as epidermis is destroyed,
infection easily occurs. Staphylococci and certain fungi are able to penetrate the hair follicles and cause
disease in the deeper tissues.
Disc method:- This method is employed to see whether the bacteria are inhibited by the particular
concentration of detergent or soap, or not. By using this method we can find out the least inhibitory
concentration of detergent or soap for particular bacteria
• Requirements:- Mannitol salt agar media, Nutrient agar medium, LB broth, MacConkey agar,
EMB agar, soaps and detergents, sterile cotton-swab, Inoculating loop, Whatman filter paper discs,
Wax marking pen, 1ml sterile pipette.
• Procedure :-
Isolation of Pathogenic Bacteria:- The collected sample were spread plated on different culture
media including selective media, MacConkey agar, EMB agar, Bismuth sulfate agar incubated at
37℃ for 24 to 48 hours. And isolated colonies were then identified by microscopic view, Gram
staining and various biochemical tests. The isolated colonies were streaked on nutrient agar slants and
pure culture was maintained.
The antimicrobial susceptibility test used was the Kirby-Bauer modified disk diffusion method as
described by Bauer. The test organisms from an overnight culture plate, incubated at 37°C were
suspended in saline solution (0.85% NaCl) and adjusted to match a turbidity of 0.5 McFarland
Standard.
Sterilized Whatman filter paper disks of 6mm were soaked in the different detergent and soap
solutions of different concentration for a period of 1 hour to ensure that the disks were fully saturated.
The impregnated discs were aseptically transferred into the sensitivity plates with the aid of a sterile
forceps. Subsequently, the plates were inverted, incubated at 37°C for 24 hours for bacteria and at
30℃ for 24-48 hours for fungi and then examined for zone of inhibition around the disk.
Note:-McFarland turbidity standards are prepared by mixing various volumes of 1% sulfuric acid and
1% barium chloride to obtain solutions with specific optical densities. 0.5 McFarland turbidity
standard provides an optical density comparable to the density of a bacterial suspension 1.5x 10^8
colony forming units (CFU/ml). 0.5 McFarland standard is commercially available.
For performing antimicrobial susceptibility testing using Kirby Bauer disc diffusion method, a
cell suspension of organisms equivalent to a 0.5 McFarland standard is used.

Disc impregnated with

Soap & Detergent solution Media with test
organisms

fig. KIRBY-BAUER DISK DIFFUSION METHOD

• Observations:-
Table.1: Variation in growth of different species of bacteria under different concentration of detergent

Microorganism Control 100mg/ml 200mg/ml 300mg/ml 400mg/ml

Escherchia coli Maximum High Moderate Low Very little

Pseudomonas aeroginosa Maximum High Moderate No growth No growth
Bacillus subtilis Maximum Little No growth No growth No growth
Staphylococcus aureus Maximum No growth No growth No growth No growth
Micrococcus sp. Maximum High Moderate Low Very little

Table.2: Variation in growth of different species of bacteria under different concentration of soap
Microorganism Control 100mg/ml 200mg/ml 300mg/ml 400mg/ml

Escherchia coli Maximum High Moderate Low Very little

Pseudomonas aeroginosa Maximum High Moderate Low Very little

Bacillus subtilis Maximum No growth No growth No growth No growth

Staphylococcus aureus Maximum High Moderate Low Very little
Micrococcus sp. Maximum Low No growth No growth No growth
•Result:- The effect of different concentrations of surf excels on growth of different species of
bacteria showed remarkable variations (Table 1).
Among five strains of bacteria, Staphylococcus aureus unable to grow in any of the detergent
concentrations indicates it is good bactericidal. For E.coli and Micrococcus sp. the MIC was
400mg/ml while at other concentrations (lower levels) luxurious colonies were observed.
In case of Bacillus subtilis the MIC was 100mg/ml and for Pseudomonas aeroginosa it was
200mg/ml. Among bacterial strains the growth of Staphylococcus aureus was completely inhibited
even at 100 mg/ml of Lifebuoy Green. Among other species Micrococcus sp.,E. coil and
Pseudomonas aeroginosa the MIC was 100mg/ml, 300mg/ml 200mg/ml respectively.
Comparing to control, the higher growth was observed in the plates inoculated with E.coli,
Pseudomonas and Micrococcus at 100mg/ml concentration, while other species showed either
moderate, little or no growth.
 On other hand 100mg/ml soap showed higher colony growth of E.coli, Pseudomonas and
Staphylococcus, whereas 300mg/ml soap inhibited the growth of all bacteria except E.coil and
Micrococcus sp. (Table 2).
• Interpretation:-
In the present study the bacteria which were used to study their role against soaps and
detergents showed wide variation. Such variations indicate their survival in the particular
constituents and their antimicrobial property. Of these five bacteria, E. coli showed lesser
antimicrobial property against both soap and detergent. This may explain its wide distribution
as well as number of strains.

• Precautions:-
1. Those areas of the hand should only be marked which have no cut or abrasion, i.e. where the skin is
intact.
2. Discard the swabs after use into a container with disinfectant to avoid contamination of the
laboratory.
3. Hands should immediately be washed with soap and water and rinsed with disinfectant after use to
avoid risk of infection. 
4. Care should be taken while making suspension of test organism, soap & detergent.s
 Experiment No. 2
• Aim -Determination of quality of milk samples by Methylene Blue Reductase Test method
(MBRT).
• Principle:- Milk is a white nutritious liquid secreted by a mammals and used as a food by human
being. Large number of bacteria present in raw milk, especially, E.coli, streptococcus lactis . They are
indicative of poor methods of production handling . The reduction test is based upon the oxidation-
reduction activities of the bacteria present in the milk.
The principle behind this test is that the quality of the milk is determined by observing the color that
appears in the milk after addition of dye like methylene blue. Because methylene blue is a redox
indicator, which loses its color when it comes under the effect of lack of oxygen.
Simply, the basic principle is that the methylene blue (an oxidation-reduction dye or indicator) which
is blue in its oxidized state, is reduced to colorless compound “leuco” as a result of the metabolic
activities of bacteria in milk.
The time taken by the dye to get reduced determines the amount of microbes in the milk and the
metabolic reactions in the microorganisms.
• Requirements:- Samples milk from various sources, Methylene blue solution, sterile screw, Sterile
10 ml. pipettes, Water bath at 37 °c, Sterile distilled water, Bunsen burner.
• Procedure:-
1. Methylene blue solution was prepared (1 mg MB) in 250 ml. of sterile distilled water.
2. Each milk sample was thoroughly mixed and transferred 9ml. of each type of milk into the labeled
test tubes 1 and 2, using separate sterile 10 ml. pipettes each time.
3. 1ml Methylene blue was added to each test tube and the tubes were closed with the stoppers.
4. The contents of each tube were mixed by gently inverting tube 2-3 times and incubated in a water
bath, at 37 °C.
5. After 30min. examine the sample, checking for decoloration. The total time gives MBRT time.
• Observation:-
Decolorization Time Milk Quality

30 minutes or less Poor {very bad}

Initial (0min.) After 150min.
1 to 2 hrs Fair Final
3 to 4 hrs Good

5 hrs and above Excellent

• Result:-The shorter the decolorization time, the higher the number of bacterial flora present in milk,
and the poor quality of milk.
• Interpretation:-
Positive Result: If the viable bacteria decolorized the milk within 30 minutes, the milk is considered
unsatisfactory.
Negative Result: If the milk is not decolorized within 30 minutes, the milk is considered as good
quality.
If dye color is disappear in 30 minuets or less , its means that the milk has Poor quality.
If dye color is disappear in 1-2 hours , its means that the milk has Fair quality.
If dye color is disappear in 3-4 hours, its means that the milk has Good quality.
If dye color is disappear in 5 hours and above, its means that the milk having Excellent quality.
• Precautions:-
1. Always used fresh, unrefrigerated, raw milk of testing.
2. Use proper sterilized glassware and washing glassware.