SCHOOL OF MEDICINE

DEPARTMENT OF MICROBIOLOGY

INTRODUCTION TO MICROBIOLOGY

MBMM2411

MICROBIOLOGY LAB REPORTS

NAME: HASSAN MOHAMED

REG NUMBER: BMS/2022/30250

1) STERILIZATION BY AUTOCLAVING
Objective:To perform sterilization of various equipment through autoclave.
Introduction:

Sterilization is the process by which a surface or object is freed of all living

microorganisms either in the vegetative or in the non-vegetative state. Materials
that have been subjected to this process are sterile. Although most sterilization is
performed with a physical agent such as heat, a few chemicals such as high ethanol
concentration alcohol can be classified as sterilizing agents because of their ability
to destroy microorganisms.

Methods of sterilization can be classified as physical and chemical methods.

Physical methods involves use of direct sunlight, heat (moist and dry heat), filters
(depth and membrane filters), radiations and sound waves. Chemical methods
involve use of chemical agents that are used as antiseptics as well as disinfectants,
for example alcohols and formaldehyde.
Principle:
Autoclave is a type of physical method of sterilization that utilizes moist heat at
temperatures higher than 1000C. The autoclave is a cylindrical metal chamber with
an airtight door at one end and racks to hold materials. The lid is fastened by screw
clamp and rendered airtight by asbestos washers. It has a discharge tap for air and
steam at the upper side, a pressure gauge and a safety valve that can be set to blow
off at any desirable pressure. Heating is usually carried out by electric means.
Steam circulates within and is supplied under pressure to the inner chamber where
materials are loaded for sterilization. The water in the autoclave boils when the
vapor pressure equals that of the surrounding atmosphere. Following the increase
of pressure inside the closed vessel, the temperature at which the water boils inside
the autoclave also increases. The saturated steam that has a higher penetrative
power, on coming in contact with a cooler surface condenses to water and releases
its latent heat to that surface. The gross reduction in volume of steam sucks in
more steam to the area and this process continues till the temperature of that
surface is elevated to that of the steam. Sterilization is achieved when the steam
condenses against the objects in the chamber and gradually raises their
temperature. The most efficient pressure-temperature combination for achieving
sterilization by autoclave is 15 psi, which yields 1210C.
Materials:
  Autoclave
 Water
 Wire bottles
Procedure:
Correct volume of water was added to the autoclave. The wire bottles containing
bottles with loose caps (to be sterilized) were placed in the inner chamber of the
autoclave. The lid was then secured, air lock opened and the draw off knob closed.
The valve was then adjusted to the required pressure. The autoclave was then
switched on and the water started to boil with air and steam coming out through the
air lock. The air lock was then closed a minute after all the water droplets were
expelled and only steam was emerging. When the required pressure was reached
and excessive steam began to be released through the safety valve, the heat was
reduced and timing was set at 20 minutes. At the end of the timing, the heat was
turned off and the autoclave was allowed to cool until the pressure gauge showed a
reading of zero.
Discussion:
The autoclave kills all the vegetative as well as non-vegetative forms of bacteria. It
also has many uses such as;
1. It is a good method to sterilize heat-resistant materials such as glassware,
cloth, rubber, metallic instruments, liquids, paper, some media and some
heat-resistant plastics.
2. It is also useful for sterilization of heat-sensitive items, such as plastic petri
dishes .
3. It is useful for sterilization of materials that cannot withstand the higher
temperature of the hot-air oven.
However, the autoclave is ineffective for sterilizing substances that repel moisture
such as oils, waxes or powders. Various sterilization controls are used to determine
the efficacy of sterilization by the autoclave. Brown’s tube is the most commonly
used chemical indicator. It contains red solution that turns green when exposed to
temperature of 1210C for 15 minutes in the autoclave.
References:
1. Subhash Chandra Parija – Textbook of Microbiology & Immunology –
Second edition; Published by Elsevier.
2. https://jcp.bmj.com
3. https://www.ncbi.nlm.nih.gov
1) CULTURE MEDIA PREPARATION
Objective:To prepare various culture media .
Introduction:
A culture media refers to a liquid or solid substance that contains nutrients to
support the growth and survival of microorganisms. The basic ingredients of
culture media include water and electrolytes such as sodium chloride, peptone, 2-
3% concentrated agar, meat extract, yeast extract and blood and serum.
Culture media can be classified in several ways. They can be classified either as
liquid, solid or semisolid media. Liquid media provide greater sensitivity for the
isolation of small numbers of microorganisms and include nutrient broth, sugar
media and enrichment media. By varying the concentration of agar, it is possible to
make a solid or semisolid media. Solid media is prepared by addition of 2% agar
while a semisolid media is prepared by addition of reduced concentration of agar .
Culture media can also be classified as either simple, complex, defined or special.
Simple or basal media such as nutrient broth form the basis of other media.
Complex media have some complex ingredients which consist of a mixture of
many chemicals in unknown proportions. A defined or synthetic media contains
known quantities of all ingredients. Special media include enriched media,
enrichment media, selective media, indicator or differential media, transport media
and sugar media.
Principle:
Preparation steps for various culture media are usually provided on the culture
media continue. The general steps are weighing and dissolving, addition of heat
sensitive ingredients, dispensing, sterilization and sterility test; and storage. The
commonly used culture media in the laboratory are nutrient agar and MacConkey
agar. Nutrient agar is composed of nutrient broth and 2% agar and is used as a
routine culture.MacConkey agar is composed of peptone, lactose, sodium
taurocholate, agar and neutral red .
Materials and reagents:
 Solid Nutrient Agar and MacConkey Agar in containers
 Petri dishes
 Distilled water
 Heat source
 Autoclave
  Measuring cylinder
 Weighing machine
 Conical flask
Procedure:
a) Preparation of nutrient agar:
2.8g of the powder was scooped from the container, measured and suspended in
100ml of distilled water in the conical flask. The mixture was heated to boiling
point for it to dissolve completely and then sterilized by autoclave at 15lbs
pressure for 15 minutes. The mixture was then cooled to 45-50 0C. The mixture
would then be poured on the sterile petri dish.
b) Preparation of MacConkey agar:
5.155g of the powder was scooped from the container, measured and suspended
in 100ml distilled water in the conical flask. The mixture was then heated to
boiling point for it to dissolve completely and then sterilized by autoclave at
15lbs pressure (1210C) for 15 minutes. The mixture was then cooled to 45-50 0C
and then poured into the sterile petri dish.
Discussion:
Nutrient agar is the preferred medium for;
1. Performing biochemical tests for bacteria such as oxidase test, catalase test and
slide agglutination test etc.
2. Studying colony morphology.
3. Pigment demonstration.
MacConkey agar is a differential and low selective medium commonly used for the
isolation of enteric gram-negative bacteria. It differentiates organisms into lactose
fermenting and non-lactose fermenting . Most laboratories use a combination of
blood agar and MacConkey agar for routine bacterial culture.
References:
1. Subhash Chandra Parija – Textbook of Microbiology and Immunology -
Second edition; Published by Elsevier.
2. Apurba S. Sastry, Sandhya Bhat – Essentials of Medical Microbiology –
Third edition; Published by Jaypee Brothers Medical Publishers.
2) INNOCULATION AND INCUBATION OF CULTURE MEDIAS
Objectives:To perform various culture methods.
Introduction:
Culture methods involve inoculating the specimen onto appropriate culture media,
followed by incubating the culture plates in appropriate conditions. Culture
methods are very crucial in a microbiology laboratory. Various culture methods are
carried out to;
1. Isolate bacteria in pure cultures and identify the same by performing various
tests.
2. Demonstrate biochemical, antigenic and other properties of the isolated
colonies.
3. Demonstrate susceptibility of the isolated bacteria to antibiotics,
bacteriophages, and bacteriocins
4. Prepare antigens for various uses.
5. Maintain stock culture.
Various methods are used culturing bacteria such as streak culture, lawn culture,
pour-plate culture, stroke culture, stab culture and liquid culture. Streak culture is
the most common inoculation method and the most useful in obtaining discrete
colonies of the bacteria.
Principle:
Streak culture is carried out by streaking on the surface of a solid media plate using
a platinum loop of wire 2-4mm in diameter. A loop full of the specimen is smeared
onto the solid media to form round-shaped primary inoculum, which is then spread
over the culture plate by streaking parallel lines to form the secondary and tertiary
inoculum and finally a feathery tail end. The loop is flamed and cooled in between
the different set of streaks to get isolated colonies on the final streaks. The
inoculated culture plate is incubated at 37 0C overnight for demonstration of
colonies.
Materials:
 Inoculum
 Culture plate
 Source of heat
 Inoculating wire/loop
 Bacteriological incubator
Procedure:
A loop full of the inoculum was collected and placed near the peripheral end of the
culture plate using the inoculating wire to form the primary inoculum. The wire
was then sterilized by flaming and cooled before proceeding with the inoculation.
From the primary inoculum, parallel lines were drawn and their ends formed the
secondary inoculum. The wire was re-sterilized by flaming and then left to cool
down. From the secondary inoculum, parallel lines were drawn and their ends
formed the tertiary inoculum. The wire was sterilized again by flaming and left to
cool. From the tertiary inoculum, parallel lines were drawn and their ends formed
the feathery tail end. The wire was sterilized again by flaming and left to cool.
From here, zigzag lines were drawn. The wire was finally sterilized to prevent
spread of the inoculated bacteria. The culture plate was then incubated overnight
and observed on the next day.
Results:
Confluent growth occurs at the primary inoculum but becomes progressively
thinner and well-separated colonies are demonstrated on the final streaks of the
inoculum. The single colonies obtained are very useful to study various properties
of bacteria such as size, shape, consistency, density, color, pigmentation and
motility tests.
Discussion:
Most bacterial pathogens are capable of growing on artificial media in the clinical
laboratory. The minimum requirements for growth of bacteria include water, a
source of carbon, a source of nitrogen and certain inorganic salts. Bacterial growth
can be defined as an orderly increase of all the chemical components of the cell
and can be equated to the cell number. The growth rate is measured by measuring
the change in bacterial number per unit time. The number of bacteria at a given
time can be estimated by performing a total count or viable count. When a broth
culture is inoculated with a small bacterial inoculate, the population size of the
bacteria increases showing a classical pattern. A variety of factors affect growth of
bacteria. These factors include oxygen, carbon dioxide, temperature, pH, light and
osmotic pressure.
Some bacteria grow in a variety of simple media like inorganic salts example is
E.coli These inorganic salts provide major essential elements of carbon, hydrogen,
oxygen, nitrogen, phosphorus and sulfur. These chemicals are usually present in
the media and are not added specifically.
Some such as Haemophilus influenza are very fastidious and have certain growth
requirements. They require amino acids, vitamins and other growth factors that are
supplied by adding yeast extract . They also require addition of blood or serum for
growth.
Certain lower forms of bacteria even fail to grow in cell-free culture media and
require living cells for their growth. Treponema pallidum and Mycobacterium
leprae cannot grow in artificial culture media, but can only be cultured when
inoculated in living animals. Such bacteria are known as obligate intracellular
pathogens.
Conclusion:
It is not always possible to identify microorganisms by microscopic methods alone,
due to similarity of bacteria in their morphology and staining characters. Hence,
further study of microorganisms is essential for their identification. For this
purpose, organisms have been grown artificially in the laboratory through the
culture method.
References:
1. Subhash Chandra Parija – Textbook of Microbiology and Immunology -
Second edition; Published by Elsevier.
2. https://www.ncbi.nlm.nih.gov
3. https://bio.libretext.org

3) GRAM STAINING
Objective:To identify and differentiate gram-positive and gram-negative bacteria.
Introduction:
Structural details of bacteria cannot be seen under a light microscope due to lack of
contrast. Hence, it is necessary to use staining methods to produce color contrast
and thereby increase the visibility. The common staining techniques used in
diagnostic microbiology include simple staining, negative staining, impregnation
staining and differential staining. The commonly used differential stains include
Gram stain, acid-fast stain and Albert’s stain.
Principle:
Before staining, the smears are fixed so that they are not displaced during the
staining process. Fixation also protects the internal structures of the cells in a fixed
position. It is done by two methods; heat fixation by gently flame heating the air-
dried film, for bacterial smears; and methanol fixation for blood smears.
The gram-staining method essentially consists of 4 steps;Primary staining with
basic dyes such as crystal violet:Application of mordant in the form of dilute
solution of iodine,decolourization with ethanol or acetone, and counter-staining
with acidic dyes such as neutral red.
Materials and Reagents:
 Crystal violet stain
 Lugol’s solution
 Acetone alcohol
 Neutral red
 Alcohol
 Glass slide
 Microscope
 Oil immersion
Procedure:
Isolated colonies were obtained from inoculated bacteria and spread on the glass
slide with a stick to form a smear. The smear was air dried. The smear was then
flooded with alcohol and left to dry for 2 minutes. The smear was then covered
with crystal violet stain for 60 seconds and then washed with running water. The
smear was then tipped to one side on a lever and flooded with Lugol’s solution for
60 seconds. The smear was then washed again with running water. The smear was
then decolourized with acetone-alcohol and immediately washed with clean water.
The smear was then covered with neutral red for 2 minutes. The stain was then
washed off with clean water. The slide was then air dried. The slide would then be
examined microscopically using oil immersion objective.
Results:
Many gram positive cocci bacteria were observed.
Discussion:
Gram-positive bacteria resist decolourization and retain the primary iodine
complex, appearing violet. They have a relatively thick amorphous cell wall and
more acidic protoplasm which retain the basic violet dye and iodine complex
within the cell.
Gram-negative bacteria are decolourized by the organic solvents and appear red.
The decolourizing agent used during the staining disrupts the membranous
envelope, and the dye and the iodine complex are washed out of the bacteria.
There are certain groups of bacteria such as Mycobacterium tuberculosis and
Mycobacterium leprae that cannot be considered typical gram-negative or gram-
positive bacteria. This is because these bacteria either do not take up the Gram
stain or they have a different type of envelope. These Mycobacteria possess a waxy
envelope containing complex glycolipids that make them impervious to the Gram
stain.
Conclusion:
Gram staining is an essential procedure that is used in the identification of bacteria.
This gives a clue of the corresponding biochemical tests for further identification
of the bacteria. Gram-staining also gives a preliminary clue about the bacteria
present so that the empirical treatment with broad- spectrum antibiotics can start
early enough before the culture report is available.
References:
1. Subhash Chandra Parija – Textbook of Microbiology and Immunology - Second
edition; Published by Elsevier.
2. Apurba S. Sastry, Sandhya Bhat – Essentials of Medical Microbiology – Third
edition; Published by Jaypee Brothers Medical Publishers.
3. https://www.ncbi.nlm.nih.gov