I B.SC.

I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

I SEMESTER , PRACTICAL – 1
GENERAL MICROBIOLOGY
Experiments based on theory syllabus
15 Practicals (one practical of 4hrs/week)
Expt. No.
1. Microbiological laboratory standards and safety protocols.

2. Standard aseptic conditions of Microbiological laboratory.

3. Operation and working principles of Light/ Compound microscope.
4. Working principles and operations of basic equipments of
microbiological laboratory (Autoclave, Oven, Incubator, pH
meter, Spectrophotometer, Colorimeter, Vortex, Magnetic
stirrer).
5. Applications of basic microbiological tools (Pipettes,
Micropipette, Bunsen burner, Inoculation loop, Spreader).
6. Demonstration and observations of microorganisms from natural
sources under light microscope (Algae, Yeast and Protozoa).
7. Demonstration of bacterial motility by hanging drop method.
8. Simple staining.
9. Negative staining.
10. Differential staining - Gram staining.

11. Acid fast staining.

12. Structural staining - Flagella and Capsule.

13. Bacterial endospore staining.

14. Staining of reserved food materials.

15. Staining of fungi by Lactophenol cotton blue.

Department of Microbiology, Government Science College (Autonomous), Hassan 1

 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

EXPERIMENT No. 01
MICROBIOLOGICAL LABORATORY STANDARDS AND
SAFETY PROTOCOLS.
Microbiology is a branch of biology which deals with microorganisms,
which are omnipresent but are invisible to naked eyes of human beings. They will
be seen through microscopes. Some of the important safety measures must be
strictly followed in the microbiology laboratory to avoid the occurrence of
incidents which may be harmful. The rules, regulations and safety measures which
should be taken in a Microbiology laboratory is as follows:

 It should be assumed that the microorganisms with which we are working are
capable of producing diseases, allergies, itching, etc. Therefore, it is necessary to
take care of while handling the cultures containing test tubes, conical flasks
containing the media, and other equipment like microscopes etc.
 The glass slides, coverslips, pipettes, petri plates etc. should be cleaned before
using and discard into the jar of disinfectant solutions after use or completion of
the experiments.
 The laboratory coat or Apron with name plate should wear compulsorily while
entering into the lab. Practical record, pencil, pen, drawing aids and other
requirements should be brought to the laboratory.
 Working tables, laminar air flow system and hands are cleaned with water and
then swabbed with alcohol
 Inoculation needles, loops should be properly sterilized before and after use by
incineration.
 Microbial cultures containing test tubes, conical flasks containing the media,
physiological saline etc. are plugged with sterile cotton and wrapped with craft or
brown paper.
 The test tubes should be kept upright in the test tube racks, never lay them on the
table. Never moist the labels with tongue.
 Students should not laugh, talk, eat, drink or smoke inside the laboratory.
Accidents such as spilled cultures, breakage of glass wares, any cuts or damages,
should be reported to the staff members immediately.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

 Use instruments like hot air oven, incubators, balance, microwave oven and
microscopes etc. carefully. Hands should be thoroughly washed before leaving the
laboratory.
 Practical observation books should be completed in the laboratory itself and get
signed by batch teacher without fail.
 Practical record books should be completed in the laboratory itself or should be
submitted in next lab and get signed by batch teacher without fail.

Department of Microbiology, Government Science College (Autonomous), Hassan 3

 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

EXPERIMENT No. 02
STANDARD ASEPTIC CONDITIONS OF
MICROBIOLOGICAL LABORATORY.

Aseptic technique is a set of routine measures that are taken to prevent cultures,
sterile media stocks, and other solutions from being contaminated by unwanted
microorganisms. It is called “sterile technique,” that terminology is appropriate
only in reference to preventing the introduction of any organisms to the
laboratory or medical equipment and reagents.
Since the goal of a microbiologist is to grow microorganisms or
eukaryotic cells without the introduction of extraneous organisms, aseptic
techniques are crucial for accurate and meaningful experimentation. One should
always remember that a completely sterile working environment does not exist.
However, there are a number of procedures that will reduce the risk of culture
contaminations.
The aseptic techniques control the opportunities for contamination of cultures
by microorganisms from the environment, or contamination of the environment
by the microorganisms being handled.

Examples of aseptic technique are:

 Cleaning and disinfecting lab surfaces prior to use
 keeping petri dishes closed whenever possible
 effectively sterilizing inoculating loops and other equipment that comes into
contact with cultures or media, and
 avoiding breathing on cultures or sterile instruments.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

Some general rules to follow for any aseptic technique.

 Make transfers over a disinfected surface. Ethanol disinfection is recommended
because of its rapid action
 Vessels must be open for the minimum amount of time possible. While vessels
are open, all work must be done close to a Bunsen burner flame where air
currents are drawn upwards.
 On opening a test tube or bottle, the neck must be immediately warmed by
flaming with the vessel held as near to horizontal as possible and so that any
movement of air is outwards from the vessel.
 The parts of sterile pipettes which will be put into cultures or sterile vessels must
not be touched or allowed to come into contact with other non-sterile surfaces,
such as clothing, the surface of the working area, or the outside of bottles/ test
tubes.
 All items which come into contact with microorganisms must be sterilised before
and after each such exposure.
Sterile Handling
 Always wipe your hands and work area with 70% ethanol.
 Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids,
and use each pipette only once to avoid cross contamination. Do not unwrap
sterile pipettes until they are to be used. Keep pipettes at the work area.
 Always cap the bottles and flasks after use and seal multi-well plates with tape
or place them in resalable bags to prevent microorganisms and airborne
contaminants from gaining entry.
 Never uncover a sterile flask, bottle, Petri dish, etc. until the instant you are ready
to use it and never leave it open to the environment. Return the cover as soon
as you are finished.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

Inoculating agar plates, slopes and cultures

 Normal practice is to open agar plates away from the body and without removing
the lid completely from the base.
 In instances when the lid of the Petri dish may be removed for longer periods
than normal, work very close to the Bunsen burner flame to reduce the chances
of contamination.
 If you experience frequent contamination of plates with fungal spores, reduce
the chance of draughts further, and consider inoculating plates from below with
the agar surface facing downwards. In this way there is perhaps less chance of
spores settling onto the plate from the air.

Using a inoculation loop

 Sterilise a inoculation loop by heating to red hot in a roaring blue Bunsen burner
flame before and after use.
 The flaming procedure should heat the tip of the loop gradually.
 The loop should be red hot.
 Ensure the full length of the wire receives adequate heating.
 Allow to cool for a few seconds in the air, then use immediately.
 Do not put the loop down, or wave it around.
 Re-sterilize the loop immediately after use.

Flaming the neck of bottles and test tubes

 Loosen the cap of the bottle so that it can be removed easily.
 Lift the bottle/ test tube with your left hand.
 Remove the cap/ cotton wool plug of the bottle/ test tube with the little finger
curled towards the palm of your right hand.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

 Do not put down the cap/ cotton wool plug.

 Flame the neck of the bottle/ test tube by passing the neck forwards and back
through a hot Bunsen burner flame.
 Replace the cap/ cotton wool plug on the bottle/ test tube using your little finger.
Take care! The bottle will be hot.
 If cotton wool plugs have partly lost their shape, they can be more easily guided
back into the neck of the vessel by slowly twisting the mouth of the vessel as the
plug is pushed down.

Department of Microbiology, Government Science College (Autonomous), Hassan 7

 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

EXPERIMENT No. 03
OPERATION AND WORKING PRINCIPLES OF LIGHT/
COMPOUND MICROSCOPE
Microscope is an optical instrument used in all biological laboratories to
observe the objects which cannot be seen through naked eyes of human beings.
Microscopes are of many types, namely bright field /compound microscope, dark
field, phase-contrast, stereo, Transmission Electron Microscope and Scanning
Electron Microscopes.

SIMPLE MICROSCOPE :
A simple microscope is a convex lens of short focal length. It is used to form
a virtual image of an object placed just inside its principal focus. It is made up
of a single convex lens or a combination of lenses which functions as a convex
lens. The convex lens magnifies the object and also helps to produce a
magnified image of a near object which appears to be at the distance of distinct
vision. Improved simple microscopes, used by the biologists during field work,
may magnify an object even up to 100 times.

A dissecting microscope is an example of a simple microscope used either

for dissecting the material or for viewing the magnified image of the material.
It consists of only one lens unit. This lens unit may even be an ordinary
magnifying glass.

It is used either for dissecting the material or for less magnifications, that is,
only 6X, 12X or rarely 20X. For the light adjustment purposes, a mirror is
attached to the limb under the stage. Mirror can be moved vertically with the
help of an adjustment screw. At the tip of the limb is present a folded arm, on
which a lens of definite magnification (6X, 12X, etc.) is fitted. Folded arm is
moved to keep the lens in the desired position on the stage.

The material to be viewed or dissected under a dissecting microscope is

placed on the stage. The eye is placed close to the lens. Folded arm is tilted to
bring the lens over the material. Light is adjusted by the movement of the

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

mirror. Focusing is done with the help of the adjustment screw of this
microscope

COMPOUND MICROSCOPE:
The compound microscope consists of two important parts namely, optical
parts and mechanical parts.
 The optical parts include ocular (eye piece), objective lens, condenser and mirror.
 The mechanical parts include draw tubes, body tube, nose piece, rough/coarse
adjustment, fine adjustment, arm, stage, base/foot, inclination joint etc.
THE OPTICAL PARTS:
 Ocular lens (eyepiece): It consist of two magnifying lenses through which object
will be observed. It is available in magnification with 5X, 10X, 15X and 20X.
 Objective lens: They are used to magnify the objects and are available in three
magnifications, 10X (low power) 45X (high power) and 100X (oil immersion).
 Condenser and iris diaphragm: It is present below the stage, moves up and
down and helps to open and close the shutter, so that the amount of light required
can be regulated. Abbey condenser is generally used.
 Mirror: It is fixed below the stage and is Plano-concave. The concave surface is
used for focusing.
MECHANICAL PARTS:
 Draw tube: It holds the ocular in proper position.
 Body tube: It is together with draw tube holds the ocular and objectives in proper
position at a distance from each other.
 Nose piece: It is a circular moving disk to which the objectives (10X, 45X & 100X
) are fixed. It is present at the lower end of the body tube.
 Rough/coarse adjustment: It moves the body tube up and down over a greater
distance and brings the object in focus.
 Fine adjustment: It moves the body tube gently for focusing and clear image of
the object is seen.
 Stage: It supports the slide on which the object is mounted. It has central opening
through which light is passed on to the object. There are spring clips to hold the
slide.
 Base/foot: It is horse-shoe shaped and supports the entire body of the microscope.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

 Inclination joint: It permits tilting of the upper part of the microscope towards
the eye to facilitate proper viewing.

USE OF OIL IMMERSION OBJECTIVE:

 Ensure that the eyepiece, objective, condenser and the mirror of the microscope
are cleaned with muslin cloth or tissue paper.
 Focus the microscope and observe the object under low power(10X) and high
power.
 Remove the high power objective and then place a drop of immersion oil or cedar
wood oil above the coverslip where the object is present and focus with oil
immersion objective(100X), gently using rough adjustment to view the object.
Then by using fine adjustment brilliant enlarged image of the specimen is seen.
 After viewing the image of the object, gently lift the oil immersion objective by
using rough adjustment, clean the objective lens with tissue paper.
 The specific magnification of the oil immersion objective is marked on it has
100X. The total magnification is calculated by multiplying the magnification of
the eyepiece and oil immersion objective is used.
Magnification of the eyepiece x magnification of oil immersion objective
10x X 100x = 1000x is the total magnification.

Department of Microbiology, Government Science College (Autonomous), Hassan 10

 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

EXPERIMENT No. 04
WORKING PRINCIPLES AND OPERATIONS OF BASIC
EQUIPMENTS OF MICROBIOLOGICAL LABORATORY

AUTOCLAVE:
Autoclave is an instrument which is used in microbiology laboratory to
sterilize the media and other related materials. The autoclave is used to sterilized
the glass wares, media, discarded cultures, metallic instruments, etc. There are
different kind of Autoclaves namely, horizontal and vertical. Depending upon the
requirement these can be employed for sterilization.

Principle:

Autoclave works on the principle of “latent heat”. The killing action of heat on
the organisms can be done by using increase in the steam in a closed system. The
water molecules become aggregated resulting in increase in their penetration.
General temperature employed for proper sterilization is 121.80C for 20 minutes
with the pressure of 15lb/inch2.

Construction:

The Autoclave is usually of pressure cooker type made up of gun metal sheets
which is supported in an iron case. It is closed by swing door which is fastened
by radical bolts tightly. The steam passes from below at the base. The side walls
are heated by the steam jacket. It has a provision to record the temperature and
pressure. It also consists of safety valve that guards against the accidents.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

Operation:

Sufficient water is poured in the autoclave to the required level. All materials
which has to be sterilized are kept inside in the basket provided. Door is locked,
and the exhaust valve is opened. Power cord is connected to heat up the coils.
Water boils and start to produce steam which attains the temperature around
1000C. Now the exhaust valve is closed. When the pressure of 15lb/inch2 reached
time is recorded. After 20 minutes Autoclave is switched off and exhaust valve
is opened.

After complete evacuation of steam, the door is opened. With the help of a
hand glove the materials are taken out.

Care should be taken not to open the autoclave soon after completing
sterilization. Because the outside air enters into the autoclave and possibility of
and then open the lid and taken out the materials to the working table.

HOT AIR OVEN:

It is important microbiology lab equipment, which sterilizes the material by
dry heat.

Principle: The high temperature of the dry heat causes dehydration of bacterial
cells and kills the microorganisms by inactivating the enzymes.

Construction and operation: It is consisting of an insulated cabinet which is held

at a constant temperature by means of electricity and regulated by a thermostat.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

The inner surface of the steel cabinet wall is insulated by thick asbestos lining
to reduce the radiation of heat. There are a number of adjustable perforated racks
or shells to keep the materials to be sterilized.
The electrical connection is made at the bottom. There is a thermostat, pilot
lamps, indicator on the front side. There is an air circulating fan placed inside the
unit. There is a thermometer inside the cabinet to note the temperature. At the
sides there are vents to escape excess heat.
The hot air oven is used to sterilize the glass wares, metallic instruments, Petri
plate, test tubes, pipettes, etc. the instrument is operated at a temperature of 160-
1800c for about 1-2 hours allow it to cool.
Care should be taken not to open the hot air oven soon after completing
sterilization. Because the outside the air enters into the hot air oven and possibility
of cracking the glass wares. Take out the materials to the working tables.

INCUBATOR:
It is important microbiology laboratory equipment used to incubate the plated
microbial cultures at appropriate temperature for particular days. The bacterial
growth takes 24-48 hours at 370c and fungal growth requires room temperature
250c for 4-5 days.
Microbiological incubators maintain constant temperature and operates from
room temperature to 2000C. To sterilize, the 1600C temperature is maintained for
1 hr. It consists of a steel cabinet or box plated with a door with a glass panel to
see the objects inside. Inner chamber lining of door are insulated with asbestos
which prevents the escape air outside. The inner chamber is having 4-5 adjustable
perforated racks to keep the culture plates, flasks, test tubes etc. for incubation.

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

The incubator is provided with a thermometer, temperature adjustment

indicator lamp, power button, and heating coil with electrical connection.

pH METER:
A pH meter is a scientific instrument that measures the hydrogen-
ion activity in water-based solutions, indicating its acidity or alkalinity expressed
as pH. The pH meter measures the difference in electrical potential between a pH
electrode and a reference electrode, and so the pH meter is sometimes referred to
as a "potentiometric pH meter". The difference in electrical potential relates to the
acidity or pH of the solution. The pH meter is used in many applications ranging
from laboratory experimentation to quality control.

SPECTROPHOTOMETER:
The spectrophotometer technique is to measure light intensity as a function of
wavelength. It does this by diffracting the light beam into a spectrum of
wavelengths, detecting the intensities with a charge-coupled device, and
displaying the results as a graph on the detector and then on the display device.
1. In the spectrophotometer, a prism (or) grating is used to split the incident beam
into different wavelengths.
2. By suitable mechanisms, waves of specific wavelengths can be manipulated to
fall on the test solution. The range of the wavelengths of the incident light can
be as low as 1 to 2nm.
3. The spectrophotometer is useful for measuring the absorption spectrum of a
compound, that is, the absorption of light by a solution at each wavelength.
Some of the major applications of spectrophotometers include the following:
 Detection of concentration of substances

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 I B.SC. I SEMESTER, MICROBIOLOGY LABORATORY MANUAL

 Detection of impurities
 Structure elucidation of organic compounds
 Monitoring dissolved oxygen content in freshwater and marine ecosystems
 Characterization of proteins
 Detection of functional groups
 Respiratory gas analysis in hospitals
 Molecular weight determination of compounds
 The visible and UV spectrophotometer may be used to identify classes of
compounds in both the pure state and in biological preparations.

COLORIMETER:
Colorimeter is an instrument used for the measurement of a compound or a group
of compounds present in a complex mixture. The property of colorimetric
analyses is to determine the intensity or concentration of compounds in coloured
solution.

This is done by passing light of specific wavelength of visible spectrum through

the solution in a photoelectric colorimeter instrument and observe the
galvanometric reading of reflection sensitizing the quantity of light absorbed.

Based on the nature of colour compounds, specific light filters are used. Three
types of filters are available — blue, green and red — with corresponding light
wavelength transmission rays from 470-490 nm, 500-530 nm and 620-680 nm,
respectively.

There are two fundamental laws of absorption which are highly important in
colorimetric estimation. These are Lambert’s law and Beer’s law. Lambert’s law

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states that when monochromatic light passes through a solution of constant

concentration, the absorption by the solution is directly proportional to the length
of the solution. In contrary, Beer’s law states that when monochromatic light
passes through a solution of constant length, the absorption by the solution is
directly proportional to the concentration of the solution.

VORTEX:
A vortex mixer is a simple device used commonly in laboratories to mix small
vials of liquid. It consists of an electric motor with the drive shaft oriented
vertically and attached to a cupped rubber piece mounted slightly off-center. As
the motor runs the rubber piece oscillates rapidly in a circular motion. When a
test tube or other appropriate container is pressed into the rubber cup the motion
is transmitted to the liquid inside and a vortex is created. Most vortex mixers are
designed with 2 or 4-plate formats, have variable speed settings ranging from
100 to 3,200 rpm, and can be set to run continuously, or to run only when
downward pressure is applied to the rubber piece.

MAGNETIC STIRRER:
A magnetic stirrer is a device widely used in laboratories and consists of a
rotating magnet or a stationary electromagnet that creates a rotating magnetic
field. This device is used to make a stir bar, immerse in a liquid, quickly spin, or
stirring or mixing a solution, for example. A magnetic stirring system generally
includes a coupled heating system for heating the liquid

Department of Microbiology, Government Science College (Autonomous), Hassan 16