BBCEL-144:

BASIC
Indira Gandhi National
Open University
MICROBIOLOGY
School of Sciences
(PRACTICAL)

BASIC MICROBIOLOGY
Experiment 1
Microbiology Lab Practices and Biosafety 5

Experiment 2
To Study the Principle and Applications of Important
Instruments 7

Experiment 3
Preparation and Sterilisation of Culture Media for
Bacterial Cultivation 21
Experiment 4
To Study the Shapes of Bacteria, Fungi, Algae and
Protozoa from Permanent Slides or Pictographs 27

Experiment 5
Differential Staining of Bacteria: Gram Staining Method 35

Experiment 6
Isolation of Pure Cultures of Bacteria by
Streak Plate Method 43

Experiment 7
Qualitative Detection of Antibiotic Action by Kirby-Bauer
Antibiotic Sensitivity Test 49
 PROGRAMME AND COURSE DESIGN COMMITTEE

Prof. Bechan Sharma Prof. Ranjit K. Mishra Prof. Parvesh Bubber

Dept. of Biochemistry Dept. of Biochemistry SOS, IGNOU
University of Allahabad University of Lucknow
Dr. M. Abdul Kareem
Prof. Reena Gupta Prof. Sanjeev Puri SOS, IGNOU
Dept. of Biotechnology UIET, Panjab University
H.P. University, Shimla Dr. Arvind Kumar Shakya
SOS, IGNOU
Prof. D. V. Devaraju Prof.Seemi Farhat Basir
Dept. of Biochemistry Dept. of Bio Sciences Dr. Maneesha Pandey
Bangalore University Jamia Milia Islamia SOS, IGNOU

Dr. Sunita Joshi

Dept. of Biochemistry Prof. Vijayshri Dr. Seema Kalra
Daulat Ram College School of Sciences SOS, IGNOU
Univ. of Delhi IGNOU, New Delhi

COURSE PREPARATION TEAM

Content Editor Content Writer
Prof. Geeta Shukla Dr. Sunita Joshi (Retd)
Department of Microbiology Dept. of Biochemistry
Panjab University, Chandigarh Daulat Ram College
Univ. of Delhi
Course Coordinator: Dr. Seema Kalra, Assistant Professor, SOS, IGNOU
Cover Page and Graphic Design: Dr. Seema Kalra, Email: seemakalra@ignou.ac.in
Print Production Team
Sh. Rajiv Giridhar Sh. Hemant
Assistant Registrar (Pub.) S.O (Pub.)
MPDD, IGNOU MPDD, IGNOU

Acknowledgement: Mr. Sumit Verma for CRC Preparation and Word Processing.
June, 2021
© Indira Gandhi National Open University, 2021
ISBN:
Disclaimer: Any materials adapted from web-based resources in this module are being used for educational
purposes only and not for commercial purposes.
All rights reserved. No part of this work may be reproduced in any form, by mimeograph or any other means,
without permission in writing from the Copyright holder.
Printed and published on behalf of Indira Gandhi National Open University, New Delhi by
Prof. Sujatha Varma, Director, School of Sciences, IGNOU
Further information on the Indira Gandhi National Open University courses may be obtained from the
University’s office at MaidanGarhi, New Delhi-110 068 or the official website of IGNOU at www.ignou.ac.in.
Printed and published on behalf of Indira Gandhi National Open University, New Delhi by Director, SOS,
IGNOU.
Printed at

2
 BBCEL-144: BASIC MICROBIOLOGY
Dear learners, welcome to the practical sessions of the course “Basic Microbiology”. The
lab exercises and experiments provided in this manual are based on the syllabus that you
have studied in BBCET-143. Throughout the theory course you were taught the taxonomic
classification of microorganisms and their characteristics. You learnt in details about their
life cycles, their applications in health and diseases as well as food and other industries.
Methods of culturing these tiny organisms in lab and methods of controlling their growth
were also discussed.

This lab course is worth 2 Credits and consists of seven laboratory experiments. The
experiments are designed in such a way that you are able to experience and connect the
theoretical concepts explained so far. Basic principle on which these experiments are
based has also been explained in each exercise.

Expected learning outcomes:

The broad objective of this lab course is to enable you to:

• learn and practise rules to be followed in a microbiology and biosafety lab;

• understand the Principle and applications of some important instruments used in a

microbiology lab;

• prepare and sterilize the culture media used for bacterial cultivation;

• study the shapes of bacteria, fungi, algae and protozoa from permanent slides;

• isolate pure bacterial cultures using streak plate method; and

• perform Kirby-Bauer antibiotic sensitivity test for qualitative detection of antibiotic

action.

Study Guide
We advise you to go through respective blocks of BBCET-143 before you come to attend
the practical sessions. This will enable you to easily understand the purpose of doing
experiments and their applications. You should also read the principle of each experiment
of this course along with procedure before you start performing the experiment. It is
always good to prepare all the reagents freshly and store them under appropriate storage
conditions. Adhere to all the safety measures and follow the safety instructions while
handling the reagents. One of the good laboratory practices is to maintain your log books
up-to-date i.e., enter the observations made while performing the experiments. Carry this
laboratory manual and your log book during lab sessions.

Like all other IGNOU laboratory courses, this is an intensive residential exercise requiring
one week for completing 2 credits. Everyday there will be two laboratory sessions of 4
hours each. So there will be a total of 14 sessions. The first session will be introductory
and the remaining 2nd to 12th sessions will be based on the exercises given in the course.
A schedule for laboratory exercises will be given to you in the first session. Sessions 1 to
12 will have guided exercises under the supervision of the academic counsellor. The last
two sessions i.e., 13th and 14th will be unguided sessions and that would pertain to
evaluation for the term end examination. In each session you will perform exercises for 3
hours and in the remaining 1 hour you are advised to complete your practical note book.
3
 The laboratory notebook must be submitted to the counsellor for corrections and grading.
70% marks have been assigned for doing the experiments and recording them properly.
You should be aware that due to time constraint as you will have limited access to
laboratory work; therefore, you are required not to miss any of the laboratory sessions.

Performance during the practical sessions will be graded and you will have to appear for
the viva-voce at the end of the practical session. At the end of the laboratory session you
would be asked to perform the assigned experiment, which will be graded. Final
assessment will be made based on the continuous performance during the laboratory
sessions, maintenance of log books and records followed by viva-voce. 30% marks are
reserved for the assigned experiments.

For the better understanding of how to use laboratory apparatus or perform experiments,
few video links may be provided where ever available. There might be a slight difference
in the steps or procedure being explained in the video when compared to the procedure
provided in this self-instructional material. However, the principles and reagents remain
same. Hence, there is no need to worry about slight modifications adopted in the
procedure.
We wish you best in this endeavour!!

IMPORTANT INFORMATION

• Attendance is compulsory in the Laboratory Course work held

generally at the Study Centre.
• The Laboratory Course is worth 2 credits to be completed over 7
days duration.
6 days of Guided Laboratory work
1 day for the Unguided Laboratory work
• To successfully complete the laboratory course you will have to
pass (at least 35% marks) in the Guided and Unguided
components separately.

4
Experiment 1 Microbiology Lab Practices and Biosafety

EXPERIMENT 1
MICROBIOLOGY LAB
PRACTICES AND
BIOSAFETY

Most microbiology experiments use living organisms which makes it

imperative to follow safety precautions. Although laboratory cultures have
reduced virulence (disease causing power) due to long term sub culturing and
maintenance in artificial media, yet it is advisable to treat them as potential
pathogens. Generally care must be taken to avoid contamination of pure
(axenic) cultures during transfers and to minimise laboratory accidents. All
these experiments are performed using aseptic techniques.

Some general rules that one has to know and adhere to for a safe and
productive laboratory experience include:

At the beginning and completion of every lab exercise always clean the
laminar flow and work bench with 70% alcohol or other disinfectant
provided by the instructor.

Put ‘ON’ the UV light of the laminar flow for 10min before starting aseptic
transfers. Do not keep microbial cultures in when UV light is on.

Wash your hands with a detergent before leaving the laboratory.

Do not leave contaminated used items on the work bench. Take care to
either decontaminate before discarding (disposable tips and needles;
contaminated cultures) or sterilise (inoculating loops, forceps) them prior
to reuse.

Make it a habit to wear lab coat while working to protect your clothes
from accidental microbial contamination or spilling of reagents /stains.
Wear gloves while working. Protect your eyes with glasses / goggles.
Always wear closed shoes in the lab. Remove protecting clothing (lab
coat) before leaving the laboratory.

Wear surgical masks or face shields when working with biohazardous

substances.

Eating, smoking and drinking are prohibited.

5
BBCEL-144 Basic Microbiology (Practical)

Never mouth pipette cultures or reagents. For pipetting use auto pipettes
or mechanical pipetting aid (rubber bulbs).

Label the cultures / reagents with self-stick labels. Avoid licking labels.

Plan your experiment well to avoid unnecessary movement and

discussions as these can lead to errors and at times uncalled for
accidents.

Wear disposable gloves while handling body fluids of unknown origin.

Handle alcohol / other inflammable solvents with care. Never dip a hot
loop / L-shaped glass rod into an alcohol containing beaker. It may catch
fire.

Report all spills and broken glassware immediately to your instructor and
cleanup according to his instructions.

Learn safe handling of biohazardous material and management of crisis.

Never apply cosmetics / insert contact lenses or put objects such as

pencils in the mouth.

It is advisable to tie back long hair or cover your head with a paper cap.

The laboratory must be equipped with emergency measures such as eye

wash solution, first aid kit, fire extinguisher in case of mishap. Their
location and operation should be known to everyone.

Always carry cultures in sturdy racks without overloading.

The lab must maintain a user’s log book for all equipments. Apart from
providing usual details of date, time, duration and operator’s name and
contact details, the user must inform about the nature of material or
culture for which the instrument was used.

It is equally important to let the technician / instructor know if an

instrument stops working or spillage occurred during its operation.

Record all observations meticulously in a separate note book. Do away

with the habit of recording observations on loose sheets / at a later date.

Never bolt the lab room, just close the door only.

Every student must go through these basic microbiology practices and agree
to stringently follow them. Before proceeding to work you are expected to sign
this sheet before your instructor.

----------------------------------------- ------------------------------------------

Name & signature of student Counter-signed by the instructor

6
Experiment 2 To Study the Principle and Applications of Important Instruments

EXPERIMENT 2
TO STUDY THE PRINCIPLE AND
APPLICATIONS OF
IMPORTANT
INSTRUMENTS

Structure
2.1 Introduction 2.4 Operation of Instruments

Expected Learning Outcomes 2.5 Precautions

2.2 Principle and Applications

2.3 Requirements

2.1 INTRODUCTION
A characteristic feature of every microbiology laboratory is to provide aseptic
conditions for transfer and working with microorganisms. In this exercise you
will learn about some of the common instruments needed to have a basic set
up for studying, cultivating and maintaining microbes. These include
autoclave, hot air oven, biological safety cabinet (laminar air flow bench), pH
meter, bacteriological incubator, BOD incubator and microscope. The purpose
of dealing with them separately is to spend time in understanding the principle,
operation and applications of each one of them before embarking on actual
experiments.

Expected Learning Outcomes

After studying this experiment, you should be able to:

explain the instrumentation and principle behind each instrument;

independently operate them;

indicate the application(s) of these instruments in microbiology;

describe the variations available and their specific usage, if any; and

handle and maintain them based on the instructions provided by the
manufacturer and precautions mentioned in the end. 7
BBCEL-144 Basic Microbiology (Practical)

2.2 PRINCIPLE AND APPLICATIONS

All microbiological investigations in artificial conditions require sterilised
medium, glassware, inoculation needles / loops, forceps and at times heat
labile substances. There are different ways available for sterilisation; some of
which are for specific components. The methods commonly used include dry /
moist heat, filtration (membrane filters), chemicals (ethylene oxide) and
irradiation with γ-rays.

Temperature sensitivity of biological processes is exploited quite often to

destroy microorganisms / resistant spores present in culture medium or
sticking to glassware. Both dry and moist heat is effective but dry heating
requires higher temperature (160-1800C) and longer duration (1½ to 3 hrs) as
compared to moist heat. It penetrates slowly and unevenly. The process of dry
heat sterilisation was originally developed by L. Pasteur. For steam under
pressure sterilisation, medium, glassware, autoclavable plastic tubes and tips,
equipment are autoclaved at 1210C for 15-20 min at 15lb /in2. Moist heat has
greater penetrating power; coagulates proteins and kills both vegetative cells
and resistant spores. In case the medium has components that cannot
withstand these conditions, they are sterilised separately by other means and
then added aseptically.

Dry heat sterilisation is carried out in hot air oven which is an insulated
cabinet with perforated shelves and surrounded by heating filament and fans
fixed on the walls (Fig.2.1). In this hot air is created by forced air circulation
(mechanical convection) with a fan that in turn maintains uniform temperature
throughout the oven. This prevents the rising of hot towards the top while
keeping the cool air at the bottom as compared to static hot air ovens which
take longer to heat up. A hot air oven is suitable for heat stable articles such
as glassware and metal instruments (forceps, blades, scalpel, etc). Dry heat
kills by oxidation of essential constituents and irreversible protein denaturation.

Fig. 2.1: Hot air oven.

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Experiment 2 To Study the Principle and Applications of Important Instruments

An autoclave or a pressure cooker developed by Charles Chamberland is

used for moist heat sterilisation. It is a double walled metal vessel in which
steam is pressurised in the outer jacket and then released into the inner
chamber from which air is displaced (Fig. 2.2). The desired temperature
(1210C) is achieved at pressure of 15lb /in2. Hoever, some heat labile
substances are autoclaved at 110ºC for 20min at 10lb/in2, e.g. medium
containing sugar so as to prevent carmelization of sugar.

Fig. 2.2: Types of autoclave- vertical and horizontal.

Other variations of moist heat for sterilisation include tyndallisation and

pasteurization. In tyndallisation the material is exposed to free flowing steam
at 1000C for 20min on three consecutive days with intermittent incubation at
370C.The steam kills vegetative cells while spores germinate during incubation
which is killed in the next round of exposure to steam. Repeating these two
steps ensures destruction of vegetative cells and resistant spores. The method
consumes much more time than autoclaving. It is suitable for sterilising
thermolabile chemicals. Pasteurisation, on the other hand uses lower
temperatures (710C for 15 seconds or 630C for 30min). It is used to destroy
pathogens and spoilage causing microorganisms from thermolabile products
such as milk and beer.

Biological safety cabinet (BSC) or tissue culture hood is an enclosed

workspace with a ventilated hood. It provides protection to the investigator/
laboratory personnel and environment from pathogenic organisms. The hood
minimises the formation of aerosols and are equipped with HEPA (high
efficiency particulate air) filters that filter air moving out of the cabinet. Different
types of cabinets are available which vary in the kind of protection they provide
for example class I cabinets protect the investigator and environment but not
the sample (Fig. 2.3).

Fig. 2.3: Biological safety cabinet. / airscience.com

9
BBCEL-144 Basic Microbiology (Practical)

Laminar air flow (LAF) bench or cabinet is a type of BSC that protects only
the sample and not the user (Fig. 2.4). It is an ideal flat workstation for aseptic
pouring of media and transfer of cultures. The front of the cabinet has a glass
shield or two openings for the user’s hand to enter the cabinet. To generate a
sterile chamber LAF is equipped with filter pads (pre filters), fan/ blower, UV
lamp and HEPA (high efficiency particulate air) filters. As air passes through
the filter pad, it traps dust particles and some microbes. The fan /blower allow
movement of pre filtered air towards the HEPA filter (traps remaining
microbes) and then filtered air is blown in a smooth laminar (stream line) flow
towards the user. The LAF is enclosed on the sides and positive pressure is
maintained to prevent entry of contaminants from the outside. A germicidal UV
lamp if present is used to sterilise the cabinet and contents before the
experiment.

Fig. 2.4: Horizontal and vertical laminar air flow (LAF) cabinet.

A bacteriological incubator provides optimal conditions primarily constant

temperature, for culturing microbes in the laboratory. The main body of an
incubator is double walled enclosure, available in different sizes. It is insulated
by filling the space between the two walls with glass wool. This helps to
reduce heat loss and energy consumption. The inner wall of the incubator has
projections to support adjustable perforated shelves for placing petri plates
and other culture vessels. The incubator has two doors; the inner one is made
of glass that allows us to view the contents without opening the door while the
outer opaque door closes the insulated unit. It also has air ventilators on the
sides.

The temperature is set and maintained by a thermostat via alternate on

(heating) and of no heating cycles which can be read from the thermometer
present on the outer wall. The perforated shelves facilitate movement of hot
air. It is always better to keep a beaker filled with water to prevent dehydration
10 of the medium by dry heat.
Experiment 2 To Study the Principle and Applications of Important Instruments

Many types of incubators are available that fulfil specific requirements of

organisms such as gas / humidity control / HEPA filters etc. The incubators
commonly used in microbiology are shown in Fig.2.5. As compared to a
bacteriological incubator, a BOD (biological oxygen demand) incubator has
both heating and cooling system; it is usually operated at a lower temperature
and is used for cultivation of fungi or even Protozoa like Leishmania. An
orbital shaker is also used for cultivation of microbes. As the culture vessel is
agitated it results in increased aeration, uniform transfer of heat, mixing of
cells and medium components and above all faster growth. But it can be used
only for organisms that require oxygen and are grown in broth cultures.

Bacteriological incubator BOD incubator Orbital shaker

Fig. 2.5: Types of incubators in microbiology labs.

A pH meter is a reliable and convenient method for measuring hydrogen ion

concentration of aqueous solutions to determine the acidity or alkalinity of the
solution. For most solutions this concentration is very low therefore, Sorensen
(1909) introduced the concept pH as a convenient way to express H+
concentration. It is defined as the negative log of H+ ion activity (nearly same
as concentration in dilute solutions). You can see from the pH scale (0-14), a
solution is pH neutral if it has a value of 7, below and above that value it is
increasingly acidic or basic, respectively (Fig. 2.6). At 250C pure water is
weakly ionised and the concentration of H+ is only 10-7 which corresponds to
pH 7 (neutral).

The pH scale

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Acidic Caustic

Neutral

Fig. 2.6: pH scale.

The measurement of pH is a routinely required for providing optimal conditions

for microbial growth as biochemical reactions are pH dependent. A pH meter
measures the voltage between two electrodes (one of which is ion selective)
placed in the aqueous solution (Fig. 2.7). The voltage is then translated into
pH by the instrument. 11
BBCEL-144 Basic Microbiology (Practical)

The ion selective electrode (ISE) is a glass electrode whose thin blub is made
up of high conductivity borosilicate glass, permeable to H+ ions and not other
ions. The bulb is filled with 0.1M HCl, connected to a platinum lead via Ag-
AgCl electrode. The hydrogen ions move from a solution of high to low
concentration, resulting in electric potential across the glass. The reference
electrode commonly used is calomel electrode (Hg-HgCl2 paste in saturated
KCl), encased in a glass tube impermeable to H+ ions (pH independent). Now
a day’s pH meters with both glass and reference electrodes in a single unit are
also available that can measure pH in smaller volumes.

Fig. 2.7: Digital pH meter and the two electrodes of a pH meter.

A compound microscope is indispensable for observing the microbial world

(Fig. 2.8). It is equipped with two separate lens systems (objective and ocular)
that have the effect of achieving higher magnification than a single lens in a
simple microscope. Most microscopes are parfocal that means when one lens
is in focus, the other lenses will also have the same focal length and requires
minor adjustments as we switch lenses.

The lens system nearest the specimen is the objective; it focuses rays of light
from the specimen to produce a real, inverted and magnified image inside the
body of the tube. It also plays an important role in producing a high resolution
clear image. Generally there are three objectives (10x, 40x and 100x) of
decreasing focal length (shorter the focal length of the objective, shorter is the
working distance of the lens), attached to a revolving nose piece. The
objective lens is assembled using a number of lenses that correct for
chromatic and spherical aberrations. The ocular or eye piece (10x) magnifies
the real image and produces a virtual image that is seen by the eye. The total
magnification is the product of objective and ocular magnification and the
12 maximum useful magnification of an image is usually set at 1000 x NA.
Experiment 2 To Study the Principle and Applications of Important Instruments

Fig. 2.8: Bright field compound microscope.

The microscope has two sets of knobs for coarse and fine adjustments. They
also provide stage adjustment controls for moving the stage horizontally and
vertically. In order to have full utilisation of magnification and resolution, the
microscope is illuminated with artificial light source (tungsten-halogen bulb).
The condenser is located below the stage. It has two lenses that concentrate
the light (both oblique and direct) coming from the lamp onto the sample. It
can be moved up and down. The size of the light cone is controlled by iris
diaphragm, present above the condenser. With oil immersion objective (100x),
the working distance is least (1.8mm) and the iris diaphragm is opened more.

In addition to magnification, resolution and contrast are two other factors in

generating a good quality image. At any given magnification, the resolving
ability of the microscope would determine how clearly you can see and
distinguish between two close points. It is directly proportional to the
wavelength of light used and inversely proportional to the numerical aperture
(NA) of the microscope objective. These values are related by the Abbe’s
equation (including Rayleigh’s correction)

Where, λ = wavelength of light used

n = refractive index of medium between the specimen and objective lens

α = half aperture angle in radians.

n sin α = numerical aperture (NA) of objective,

The shorter is the wavelength of light (blue light will have a greater resolution
than red light), the finer the detail revealed by the objective. You know the
visible spectrum is narrow (400-700nm) therefore decrease in wavelength to
increase resolution has a limitation. The other parameter that can be altered is
numerical aperture which is a measure of the resolving power of a microscope
objective. It is equal to the product of the refractive index (light bending power) 13
BBCEL-144 Basic Microbiology (Practical)

of the medium (n) between the specimen and the objective and the sine of the
angle (sin α) between the outermost ray entering the objective and the optical
axis. Up to a certain limit, an increase in numerical aperture of the objective
lens increases resolving power. Another way by which the percentage of light
rays passing from the specimen into the objective lens can be increased is by
using immersion oil (has same refractive index as glass; more than air)
between the slide and the lens. It works by decreasing refraction. Even then
the best compound microscope cannot resolve parts of a specimen that are
closer than 200nm

Finally the object must possess certain degree of contrast with the surrounding
medium in order to be seen clearly through the microscope. The degree of
contrast of an image can be increased by staining the specimen and / or by
optimizing the optical settings in the microscope.

2.3 REQUIREMENTS

Autoclave

Bright field compound microscope

Hot air oven

Biological safety cabinet (BSC)

Incubator

BOD incubator

pH meter

medium

Packed glassware

Permanent slide

Immersion oil

2.4 OPERATION OF INSTRUMENTS

(a) Autoclave: Follow the operating instructions given below: (include
changes depending on the model).

i. Twist the lid of the autoclave anticlockwise and then lift it. The lid has a
pressure gauge, pressure release unit (whistle) and safety valve.

ii. Fill water in the base chamber (1-1.5 litres).

iii. Load the autoclave with material to be autoclaved only after proper
washing, drying and packing (flasks, petri plates, media, water, etc).
Take care that none of the items touch the top or sides of the autoclave.

iv. Check that the rubber seal is in place and in good condition.

14 v. Close and lock the lid by turning it clockwise, ensuring it is air tight.
Experiment 2 To Study the Principle and Applications of Important Instruments

vi. Turn on the power. The heater begins to heat the water in the chamber.

vii. Open the air outlet so that air can be displaced by the incoming steam.

viii. Open the steam supply valve to allow steam to enter the inner chamber.

ix. Monitor the temperature and close the air outlet when the temperature is
1000C. Depending on the model the valve may close automatically.

x. To further increase the temperature to 1210C, let the pressure reach15lb

/in2. Present day autoclaves have adjustable pressure regulators.

xi. Once the pressure is reached the whistle blows and lifts to release
vapours from the chamber.

xii. When the thermometer shows 1210C, note the time and autoclave for
15-20min at this temperature.

xiii. At the end of 20min, close the steam water supply / simply switch off and
wait for the pressure to come to zero (indicates no more steam). If you
are not autoclaving liquids then you can open the air outlet (operating
valve) to release the pressure rapidly.

xiv. Open the autoclave door by twisting it anti clock wise and lift the lid
carefully. Stand behind the door when opening it.

xv. Take out the autoclaved material. Wear heat insulating gloves, if
required or allow it to cool for some time before removing.

xvi. Remove all water from the base chamber, dry it, close the autoclave and
disconnect.

(b) Hot air oven

i. Switch ‘ON’ the power supply.

ii. The temperature controller will display the chamber temperature.

iii. Keep the items to be sterilised in the shelves.

iv. Set the required temperature by pushing the ‘PUSH’ switch and then
release it.

v. Indicator bulb glows indicating that the heater is ‘ON’.

vi. Switch ‘ON’ the fan switch for air circulation.

vii. Once the desired temperature is achieved, sterilise for 1½ hrs or more at
the set temperature (160-1700C).

viii. Wear heat insulating gloves to offload sterilised material.

(c) Biological safety cabinet (BSC)

i. Load the cabinet with all needed material.

ii. Turn ‘ON’ the BSC and allow it to run for 15-20 minutes.

iii. Check inward air flow with a piece of paper. 15

BBCEL-144 Basic Microbiology (Practical)

iv. Begin working.

v. After completion, decontaminate all items to be taken out from the cabinet.

vi. Decontaminate the interior of BSC and allow it to run for 10-15min.

vii. Switch off.

(d) Laminar air flow (LAF) bench

i. Before putting on the UV lamp for surface sterilisation, take care that
none of the material placed on the bench is susceptible to UV rays.

ii. Close the glass shield.

iii. Put on the UV lamp for 15-20 minutes.

iv. Switch off the UV lamp and after few minutes put on the airflow.

v. Open the glass shield and put on the fluorescent light.

vi. Disinfect your hands with a disinfectant and wipe the work bench with
the same.

vii. Start working.

viii. After completion, the air flow, fluorescent lamp and glass shield are
closed. Clear the LAF work bench.

(e) Incubator / BOD

i. Switch on the incubator.

ii. Set the desired temperature and allow time for heating up, keeping the
door closed.

iii. Check the temperature from the thermometer.

iv. Place your cultures in the perforated shelves for incubation. The Petri
plates are kept in an inverted position. They should be preferably sealed
if incubation is continued for longer duration. It is always a good practice
to check in between that the temperature is maintained.

v. Close the door.

vi. Observe the plates after incubating for the required duration.

(f) pH meter

i. Put ‘ON’ the pH meter.

ii. Calibrate the pH meter using buffers (pH 4, 7 and 10) of known pH at the
temperature of the test solution.

iii. Switch the function selector from the Standby position to pH position.

iv. Set the temperature with temperature knob.

16
Experiment 2 To Study the Principle and Applications of Important Instruments

v. Press STD button for calibration.

vi. Take out the electrode from storage solution, rinse with distilled water
and blot dry.

vii. Immerse the electrode in pH 4 buffer and measure its pH.

viii. Adjust display to read 4 with calibration knob 1.

ix. Repeat calibration with pH 10 buffer.

x. Remove the electrode from standard buffer, rinse with water, blot dry.

xi. Measure the pH of the first buffer again. If it does not read 4, readjust
calibration knob 1. Switch to standby.

xii. Remove the electrodes from standardisation buffer, rinse and blot dry.

xiii. Now place the electrodes in unknown solution.

xiv. Switch function selector from Standby mode to pH.

xv. Read pH of the solution.

xvi. Return to Standby mode.

xvii. Remove from test solution, rinse and shift to distilled water for storage.

xviii. Switch off the instrument.

(g) Light microscope

i. Connect the microscope to the power supply.

ii. Place the slide on the stage and try to position its centre over the hole in
the stage using adjustment control.

iii. Adjust the light source.

iv. With the low power objective in position, lower the body tube using
coarse adjustment; stop once it is about ¼ inch from the slide.

v. Observe through the eye piece and slowly raise the objective with
coarse adjustment, until the specimen is almost focussed.

vi. Now use fine adjustment knob to bring the specimen into sharp focus.

vii. Adjust the iris diaphragm and condenser to get optimal light intensity.
Keep the condenser close to the stage especially when using oil
immersion objective.

viii. Examine the specimen in low power objective and centre the part of the
specimen for observation at higher magnification.

ix. Shift the objective lens by rotating the nosepiece until the objective is set
into position.

x. Focus using coarse and fine adjustment knobs as done with low power
objective and examine the specimen.
17
BBCEL-144 Basic Microbiology (Practical)

xi. Use the same procedure for oil immersion objective.

xii. After completing observations remove the slide.

xiii. Clean the oil from the objective lens with lens paper.

xiv. Shift to low power objective; disconnect power supply and cover the
microscope.

2.5 PRECAUTIONS

Allow the pressure to come down gradually when liquids are autoclaved
otherwise it may lead to wetting of plugs and / blowing out.

False pressure should be removed.

It is essential to measure sterilisation time when the temperature

reaches 1210C rather than from pressure. Air must be completely
removed; otherwise you will not achieve the desired temperature at 15lb/
in2 pressure.

Preferably sterilise in small units; large loads need longer time for
sterilisation.

Plug flasks / tubes with cotton plugs or paper. In case of plastic / screw
caps, do not tighten them; allow air to escape.

Do not overload the autoclave or hot air oven.

The liquids in flasks / tubes for autoclaving should only be filled ⅔ of the
total volume.

Decontaminate waste by autoclaving separately.

Aluminium foil should never be used to pack the glassware.

Place all required materials in BSC before beginning.

In LAF bench do not put on UV lamp and airflow simultaneously.

Do not work while UV lamp is ‘ON’.

The work bench must be cleared after the experiment and never leave
contaminated plates / other glass ware.

Wear lab coat and gloves while working.

The growth of organisms is temperature sensitive. Avoid opening the

incubator repeatedly as this will result in temperature fluctuations.

Do not leave contaminated cultures in the incubator.

Keep sterile water in the incubator to prevent drying of culture media.

Regularly clean the incubator.

18
Incubate the Petri plates in inverted position.

Experiment 2 To Study the Principle and Applications of Important Instruments

Calibrate the pH meter with buffers of known pH at the temperature of

the test solution.

Before use always check that the standard buffers are free of
contamination.

The pH sensitive glass electrode is extremely fragile; handle it carefully

especially when lowering it into solutions.

Observe the objective lens from the side while lowering it and then focus
by raising the body tube slowly.

Never focus downward while looking through the eye piece.

Do not touch the objective lenses; if they become dirty wipe them gently
with lens paper.

Always remove oil from the oil immersion objective after use with lens
paper. If it gets dried remove it carefully with xylol. Follow the same
procedure if by mistake oil comes in contact with lower power objectives.

While focussing oil immersion objective, do not let the objective touch
the slide.

Keep the stage of the microscope clean and dry.

Do not let the objective lens touch the slide / cover slip.

When not in use bring the lower power objective in focussing position.

SAQ 1
1. What is the difference between a bacteriological and BOD incubator?

2. Define the terms:

(a) Par focal (b) Resolving power of a lens (c) Working distance

3. What is the pH of 10-8 N HCl?

4. Is wet heat or dry heat more effective as a sterilising agent, why?

5. Which articles can be safely sterilised on a hot air oven?

ANSWERS
1. Bacteriological incubator: Used for cultivation of bacteria / has only
heating system; set temperature may rise if outside temperature
rises/ generally operated at 30- 350C / need to keep water to prevent
drying

BOD incubator: Used for cultivation of fungi; BOD test / has both
heating and cooling options; better at maintaining set temperatures /
usually operated at lower temperatures (20-250C) / temperature is
external environment independent /can humidity is controlled / 19
BBCEL-144 Basic Microbiology (Practical)

2. (a) Par focal: When one lens is in focus then other lenses will also
have the same focal length and moving from low to high power will
require at best minor adjustments.

(b) Resolving power of a lens is its ability to resolve two close-by

objects as discrete entities. It is dependent on the wavelength of
light and NA of each lens.

(c) Working distance: It is the distance between the objective lens

and the slide; it decreases as magnification of the lens increases.

3. It will be less than 7 because at such low dilutions, the contribution of H+

ions from water also becomes significant.

4. Wet (moist) heat is a better sterilising agent than dry heat because it has
greater penetrating power; coagulates proteins and kills both vegetative
cells and resistant spores. It sterilises faster (15-20min) and at lower
temperatures (1210C).

5. Glassware and metal instruments (forceps, blades and scalpel) can be

sterilised in a hot air oven.

20
Experiment 3 Preparation and Sterilisation of Culture Media for Bacterial Cultivation

EXPERIMENT 3
PREPARATION AND
STERILISATION OF
CULTURE MEDIA FOR
BACTERIAL
CULTIVATION

Structure
3.1 Introduction 3.4 Protocol
Expected Learning Outcomes 3.5 Observations
3.2 Principle 3.6 Results
3.3 Requirements 3.7 Precautions

3.1 INTRODUCTION
The growth and maintenance of microorganisms depend on availability of
nutrients and optimal environmental conditions such as temperature and pH. A
culture medium is a source of nutrients that supports the growth of either most
or specific group of microbes. It is a sterilised aqueous solution suitable for
laboratory cultivation, maintenance and to conduct various studies. In
addition, culture media differ in their physical form and chemical composition.
The former includes liquid or broth; semisolid and solid media while the latter
can be either defined (synthetic) or complex (undefined; non synthetic).

In this laboratory exercise you shall learn to (a) prepare a complex media
(liquid broth and solid) and (b) sterilise glassware (dry heating) and media
(autoclaving).

Expected Learning Outcomes

After preparing and sterilising culture medium you should be able to:

Independently prepare culture media;

Prepare both semi solid and liquid broths; 21

BBCEL-144 Basic Microbiology (Practical)

Operate the autoclave;

Sterilise glass ware and culture media;

Aseptically prepare agar slants, agar deeps and agar plates;

Indicate differences between complex and synthetic media; and

Choose media based on your experimental needs.

3.2 PRINCIPLE
A culture medium is a source of nutrients for growth and multiplication of
microbes. They provide organic carbon to generate energy; organic (partially
digested proteins) or inorganic nitrogen and salts; some have additional
supplements such as B-group vitamins and growth factors. These media are
broadly divided into two types- chemically defined or synthetic and complex or
non synthetic (undefined). As the name suggests, a chemically defined media
is composed of known amounts of chemically pure organic or inorganic
compounds. It supports the growth of specific non fastidious heterotrophs
(bacteria that grow without specific nutritional supplements), autotrophs, etc
and can be made only after knowing specific growth requirements of the
microorganism. On the other hand, a complex media generally has few
substances whose composition is not completely defined, many of which also
show batch to batch variations. Some of the common ingredients include
peptones, yeast extract and beef extract. They are rich source of B- vitamins,
growth factors and energy nutrients. It is useful when growth requirements of
an organism are not known as it supports the growth of most heterotrophs.
Microbiologists also modify existing media by adding specific nutrients
(enrichment media) that increases the number of particular species of bacteria
or a media that distinguishes colonies of microbes based on the substance(s)
added. An example of a differential media is MacConkey agar that
differentiates lactose fermenting from non fermenting Gram negative bacteria.

The medium may be in liquid state or solidified by adding agar. Agar is a

complex polysaccharide obtained from red algae, Gelidium and Gracilaria. The
usefulness of agar is due to its inert nature (not liquefied by most organisms);
melts around 950C and remains liquid till 440C; solidifies at room temperature
and remains in this state throughout the period of incubation of microbes at
370C or lower. The melted medium can be poured into Petri plates to produce
agar plates or in tubes to form agar slants / deeps each of which is suitable for
different studies / storage.

3.3 REQUIREMENTS

Petri plates

Culture tubes

test tube caps / non absorbent cotton plugs

test tube racks

1 or 2 litre Erlenmeyer flasks

10ml pipettes with pipettor

22
Experiment 3 Preparation and Sterilisation of Culture Media for Bacterial Cultivation

weighing paper

balance

Stirring glass rod

Bunsen burner / hot plate

Water bath set at 48-500C

Heat proof gloves

Beakers (250ml)

Aluminium foil

Autoclave

Glass marking pencil

Incubators

pH paper

Paper towel

Ingredients (g/l) for Nutrient Broth (pH 7.0)

Peptone 5g

Yeast extract 5g

Sodium chloride 5g

Beef extract 3g

3.4 PROTOCOL
(A) Preparation and sterilisation of Nutrient Broth

Weigh peptone (5g), yeast extract (5g), sodium chloride (5g) and beef
extract (3g) for one litre of yeast extract broth.

Dissolve each of these ingredients separately in warm water by

continuous shaking. Once they have dissolved transfer them to a 2 litre
conical flask and make up the volume to 900ml.

Adjust the pH to 7 with few drops of acid /alkali. After each addition, mix
and check pH with pH paper.

Divide the liquid into two equal parts and use one half for preparation of
semi solid medium and the other for liquid broth.

Weigh 5g agar and slowly add with stirring to 500ml medium.

Note: For completely solid medium use between 1.5 to 2% agar.

Boil the medium to melt agar.

Distribute the medium in smaller flasks and plug them tightly.

23
BBCEL-144 Basic Microbiology (Practical)

Autoclave at 15 pounds / inch2 (1210C) for 20min. You can either

sterilise wrapped glassware (Petri plates, pipettes, glass rod) by
autoclaving or dry heating in an oven (160- 1800C for 2-3 hrs). Your
instructor will demonstrate the use of autoclave and hot air oven.

Open the autoclave when the pressure comes down to zero. Unload
autoclaved material and transfer it for appropriate storage. Distribute the
culture medium aseptically either immediately or as and when required.

(B) Pouring sterilised media into sterile tubes and Petri plates

(i) Broth culture (liquid culture medium)

Add 10-15 ml of liquid culture media aseptically to sterile culture

tubes.

Leave it open for few minutes in the laminar flow bench.

Plug it tightly with cotton plugs or screw caps.

(ii) Agar slants / agar deep tubes and plates

The semi solid medium can be poured in tubes and Petri plates at around 45-
500C. The medium is kept in a water bath set at 500C. It is poured rapidly and
carefully such that the medium does not set while pouring and is free from air
bubbles. The variations resulting from setting on tubes and plates are depicted
in Fig. 3.1:

Agar slants are formed if the solid medium in the liquefied state is
poured in test tubes which are then placed in a slanted position to
harden, i.e. by keeping at an angle of 30º to 45ºC.

If the above tubes are left in an upright position they are called agar
deeps.

Agar plates are prepared by pouring molten medium (15ml) into Petri
plates. The plates are inverted after the medium has set.

An alternative to the above procedure for broth culture / agar slants and agar
deep tubes is to pour the respective media in tubes prior to autoclaving. After
autoclaving the agar slant tubes are prepared by leaving them for hardening in
a slanted position while others are left in an upright position.

Fig. 3.1: Liquid broth and forms of solid media.

24
Experiment 3 Preparation and Sterilisation of Culture Media for Bacterial Cultivation

3.5 OBSERVATIONS
Record your observations:

______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________

3.6 RESULTS
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________

3.7 PRECAUTIONS

Ensure that all media components can withstand 1210C before sterilising
them by autoclaving. Otherwise it is important to filter sterilise heat labile
substances separately and later add them aseptically into cooled
autoclaved medium.

Always wear heat proof gloves while melting agar and unloading
sterilised material from the autoclave.

While preparing solid / semisolid medium, stir the flask continuously

during melting, to avoid charring of agar that settles initially at the bottom
of the flask.

The volume of medium preferably should not exceed ⅔rd of the flask
and is in upright position during autoclaving. It should not wet the cotton
plug.

It is advisable to incubate autoclaved medium at 300C at least for 24hrs

to check for contamination.

Cotton plugs must be tightened before autoclaving so that they are not
easily displaced or loosen enough to cause contamination.

Preferably distribute the medium into smaller flasks prior to autoclaving.

Do not overload the autoclave. There should be space between packed

items for steam circulation.
25
BBCEL-144 Basic Microbiology (Practical)

SAQ 1
1. Compare complex and defined medium.

2. Why is the autoclaved culture medium cooled to around 480C before

pouring into Petri plates?

3. Indicate two ways of sterilising culture media.

4. Give reasons for suitability of agar as a solidifying medium.

ANSWERS
1. A complex medium generally has few substances whose composition is
not completely defined, many of which also show batch to batch
variations. It is useful when growth requirements of an organism are not
known as it supports the growth of most heterotrophs.

A defined media has known amounts of chemically pure organic or

inorganic compounds. As long as you use same grade and quantity of
the compounds it will have the same composition. Such a medium can
be made only after knowing specific growth requirements of the
microorganism. It supports the growth of specific heterotrophs.

2. The autoclaved culture medium containing agar is cooled to a

temperature around 480C, at which it is still liquid. At this temperature it
is poured rapidly into Petri plates and allowed to set undisturbed and you
get minimal water condensation on its lid.

3. (a) Autoclaving (steam sterilisation) at1210C for 15-20 minutes.

(b) Filter sterilisation (cellulose membrane filters) of heat labile

substances such as antibiotics.

4. (a) It liquefies at 1000C and solidifies below 400C; organisms can be

incubated at 370C or lower without liquefaction.

(b) It does not require refrigeration (unlike gelatin) to

solidify.

(c) Most organisms do not degrade (liquefy) agar

Note: If you have prepared a different media, record its composition and
find out from your instructor the kind of organisms for which it is most
suitable.

26
Experiment 4 To Study the Shapes of Bacteria, Fungi, Algae and Protozoa from
Permanent Slides or Pictographs

EXPERIMENT 4
TO STUDY THE SHAPES OF
BACTERIA, FUNGI, ALGAE
AND PROTOZOA FROM
PERMANENT SLIDES OR
PICTOGRAPHS (DRY LAB
EXERCISE)

Structure
4.1 Introduction 4.4 Observations

Expected Learning Outcomes 4.5 Results

4.2 Principle 4.6 Precautions

4.3 Requirements

4.1 INTRODUCTION
The dry lab exercise aims at acquainting you with the microbial world. You
shall be introduced to the general characteristics of bacteria, fungi, algae and
protozoa along with examples of pathogenic and non pathogenic organisms
within each group. Before coming for this class it is advisable to revise their
basic structure and other characteristics from your theory paper. It is quite
possible that the examples shown in dry lab session may differ from those
mentioned here. In that case your instructor will guide you.

Expected Learning Outcomes

After observing the permanent slides / pictographs you would:

have a bird’s eye view of the microbial diversity;

learn about the distinguishing characteristics of bacteria, fungi, protozoa

and viruses; 27
BBCEL-144 Basic Microbiology (Practical)

know examples of pathogenic and non pathogenic members from each

group; and

draw / click photographs of different microbes and comment on their

morphology and size variations.

4.2 PRINCIPLE
The microbial world is known for its diversity in shape, habitat, structural and
metabolic adaptability. The salient features of bacteria, algae, fungi and
protozoa along with examples are discussed here to help you identify these
organisms / their developmental stages. Bacteria are unicellular prokaryotes
that represent a diverse group of organisms differing in size, shape,
distribution and metabolic adaptability. There are three basic shapes known-
rod like bacillus, spherical (coccus) and curved; each of which shows variation
(Fig. 4.1). The bacilli may remain attached after division as in Diplobacilli; they
may bend at the point of division after cell division (palisade /fence like) or
have coccus like shape (Coccobacillus). Cocci may divide in one plane
(diplococci), two planes (tetrads); three planes (Sarcina) or divide randomly
(staphylococci). The spiral bacteria occur as Vibrio (curved comma shaped
rod), spirillum (thick rigid spiral) or spirochetes (thin flexible spiral).

Bacillus Coccobacillus Diplobacilli

Streptobacillus (chains) Palisade

(a) Arrangement of bacilli.

Coccus Diplococcus Staphylococci

Streptococci Tetrad Sarcina

(b) Arrangement of spherical shaped (coccus) bacteria.

28
Experiment 4 To Study the Shapes of Bacteria, Fungi, Algae and Protozoa from
Permanent Slides or Pictographs

(c) Variation in spiral morphology

Fig. 4.1: Shapes of bacteria.

Algae are eukaryotic unicellular or multicellular organisms that possess

chlorophyll and most of them carry out oxygenic photosynthesis like higher
plants, although some are chemoheterotrophic. They are widely distributed;
found commonly in water where they are either suspended or anchored to a
substratum. Some are also found on moist surfaces and in symbiotic
associations.

A typical algal cell is surrounded by has a thin rigid cell wall which may have
an outer matrix in some. Their vegetative body is called thallus that exhibits
enormous variation in size and complexity. They may be unicellular, colonial,
filamentous, sheet like or tubular (Fig. 4.2). Many are flagellated whose
position and number varies. Being eukaryotes they have membrane bound
organelles. The chloroplast may have a pyrenoid- the site of synthesis and
storage of starch. Their mitochondrial structure varies considerably among
algal groups.

(a) Chlorella vulgaris (b) Chlamydomonas reinhardtii (c) Volvox

(unicellular, non motile) (unicellular, flagellated) (green algal colony)

(d) Spirogyra (e) Red algae (f) Acetabularia crenulata

(filamentous alga) (branching filamentous) (largest single celled organism)

Fig. 4.2: Diversity in algal forms.

Sources: en.wikipedia.org (b, d, and e); phtocode.net (a); pinterest.com (c); zoology-
quest.blogspot.com (f)
29
BBCEL-144 Basic Microbiology (Practical)

Protozoa are protists that represent a large and diverse group of mostly
motile, heterotrophic, unicellular eukaryotes lacking a rigid cell wall. They
inhabit a wide range of habitats from aquatic (fresh water and marine) to
terrestrial environments. Most are free living; some are also parasitic. The
number of nucleus varies from one (amoeba) to two or more identical or two
non identical types (most ciliates). Protozoa have one or more vacuoles in
their cytoplasm which may differentiate to contractile, secretory or food
(phagocytic) vacuoles. Most protozoa can enter into a resting stage called
cyst, characterised by low metabolic activity. Their primary mode of
reproduction is asexual (binary fission; longitudinal or transverse), although
sexual mode (conjugation) is most prevalent among ciliates like Paramecium
caudatum.

(a) Paramecium caudatum (b) Amoeba proteus (c) Euglena gracilis

(d) Belantidium coli (e) Entamoeba histolytica (f) Trypanosoma species

(g) Plasmodium falciparum (h) Toxoplasma gondii (i) Eimeria

Fig. 4.3: Representative examples of pathogenic and non pathogenic protozoa.

Sources: en.wikipedia.org (b, c, d, f, h, i); pinterest.com (e); pathologyoutlines.com (g);

microscopeclarity.com (a)

The pathogenic protozoans that cause major diseases in humans and

domestic animals belong to four groups (amoebae, sporozoa, ciliates and
flagellates) that are classified on the basis of locomotion. The amoebae move
by pseudopodia (false foot); ciliates by cilia; flagellates by flagella while
30 sporozoa lack locomotor organelles in their mature stage. It is noteworthy all
Experiment 4 To Study the Shapes of Bacteria, Fungi, Algae and Protozoa from
Permanent Slides or Pictographs
sporozoans are parasitic (intra-or inter-cellular) and have a spore forming
stage in their life cycle. The pathogenic protozoa vary in their complexity and
some also have alternate host (Fig. 4.3).

Fungi are eukaryotic, spore bearing organisms that lack chlorophyll and
reproduces sexually, asexually (transverse fission, conidiospores,
chamydospores, hyphal fragmentation) or both. Most fungi are saprophytes
(absorptive nutrition) and grow in moist, dark habitats. They are grouped into
molds or yeast based on the development of their vegetative structure
(thallus). The former are unicellular fungi while the latter has long, branched
and thread like filament (hyphae) of cells that form a tangled mass (mycelium).
Many pathogenic fungi such as Candida albicans are dimorphic; they alternate
between these two extremes The hyphae may be septate (Aspergillus) or
coenocytic (non- septate such as Rhizopus) as shown in the Fig.4.4. A typical
fungal cell has a cell wall made of chitin. Fungi are pathogenic (Claviceps
purpurea, Puccinia graminis, Candida albicans, Tinea pedis) and can also
establish beneficial associations with other organisms (mycorrhizae with roots
of vascular plants and lichens with algae).

(a) Types of hyphae (b) Rhizopus stolonifer

(c) Aspergillus fumigatus (d) Agaricus campestris

(button mushroom)

(e) Foliose Lichen (f) Mycorrhizal fungi

Symbiotic fungal associations

31
BBCEL-144 Basic Microbiology (Practical)

(g) Tinea pedis (Athletes foot) (h) Puccinia graminis tritici (Stem rust of wheat)

Fig. 4.4: Diversity of fungal forms.(Sources: en.wikipedia.org (b, d, e, h); medical-labs.net

(a); cdc.gov (c); pacifichorticulture.org (f); foottalk.blogspot.org)

4.3 REQUIREMENTS

Permanent slides / pictographs

Compound microscope.

Immersion oil

lens paper

4.4 OBSERVATIONS
(a) Draw or click photographs of a representative microscopic field under
low power and oil immersion objective.

(b) Describe the structure and indicate identifying features observed under
the microscope (c) Indicate the colonial morphology; asexual / sexual
structures wherever observed.

(c) Draw the organism / developmental stages from pictographs. Note the
magnification and structural details that are clearly evident.

4.5 RESULTS
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________

4.6 PRECAUTIONS
(a) Always clean all lens systems by wiping with lens
issue.

(b) In case the oil immersion lens is sticky, moisten a piece of lens paper
with xylol to wipe it. Remove xylol immediately with 95% alcohol and
then wipe the lens with dry lens paper.
32
Experiment 4 To Study the Shapes of Bacteria, Fungi, Algae and Protozoa from
Permanent Slides or Pictographs

SAQ 1
1. Give an example each of pathogenic and non pathogenic protozoa,
bacteria and fungi.

2. Indicate examples of beneficial bacteria, fungi, algae and protozoa

3. Enumerate the basic bacterial shapes.

ANSWERS
1.

Category Non pathogenic Pathogenic

Bacteria Lactobacillus lactis Vibrio cholerae

Fungi Agaricus campestris Tinea pedis

Protozoa Entamoeba coli Entamoeba histolytica

2. (a) Bacteria: Bifidobacterium bifidum; Lactobacillus acidophilus

(b) Fungi: Foliose Lichen; Mycorrhizal fungi; Penicillium chrysogenum

(source of antibiotic penicillin)

(c) Algae: Sargassum (biofuels); blue green algae (biofertilisers)

(d) Protozoa: Euglena gracilis (neutraceuticals; biofuels);

3. There are three basic shapes known- rod like bacillus, spherical
(coccus) and curved; each of which shows variation.

33
BBCEL-144 Basic Microbiology (Practical)

34
Experiment 5 Differential Staining of Bacteria: Gram Staining Method

EXPERIMENT 5
DIFFERENTIAL STAINING OF
BACTERIA:
BACTERIA GRAM
STAINING METHOD

Structure
5.1 Introduction 5.4 Protocol

Expected Learning Outcomes 5.5 Observations

5.2 Principle 5.6 Results

5.3 Requirements 5.7 Precautions

5.1 INTRODUCTION
Gram staining is a differential staining method that was devised by a Danish
physician Hans Christian Gram in 1884 while working in Berlin. He was
examining lung tissue sections from patients who died of pneumonia and
wanted to differentiate Pneumococci (now known as Streptococcus
pneumoniae) from Klebsiella pneumoniae. During these investigations he
observed that some bacterial species readily lose the primary stain on
treatment with ethanol while others resist decolourisation which led to
development of this two step staining procedure with an intervening ethanol
step. It is simple staining method that allows investigators to identify bacteria
under the microscope and easily subdivide them into two broad groups- Gram
positive and Gram negative. Some bacteria such as Mycobacterium species
that cannot be detected by Gram reaction are classified as Gram
indeterminate. The method is still in use as a first step in identification of
generally pathogenic bacteria.

In this exercise you shall learn about Gram staining method used for
identification of bacteria. 35
BBCEL-144 Basic Microbiology (Practical)

Expected Learning Outcomes

After studying this experiment, you should be able to:

prepare thin smears of bacterial cells and fix them;

identify the given bacteria as Gram positive or negative;

record differences in size and shape of bacteria;

independently perform Gram staining with unknown bacterial specimens;

and

indicate the role(s) of each reagent used.

5.2 PRINCIPLE
Gram reaction is one of the commonly used methods to characterise bacteria,
based on differences in cell wall composition. Gram positive bacteria have a
cross linked multilayered peptidoglycan followed by lipoteichoic acid polymers.
On the other hand Gram negative bacteria have a thin single layer
peptidoglycan and an outer membrane of lipopolysaccharides (LPS), within or
without a capsule.

The staining technique uses two basic stains that are called primary and
counter stain. The former is crystal violet or methylene blue while the latter is
safranin (or basic fuchsin dye). Between the two staining steps is addition of
mordant (Gram’s iodine) for better interaction between the cell wall and the
primary stain and decolorization with ethanol. The decolorizing agent is both a
protein dehydrating agent and lipid dissolver. It increases the porosity of the
cell wall by dissolving the outer LPS layer of Gram negative bacteria and
effectively dehydrates the thick Gram positive cell wall that results in cell wall
shrinkage and reduced permeability.

All bacterial cells are stained with crystal violet and appear purple colored. The
Gram positive bacteria retain the primary stain after decolorization whereas it
is easily removed from Gram negative bacterial cells. In the final step only
Gram negative bacteria are stained with the counter stain and appear reddish /
pinkish. The difference in color between two types of cells allows
characterisation (Fig. 5.1).

Gram-positive bacteria Hans Christian J. Gram Gram-negative bacteria

36 (Source: astu.edu en.wikipedia.org astu.edu)
Experiment 5 Differential Staining of Bacteria: Gram Staining Method

Fig. 5.1: (Top) Gram positive and negative bacteria; (bottom) Schematic view
of bacterial cell wall structure.

(Source: hemtecks.wordpress.com)

5.3 REQUIREMENTS

Fresh bacterial cultures

Glass slides

Inoculating loop / needle

Burner or spirit lamp

lens paper

Pasteur pipette / dropping bottle

Compound microscope

Immersion oil

You can either work with commercially available ready to use reagents
or prepare them by following the instructions below:

Crystal violet.

Solution A

Crystal violet (90% dye content) 2g

Ethyl alcohol (95%) 20 ml

Solution B

Ammonium oxalate 0.8g

Distilled water 80ml

Mix solutions A and B; allow them to stand overnight at RT; filter and
store.

Gram’s iodine (KI3)

Iodine 1g

Potassium iodide 2g
37
BBCEL-144 Basic Microbiology (Practical)

Distilled water 300ml

Grind them in a mortar by slowly adding water. Store the reagent at RT

in a brown colored bottle. .

Ethyl alcohol 95%

OR

Prepare 95% ethanol and acetone mix (1:1 v/v).

Safranin O (counter stain) 0.25ml

Ethyl alcohol (95%) 10ml

Distilled water 100ml

Store at RT.

5.4 PROTOCOL

Clean 3-4 slides with 70% alcohol and allow them to air dry. Do not
touch the cleaned surface; hold them from the edges.

Prepare a smear of each of the given cultures using aseptic technique.

A smear may be prepared either from broth cultures or colonies on

semisolid medium.

A broth culture is first resuspended by mild tapping and then a loopful of

the culture is aseptically picked with an inoculating loop. Next it is
transferred to the centre of a clean slide and spread evenly.

In case of cultures from solid medium they need to be diluted before

spreading. For this one or two loopful of water is placed on the slide.
Then with the tip of a sterile inoculating needle the culture is touched
and diluted by mixing it with the drop of water on the slide. The
suspension is now ready for spreading.

Leave the smear to air dry.

Fixed the dried smear by passing the slide rapidly few times over the
flame or use 70% methanol.

Immerse / flood the smear in crystal violet for 1min.

Gently wash off excess stain with water (Fig. 5.2).

38 Fig. 5.2: Staining and washing step.

Experiment 5 Differential Staining of Bacteria: Gram Staining Method

Carefully add Gram’s iodine and leave the slide for 1-1½ min.

Gently wash with water.

Decolorise with alcohol or alcohol: acetone mix (1:1). Add it drop wise
until alcohol drippings are no longer colored or only faintly blue.

Wash gently with water.

Gram positive Gram negative

Fixation

Crystal
violet
(Primary stain)

Iodine treatment

Decolourisation

Counter stain with safranin

Fig. 5.3: Flow chart of Gram staining method.

Counter stain with safranin for 30 seconds.

Finally wash with water, blot dry and examine the slide(s) under oil
immersion.

Record your observations and classify the given bacterial cultures based
on Gram reaction.

5.5 OBSERVATIONS
Record your observations:

(i) Draw or take pictures of well spread and stained bacterial cells.

(ii) Note the color of stained cells.

(iii) Describe the morphology and arrangement of cells.

5.6 RESULTS
(a) Interpret your results. Ask your instructor the names of organisms given.
(b)If you do not get the expected Gram reaction, reason out why it would have
failed. (c) Repeat the experiment based on your discussions.
39
BBCEL-144 Basic Microbiology (Practical)

5.7 PRECAUTIONS

Avoid using older cultures. They tend to give Gram variable reaction. It is
best to subculture before the experiment.

Decolorization step must be rigidly controlled.

The smear should be thin, less dense and uniformly spread. Try to use
only small amount of culture.

Take care not to overheat the smear during fixation as this may kill the
cells and affect your results. One simple way to feel the temperature is to
touch the slide on back of your palm.

In case of bacteria that are poorly stained with safranin such as

Haemophilus species or anaerobic bacteria, try using basic fuchsin.

Sterilise the inoculation loop after each transfer. Use cooled sterilised
loop.

Always label the slides.

SAQ 1
1. Indicate the role of mordant and decolorizing agent in Gram staining.

2. Why is a smear fixed prior to staining / other treatments?

3. Describe two differences in the cell wall structure of Gram positive and
Gram negative organisms.

4. Give two examples each of Gram positive and Gram negative

organisms. Indicate whether they pathogenic or non pathogenic.

ANSWERS
1. (a) A mordant increases the affinity of cells for the stain by forming an
insoluble complex with the primary stain. The net result is
enhanced color intensity.

(b) The decolorization step is the key deciding step of classifying

bacteria as Gram positive or negative. Due to differences in their
cell wall composition only. Gram negative bacteria are decolorized.

2. Fixation minimises the washing away of cells during staining and

washing steps.

3. (a) The cell wall of Gram positive bacteria has multilayered and cross
linked peptidoglycan, surrounded by lipoteichoic acid polymers.

(b) In Gram negative bacteria peptidoglycan is single layered followed

by an outer membrane made up of LPS.
40
Experiment 5 Differential Staining of Bacteria: Gram Staining Method

4. (a) Gram positive: Bacillus anthracis (pathogenic); Corynebacterium

diphtheriae (pathogenic); some Lactobacillus species are
probiotics (non pathogenic).

(b) Gram negative: Helicobacter pylori (pathogenic); E. coli (generally

non - pathogenic); Azotobacter vinelandii (non pathogenic).

41
BBCEL-144 Basic Microbiology (Practical)

42
Experiment 6 Isolation of Pure Cultures of Bacteria by Streak Plate Method

EXPERIMENT 6
ISOLATION OF PURE CULTURES
OF BACTERIA BY STREAK
PLATE METHOD

Structure
6.1 Introduction 6.4 Protocol

Expected Learning Outcomes 6.5 Observations

6.2 Principle 6.6 Results

6.3 Requirements 6.7 Precautions

6.1 INTRODUCTION
Microorganisms exist in nature as a mixture of many different kinds of
organisms whose composition varies from one environmental niche to
another. Traditionally microbiologists have studied the characteristics (cultural,
morphological or biochemical) of a particular microbe only after obtaining a
pure culture. This has led to development of techniques for isolation of pure
cultures. To begin with, discrete colonies on semi solid agar plates are isolated
using streak- plate, spread plate or pour plate method and then cells are
picked from individual colonies for isolation of pure cultures. A lot of
information has been gathered since then for microbes that could be isolated
from mixed populations. But detailed analysis of molecular processes and
their use as reactors in recombinant DNA technology has been limited to a
handful of microbial systems.

We all recognise that microbes are extremely versatile and are found in every
possible environment. But many of them could not be cultured and maintained
in laboratory conditions by usual techniques, making it almost impossible to
study them in isolation. With the development of metagenomic approach
(molecular tool to analyse DNA from an environmental community) the
prerequisite condition of having pure cultures to initiate investigations is no
longer there. This has greatly complimented the earlier techniques and has
yielded information about a large number new microbes and novel metabolic
pathways. 43
BBCEL-144 Basic Microbiology (Practical)

In this exercise you shall learn to isolate pure cultures of microorganisms from
a mixed population using four-quadrant streak plate method.

Expected Learning Outcomes

After studying this experiment, you should be able to:

Perform streak plate technique to dilute bacterial cultures;

Isolate pure cultures from discrete colonies; and

Identify some of the isolated organisms from their cultural morphology.

6.2 PRINCIPLE
The isolation of microorganisms (most often bacteria) by streaking a loopful of
culture on agar surface spread in Petri dishes was first developed by Loeffler
and Gaffky in Koch’s laboratory. It is a rapid qualitative method requiring
minimal skills. In essence streaking brings about dilution of the initial inoculum
such that individual discrete colonies can be formed, allowing isolation and
identification of different species of bacteria from a given environmental
sample. Each colony represents a single organism that has multiplied many
times. A pure culture of bacteria can be also be streaked to get individual
colonies which can be picked and used for conducting various studies.

Many variations of streak plate methods are available that are essentially
based on the number of sectors in which the agar plate is streaked. These
include continuous streaking, T-streak (three-sector streak) and four streak
quadrant method (Fig. 6.1).

Fig. 6.1:Streaking methods.

44
Experiment 6 Isolation of Pure Cultures of Bacteria by Streak Plate Method

This method cannot be used in case of anaerobic organisms and only viable
organisms in the mixed population can multiply to form visible colonies.

6.3 REQUIREMENTS

Sterilised semi solid nutrient agar plates (or some other nutrient medium)

Inoculating loop

Ethyl alcohol (95%)

500ml beaker

Burner / spirit lamp

Fresh nutrient broth cultures from environmental sources.

Glass marking pencil

Liquid broth

Agar slants / broth culture tubes.

6.4 PROTOCOL
(A) Isolation of discrete colonies from a given mixed culture

The four streak quadrant method is a discontinuous streaking method

involving four individual streaks. As the name suggests in this method the Petri
plate is divided into four quadrants / areas (1-4). The expected result is shown
in Fig. 6.2.The steps for four-quadrant streak plate is as follows:

Sterilize the inoculating loop by heating it in the blue flame of the Bunsen
burner until it is red hot. Allow it to cool.

For convenience invert the agar plate and divide it into four sectors with
a glass marker.

Hold the agar plate in the left hand between the fingers and the thumb
close to the flame.

Loosen the cap / cotton wool plug of the tube containing the inoculum.

Remove the cap of the test tube with the little finger of the right hand.
Flame the rim of the tube and work faster and closer to the flame.

Aseptically pick inoculum by dipping the cooled loop into the bacterial
suspension or from a colony on solid medium.

Open the lid of the Petri plate and streak the first quadrant.

Reflame the loop and allow it to cool. You can even touch the surface of
uninoculated agar medium.

Rotate the plate 900.

Now streak the second quadrant, starting from the edge of the first
streak. 45
BBCEL-144 Basic Microbiology (Practical)

Repeat the preceding three steps for quadrant 3.

Without reflaming the loop, turn the dish 900.

Streak area 4 with inoculum from the corner of area 3.

Close the lid of the Petri plate and invert it.

Incubate the plates overnight at 370C for 48hrs at 250C.

Observe the plates and record.

Fig. 6.2: Four streak quadrant method. (from: microbeonline.com)

(Observe the progressive dilution of the culture from area 1 to area 4.)

(B) Isolation of pure cultures

The next step is to pick one by one aseptically cells from discrete well
separated colony from streaked plates and transfer them to separate
nutrient agar slant / broth.

Incubate the cultures overnight at 250C.

Each of these slant / broth cultures has descended from a single

bacterial species. They are pure / stock cultures.

Store the cultures. Depending on the storage conditions, the time

between sub culturing would vary.

6.5 OBSERVATIONS
Observe the streaked plated next morning and report:

(a) The cultural morphology of the colonies. Your instructor would help you to
analyse.

--------------------------------------------------------------------------------------------------------
--------------------------------------------------------------------------------------------------------
--------------------------------------------------------------------------------------------------------
46
Experiment 6 Isolation of Pure Cultures of Bacteria by Streak Plate Method

(b) Assess the quality of your plate; either draw it or take pictures.

6.6 RESULTS
(a) Compare the distribution and amount of growth in the four quadrants.
Based on your observations discuss your results

(b) If you have not obtained the expected dilution of the culture by four
quadrant streak plate method, can you suggest possible reasons for
your failure?

(c) Indicate the names of the isolated organisms.

(i)---------------------------------------------------------------------------

(ii)----------------------------------------------------------------------------

(iii)-----------------------------------------------------------------------------

6.7 PRECAUTIONS

Best results are obtained when the loop is flamed between streaks. This
will further reduce the number of microorganisms as we move from one
area to the next.

Always cool the flamed inoculation loop before picking the inoculum.

Invert the streaked plates during incubation to prevent condensed water

droplets from falling on agar surface. This may spread the colonies and
even cause contamination.

Perform the experiment using aseptic techniques to prevent

contamination.

Label the plates. It should carry complete information about the

organism , date of experiment, your name, etc

Apply light pressure while streaking to avoid digging into agar.

Take care not to let the loop touch any of the previously streaked areas.

SAQ 1
1. Indicate two applications of streak plate method.

2. What is the purpose of flaming the inoculation loop between streaks in

different areas?

ANSWERS
1. (a) It allows isolation of specific microorganisms from mixed cultures.

(b) Bacterial cultures can be stored (short term) in an inverted position

and after covering the Petri plates. 47
BBCEL-144 Basic Microbiology (Practical)

2. Flaming kills bacteria and reduces the number of microorganisms as we

streak from one quadrant to the next. This in turn increases the chances
of obtaining discrete isolated colonies.

48
Experiment 7 Qualitative Detection of Antibiotic Action by Kirby-Bauer Antibiotic Sensitivity Test

EXPERIMENT 7
QUALITATIVE DETECTION OF
ANTIBIOTIC ACTION BY
KIRBY
KIRBYBAUER
ANTIBIOTIC SENSITIVITY
TEST

Structure
7.1 Introduction 7.4 Protocol

Expected Learning Outcomes 7.5 Observations

7.2 Principle 7.6 Results

7.3 Requirements 7.7 Precautions

7.1 INTRODUCTION
We are all aware that some microorganisms are pathogenic and cause
diseases which may even be fatal. The emergence of chemotherapeutic
agents especially antibiotics has dramatically changed the way these
infections are handled and this in turn has tremendously improved quality of
life and average life expectancy. They act on other microbes by either
inhibiting key pathways or killing them. Initially antibiotics were only obtained
from specific bacterial species, actinomycetes and fungi. These days some of
them are not only synthesized but modified to make them more potent and / or
to manage the issue of drug resistance. This poses the need to have a reliable
and faster system to assess new chemotherapeutic agents for the range of
anti microbial activity.

There are three basic ways available for determining antimicrobial

susceptibility namely, broth dilution, agar dilution and disc diffusion methods.
Due to its simplicity, reproducibility and ease of testing multiple anti microbial
agents in a single plate has led preferential use of disc diffusion method over
others in clinical laboratories. 49
BBCEL-144 Basic Microbiology (Practical)

In this laboratory exercise you would test the sensitivity of the given antibiotics
on at least three or four test organisms and evaluate your results based on
standard diameter of zone of inhibition provided by your instructor for each
antibiotic.

Expected Learning Outcomes

After studying this experiment, you should be able to:

perform antibiotic sensitivity testing by Kirby-Bauer disc diffusion

method;

accurately measure diameter of each zone of inhibition; and

Evaluate antibiotic sensitivity of the test organism (s).

7.2 PRINCIPLE
Kirby-Bauer disc diffusion method (1966) is a widely used qualitative in vitro
technique for determining antibiotic susceptibility that in turn is helpful in
deciding the choice of antibiotics and other chemotherapeutic agents, for
treating infections. It is a simple, rapid tool for testing the susceptibility of the
test organism to multiple antibiotics on a single plate. In this method the
inhibition of bacterial growth is seen as clear zones around the antibiotic
coated disc. The diameter of the zone of inhibition is compared with a
standardised chart to evaluate susceptibility of the test organism. Based on
the diameter of inhibition zones organisms have been classified as sensitive,
intermediate or resistant to given antimicrobial agents. Quite often colonies are
observed within the zone of inhibition in case of resistant strains. The antibiotic
susceptibility patterns are called antibiograms. This method can also be used
to study additive (combined effect is not greater than sum of their individual
effects) and synergistic (enhanced) effect of drugs.

The test is affected by many factors such as medium and its pH, drug
concentration, thickness of agar, ability and rate diffusion of antibiotic and rate
of bacterial growth and inoculum density. In order to obtain reproducible
results these factors need to be controlled; for example the recommended
medium is Mueller-Hinton agar and to have confluent growth.

7.3 REQUIREMENTS

forceps

Whatman paper 3 discs

Petri plates

ethyl alcohol

Bunsen burner / spirit lamp

Incubator

Metric ruler

50
Glass marking pen

Experiment 7 Qualitative Detection of Antibiotic Action by Kirby-Bauer Antibiotic Sensitivity Test

Antibiotics (pre-coated discs / aseptically add antibiotics to sterile discs)

Amount of some common antibiotics/ disc are: Ampicillin (10µg);

Gentamicin (10 µg); Streptomycin (10 µg); Tetracycline (30 µg);
Chloramphenicol (30 µg) or any other. Prepare aseptically 2mg/ ml
antibiotic solution and add 5µl / disc for 10 µg. You can purchase
injection vials for some of them.

The standardised zone diameters are based on these amounts.

Broth cultures / saline suspensions of test organisms (Escherichia coli;

Klebsiella pneumoniae or any other organism).

Cotton swabs / L-shaped spreader

Mueller-Hilton agar medium (pH 7.2 - 7.4)

You can prepare the medium either from commercially available powder
or by weighing each ingredient (Beef infusion, 300g; casamino acids,
17.5g; starch, 1.5g and agar, 17g) and mixing them in 1litre distilled
water.

Eppendorf pipettes; sterilised disposable tips

Sterile water

7.4 PROTOCOL

Pour melted Mueller-Hilton medium aseptically into sterile Petri plates.
Spread the medium and leave it undisturbed for setting. Prepare a
separate plate for each organism.

Invert the Petri plate and divide it into sectors (6 or 8); one for each
antibiotic.

Label the covers of the plates with the name of the test organism.

Mix the culture and aseptically dip a sterile cotton swab into it. Remove
excess inoculum by pressing it against the wall of the culture tube.

Inoculate heavily a Mueller-Hilton agar plate by moving the soaked swab

uniformly over the entire surface.

OR

Aseptically pipette 100µl of test organism; place it on the centre of the

Petri plate and spread it using a sterile L-shaped spreader.

Repeat the above two steps with each test organism.

Leave the plates for 10min to allow the culture to soak in and dry the
surface.

In case of pre coated antibiotic discs, carefully place a disc in each

sector. You can use either a sterile forceps or a disc dispenser. Each of
these discs is coated with the recommended amount of different
antibiotics.

OR
51
 BBCEL-144 Basic Microbiology (Practical)

In the absence of ready to use pre coated discs you can prepare discs of
Whatman No.3 paper; place them in a Petri plate, pack it and autoclave.

Prepare antibiotic solution using sterile water. Next place a disc on each
sector of inoculated plate. Finally carefully pipette 5µl of the respective
antibiotic on each disc and add slowly to sterile discs.

Gently press each disc with a sterile forceps so that they adhere to the
agar surface.

Leave the plates in the laminar flow for 30min or more at RT until dry.
Invert the plates and incubate them for 24-48 hrs at 370C.

Observe the plates. Measure the diameter of the zone of inhibition in

mm.

Evaluate sensitivity of the test organism to a given antibiotic by

comparing the size from a standardised chart.

Pipette out aseptically

5µl of the respective
antibiotic on and add
slowly to a sterile disc
placed on inoculated
plates.

Measure the diameter of

each zone of inhibition
using mm ruler,
following incubation.

Gently touch each disc

with a sterile forceps /
glass rod.

Representative result

Dispense antibiotic discs with (a) dispenser or (b) place them at equal
distance with a sterile forceps or (c) coat Whatman paper 3 discs with
antibiotic.
Fig. 7.1: Kirby-Bauer antibiotic sensitivity method.
(Adapted from Microbiology- A laboratory manual; J.G. Cappuccino & N.
52 Sherman)
Experiment 7 Qualitative Detection of Antibiotic Action by Kirby-Bauer Antibiotic Sensitivity Test

7.5 OBSERVATIONS
(a) Record the following observations in a tabulated form:

For each organism indicate:

(i) Presence /absence of the zone of inhibition

(ii) Zone size (mm);

(iii) Compare zone size with standardised chart to evaluate sensitivity /

resistance based on your results

(iv) Presence / absence of colonies within the zone of inhibition

Note: Sensitive (S); Intermediate (I) and Resistant (R)

Organism

Antibiotic Zone S/I/ Zone S/I/R Zone S/I/R Zone S/I/R

size R size size size

(a) Did any organism showed colonies within the zone of inhibition?

-------------------------------------------------------------------------------------------------

(b) In presence of which antibiotic?

-------------------------------------------------------------------------------------------------

(c) Was it resistant or sensitive to that antibiotic based on your results?

-------------------------------------------------------------------------------------------------

(b) Take pictures of test plates and paste them in the space below.

7.6 RESULTS
Indicate the sensitivity of each organism to the antibiotics tested. .

(a)___________________________________________________________

(b) ___________________________________________________________

(c) ___________________________________________________________

(d) ___________________________________________________________ 53
BBCEL-144 Basic Microbiology (Practical)

7.7 PRECAUTIONS

The antibiotic coated discs placed on agar surface should be gently
pressed with a sterile forceps / applicator to establish contact between
the disc and the culture.

Ensure that each plate has heavy growth that covers the entire surface.

Accurately pipette antibiotic solution and add slowly onto each sterile
disc placed on agar plate (in case you are not using pre-coated discs).

Leave enough space between discs to avoid merging of the zones of

inhibition.

Many factors need to be controlled to be able to evaluate satisfactorily

the degree of susceptibility of the organism to an antibiotic.

Place a ruler across the zone of inhibition at the widest diameter and
measure from one edge to the other edge.

SAQ 1
1. Indicate the factors that can affect the results of Kirby-Bauer disc
diffusion method?

2. What is an antibiotic?

3. Differentiate between bacteriostatic and bacteriocidal agents. Give an

example of each.

ANSWERS
1. (i) Medium used; (ii) pH of medium; (iii) thickness of agar; (iv) drug
concentration; (v) rate diffusion of antibiotic / anti microbial agent; (vi)
inoculum density.

2. Antibiotics are antibacterial agents that produced and secreted by some

bacteria, actinomycetes and fungi. They act by either killing or inhibiting
the growth of other microorganisms and are used to treat variety of
bacterial infections. Their spectrum of anti bacterial activity is variable;
some are broad while others are narrow spectrum. Many of the natural
antibiotics have been have modified to make them more potent /
overcome bacterial resistance, for example ampicillin is a semi-synthetic
version of penicillin.

3. Both agents interfere with microbial metabolism.

A bacteriostatic agent inhibits bacterial growth while a bactericidal

substance kills them.

Examples include:

Bacteriostatic agents: tetracyclines (antibiotics); sulphonamides.

Bacteriocidal agents: β-lactam antibiotics (ampicillin); glycopeptides

54
antibiotics.
Experiment 7 Qualitative Detection of Antibiotic Action by Kirby-Bauer Antibiotic Sensitivity Test

SUGGESTED READINGS
1. Harley, J.P. and Prescott, L.M. (2002) Laboratory Exercises in
Microbiology, 5th Ed, McGraw-Hill companies.

2. Cappuccino, James G. and Welsh, C (2020) Microbiology: A Laboratory

Manual, 12th Ed, Pearson Education, Inc.

3. Sharma, K (2007) Manual of Microbiology: Tools and techniques, 2nd Ed,

Anne Books, India.

4. Prescott, L.M and Harley, J.P and Klein, D.A (2005) Microbiology, 6th Ed,
McGraw-Hill Publishers, USA.

5. Seeley, Jr. Harry. W and Van Demark, Paul. J. Microbes in action: A

Laboratory Manual of Microbiology, 2nd Ed, D.B. Taraporevala Sons and
Co. Private LTD, India

6. microbenotes.com/ instruments-used-in-microbiology-lab

55