UNIBERSIDAD DE MANILA

659-A Cecilia Muñoz St, Ermita, Manila, 1000 Metro Manila

MICROBIOLOGY
laboratory manual

Abucay, Janah Mae D.

CHS/ BSN NR-11
 INTRODUCTION TO
MICROBIOLOGY &
PARASITOLOGY

Importance to Nursing:
Obtaining and practicing the knowledge in Microbiology & Parasitology
for nursing students is essential. Nursing students are expected to take
care of a patient and to keep oneself safe from harmful microbes.
When caring for patients or performing procedures, nurses use
microbiology ideas. To avoid infection spread, nurses should be aware
of the several ways in which infections spread.

MICRO- PARASI-
BIOLOGY TOLOGY
The study of micro-organisms The study of the biological
such as bacteria, viruses, archaea, aspects of parasites and
fungus, and protozoa is known as parasitic diseases, including
microbiology. Fundamental study their epidemiology,
on the biochemistry, physiology, biochemistry, physiology,
cell biology, ecology, evolution, and molecular biology, ecology,
pathophysiology of microbes, as evolution, and clinical aspects,
well as the host response to these as well as the host response to
agents, is included in this field. these organisms, is known as
parasitology.
 3

C O N T E NT S
L A B O R A T O R Y 1

F O
M i c r obiology Lab o r a t o r y : B a s ic
R u l e s and Requ i r e m e n t s

E L B A T
12 L A B O R A T O R Y 2

T h e Microscope

22 L A B O R A T O R Y 3

M e th ods in Mic r o b i o l o g y

30 L A B O R A T O R Y 4

C u l t ural Charact e r i s t i c s o f
M i c r oorganisms

42 L A B O R A T O R Y 5

C u l t ure Media

49 L A B O R A T O R Y 6

M e d ia Preparat i o n

60 L A B O R A T O R Y 7

P l a t ing Techniqu e s a n d M e t h od s
o f I solation Pur e C u l t u r e

66 L A B O R A T O R Y 8

C u l t ure Preserva t i o n T e c h n i q ue

70 L A B O R A T O R Y 9

B a c t erial Identi f i c a t i o n a n d
B i o c hemical Te s t s f o r I d e n t i f i c a t i o n
o f B acteria

102 L A B O R A T O R Y 1 0

F u n g i and Identi f i c a t i o n O f F un g a l
C o n taminants i n P l a n t T i s s u e
C u l t ure Lab

111 L A B O R A T O R Y 1 1

Identification of Disease Causing

Fungal Pathogen of Passion Fruit
Nursery Plants and Identification of
Disease Causing Pathogens in Passion
Fruit Plants from Field

118 L A B O R A T O R Y 1 2

M i c r obiologica l E x a m i n a t i o n o f M i l k
LABORATORY 1

Basic Rules
and
Requirements
 MICROBIOLOGY LABORATORY:
BASIC RULES AND
REQUIREMENTS
CLOTHING READ FIRST
Wear appropriate clothing to class and Read through the instructions
always wear personal protective gear first and prepare the materials
such as safety goggles, lab aprons or needed before starting any
coats, and protective gloves. activity.

FOLLOW INSTRUCTIONS MONITOR

Personally monitor all experiments
Follow verbal and written instructions
and do not leave them unattended
carefully. If you are unsure about any part
to avoid accidents. Report any
of the activity, ask your teacher before
personal accidents and breakage
taking any action.
of equipment to the instructor.

DON'T EAT OR DRINK BE CAREFUL

Do not eat, drink, or chew gum in the lab. Handle chemicals, glassware,
Do not bring in food or drinks and do not and equipment with care.
store or place them inside any lab Do not sniff or taste any
glassware or equipment. chemicals or substances.

HYGIENE TIDY UP
Wash your hands with disinfectants Keep your work area neat and
when you arrive at the lab and again tidy at all times. Store bags,
before you leave. purses and books in appropriate
Sterilize equipment and materials. areas and keep the aisles clear.

SAFETY EQUIPEMENT BE RESPONSIBLE

Use the lab responsibly. Only
Know where the safety equipment
conduct authorized experiments
are located and know what to do
under the supervision of a teacher.
in the event of an incident.
Dispose off all solid waste
Immediately report spills or
materials in a biohazard bag and
accidents to the teacher.
autoclave it before discarding in
the regular trash.

Microbiology Laboratory Manual 03

 BASIC RECORD AND FIELD
BOOK/LAB BOOK
They are the permanent record of your work and hence should contain all the works related to the
project. Basic Record is the whole systematic record of the work/study/project in full detail. Field
book/lab book is for your daily use in the lab/field and for rough works, calculations, plan schedules,
memoirs, etc. You should record your work in these books systematically and regularly. All the
experiments conducted in the lab must be recorded in these books. It is a compilation of whole work
done by the researcher, so it must be well maintained. Also it can be a good reference book for those
who come along. These are the property of the research station and hence you are not supposed to
keep those books in your home. When you resign from the job you should submit them up-to-date to
the lab in charge without any delay

You should note the following points while dealing with field book.

Next you mention the requirements

Keep the book neat and tidy.
for the experiment.

Utilize the book efficiently preserving Summarize the theory and principle. This
the legibility of your writing should be followed by the procedure.

Name of the experiment should be Mention the general calculations for the
entered along with the date of experiment. It should contain all the related
works of the project for which it is meant to.
carrying out that experiment.

The following points are to be taken care of:

Complete the index, indicating the experiment,

Do not tear pages from the field book.
its serial number, page number on which it is
Number the pages of field book.
written.

Do not over write if a mistake has The notebook should always be up to

been committed in recording, put a line date and may be collected by the lab in
over it and write the correct word again. charge at any time.

You have to submit the field book and

basic record at the end of every
month on the date assigned.

Microbiology Laboratory Manual 04

 Mandatory Details Required
the Basic Record

Index: An index containing the title of each experiment

1 with page number and Sl. No.

Brief title of the experiment and date: Every experiment

2 should have a descriptive title.

3 Aim/Objective: A clear objective should be there.

Technical Programme: This section should include any

materials required, reagent composition, protocol and
formulae. Procedure in the form of flow charts is helpful
if it involves several parts. If an experiment is a repeat
of an earlier experiment, you do not have to write down
each step, but can refer to the earlier experiment by
page or experiment number. If you make any changes,
4 note the changes and reasons why.

Observations: Periodical or quantitative or qualitative

5 observations.

Results: This section should include the final result of

the experiment in accordance with the aim, organized
in statistically valid tables and figures and discussed
logically and justifiably. All raw data, including gel
photographs, printouts, graphs, autoradiographs, etc if
6 present are to be included.

Inference: The results obtained should be interpreted in

7 accordance with the principle of the experiment.

Future Line: This section includes any suggestions from

the protocol done, any refinements required etc. It is
mandatory to have clear and accurate records of all
8 experiments conducted in the laboratory.

Microbiology Laboratory Manual 05

Basic requirements of a microbiology laboratory
A microbiology laboratory requires well-built rooms equipped with glassware, tools and
equipments. Some of the most important items of equipment are the following.

I) Common Glassware
The most important glassware used in a microbiological laboratory are test tubes,
culture tubes, Petri dishes, Measuring cylinder, pipettes, glass spreader, Flasks, screw-
capped glass bottles, haemocytometer etc.

1) Test Tubes, Culture Tubes and Screw-Capped Tubes

These are made up of glass, one end of which is closed and other end is open.
If the side wall of the open end is slightly curved outside, it is called test tube; if the
side wall is smooth, it is called culture tube. When the side wall of the tube has
screws so that a plastic cap may be fitted, it is called screw-capped tube.
The test tubes are used for testing the chemicals such as pH etc., culture tubes are
used for preparation of agar slants and purification of microorganisms. The open

end is plugged with non-absorbent cotton plug.

Sometimes the microorganisms are purified and preserved in screw-capped tubes.
2) Petri dish
R. J. Petri, a student of the most renowned bacteriologist Robert Koch devised this
dish, hence called “Petri dish”.
It consists of two shallow glass dishes, the upper half or lid and the lower half.
For the isolation and cultivation of different types of microorganisms these dishes
are used in all microbiological laboratories.
According to the requirement, its diameter varies.
Molten agar medium is aseptically poured on the lower half of the sterilized Petri
dish and then covered with the upper half.
The petri dishes are sterilized by putting them in a Petri dish container and in turn in
an oven or autoclave.
3) Pipette
It is a cylindrical and graduated glass apparatus.
Its one end (lower side) tapers, while the other end (mouth piece) is normal. The
middle portion is wider or of the same size as mouth end.
It is graduated with numbers 1, 2 . . . n. . . . . . . 10.
It has different measuring capacity such as 0.1, 0.5, 1, 5, 10 ml etc. Hence measures
different quantities.
It is used for transferring appropriate amount of liquid into other containers.
It should be sterilized in an oven or autoclave before use by keeping in pipette
container after being plugged with cotton.
For safety point, liquid should be sucked by attaching pipette-sucker at the normal
end of the pipette.
Pipettes should be sterilized by keeping them first in a steel can and is sterilized at
121°C for 30 minutes.
Microbiology Laboratory Manual 06
4) Glass Spreader
It is made by bending a glass rod and making an L-shaped structure.
It is used to spread evenly the microorganisms on agar surface present in liquid
medium.
The long arm is held in hand and the small arm is flame-sterilized and put on agar
surface.
It is brought forth and back so that microorganisms present in liquid may be
dissociated and evenly spread on the entire surface of agar.

5) Haemocytometer
This is a device used to measure the blood cells.
This is also used for counting other cells viz., spores, bacteria etc.
It consists of a number of chambers. Each big chamber has 1 X 1 X 0.1 mm =
0.1mm3 volume with an area of 1 X 1mm = 1mm2 . The depth of chamber is 0.1mm.

(1 X 1 X 0.1 mm = 0.1mm3 = 0.0001cm3 = 10-4 cm3 = 10-4 ml) Hence, the bacterial
cell count in the large chamber will be multiplied by 104 to give an estimate of
bacterial cell number/ml.
Each large chamber has 9 medium-sized chambers with 0.2 mm length, 0.2 mm
width and 0.1mm depth with a volume of 0.004 mm3 .
Each medium sized chamber is divided into 25 small chambers with 0.04 mm length,
0.04 mm width and 0.1mm depth with a volume of 0.00016 mm3 .

6) Cleaning of Glass wares.

Care should be taken that the glassware used in microbiology laboratory are neat and
clean. After receiving and before start of work, the glassware must be chemically
cleaned so that there should not be chemical deposits on its surface. Moreover, dirty
form, then by soaking overnight in chromic acid. If the latter is not effective, a mixture of
concentrated nitric acid and sulphuric acid can be applied. All traces of the cleaner are
then removed by repeated rinsing in tap water followed by distilled water. Sometimes,
new glassware may contain some bacterial/fungal spores that come with packing
materials. The glassware from factory also contains some amount of alkali, hence to
remove the alkali, 2-3 % HCl is applied for 24 hours for neutralization.

After the use of glassware the medium is removed and the former is treated with 3%
commercially available Lysol solution followed by washing with boiling water. After
drying, it can be kept in oven for 3-6 hours at 140-180°C. The used pipettes, slides,
cover slips, petri dishes, etc. are also cleaned by this method.

Chromic acid is widely used as cleaning agent for glassware. It is a mixture of sodium
dichromate and concentrated sulfuric acid and possesses powerful oxidizing and
solvent properties.

Microbiology Laboratory Manual 07

Preparation of Chromic acid
(i) First Method: Weigh 5g of sodium potassium dichromate and dissolve in 5ml distilled
water in a beaker (250ml). Add 100ml concentrated H2SO4 slowly and stirring it
constantly. The mixture is allowed to cool at about 40°C and then stored in a dry glass
stoppered bottle.
(ii) Second Method: Weigh 1g of K2Cr2O7 or Na2Cr2O7 and mix with 100ml of
concentrated H2SO4. After stirring several times (preventing from cake formation) allow
the mixture to cool at 40°C and store in a clean, dry stoppered bottle.

II) Tools in Microbiology Laboratory

The most common equipment are inoculation needle, inoculation/transfer loop, Bunsen
burner, autoclave (or pressure cooker), incubator, hot air oven, refrigerator, centrifuge,
spectrophotometer, magnetic stirrer, orbital shaker, hot plate, Distillation water still, UV-

dioxide cylinder, single-pan balance with weights (for rough
lamp, water-bath, carbon
use), chemical balance, pH meter, colony counter, laminar air flow, electrophoretic
apparatus, microscopes etc.

1) Inoculation Needle & Inoculation Loop

These are the most commonly used tools.
Inoculation needle/loop is made up of a long
platinum wire fixed into a metallic rod.
A wire loop has a handle with steel screw
shaft in which nichrome or platinum wire is to
be fitted.
The straight wire needle is used for
transferring culture from solid medium. Even
smaller amount of liquid culture can be
manipulated by using straight needle.
The loop and wire are also used for picking
small quantities of solid materials from a
microbial colony and can be used to inoculate
either a liquid or a solid medium. Both the loop
Figure 1.1 and straight wire must be flamed immediately
after use to avoid contamination.

Microbiology Laboratory Manual 08

2) Bunsen Burner
Sterilization of tools by using spirit lamp is called
incineration.
Gas enters the burner at the base, and its supply is
regulated externally by the gas cock.
The amount of air can be controlled by rotating a
sleeve that fits over the holes in the body of the burner.
To keep the flame from blowing out special tips are
frequently used to fit over the top end of the barrel.
The proper method of lighting the burner is to close off
the air supply, turn on the gas and light. The flame will
be large and yellow. Gradually open the air intake until
the flame takes a blue colour.
Figure 1.2

3) Water Bath
Water bath is an instrument that is used to
provide constant temperature to a sample.
It consists of an insulating box made up of steel
fitted with electrode heating coil.
The temperature is controlled through a
thermostat.
In some of the water baths, plate form rotates,
then it is called water bath shaker. It is more useful
to microbiologists because it provides a uniform
heat to the sample material meant for incubation.
The main use of water bath is the incubation of
Figure 1.3
samples at a desired and constant temperature.

4) Laminar Air Flow Chamber

Laminar air flow is an apparatus consists of
an air blower in the rear side of the
chamber which can produce air flow with
uniform velocity along parallel flow lines.
There is a special filter system of high
efficiency particulate air filter (HEPA) which
can remove particles as small as 0.3 mm.
In front of the blower, there lies a
mechanism through which air blown from
the blower produces air velocity along
parallel flow lines. Figure 1.4

Microbiology Laboratory Manual 09

 The laminar air flow is based on flow of air current of uniform velocity along parallel
flow lines which help in transferring microbial cultures in aseptic conditions. Air is
passed through the filters into the enclosure and the filters do not allow any kind of
microbe to enter in to the system.
Inside the chamber one fluorescent tube and another UV tube are fitted. Two
switches for these tubes and a separate switch for regulation of the air flow are
fitted outside the LAF. Due to uniform velocity and parallel flow of air current,
pouring of media, plating, slant preparations, streaking etc. are performed without
any kind of contamination.
Initially, dust particles are removed from the surface of the laminar air flow with the
help of smooth cloth containing alcohol. Switch on the UV light for a period of 30
minutes so as to kill the germs, if any present in the area of working space.
The front cover sheet of the apparatus is opened to keep the desired material inside.
The air blower is set at the desired degree so that the air inside the chamber is
expelled because the air inside the chamber may be contaminated / bring
contaminants.
Sit properly in front of the chamber and wipe the working table with alcohol to
reduce the contaminants. All the work related to pouring, plating, streaking etc. are
to be carried out in the flame zone of the burner or spirit lamp.
In microbiology laboratory, horizontal type of laminar air flow is used to supply the
air through filter.

Precautions: Put off the shoes before entering to operate the apparatus. Wash the
hands with detergents or soap. One should not talk inside the chamber while performing
microbial culture transfer, failing which chances of contamination may be more which
may come either through mouth, sneezing or air.

5. Incubator
An incubator is an instrument that consists of
copper/steel chamber around which warm
water or air is circulated by electric current or
by means of small gas flame.
The temperature of the incubator is kept
constant due to its control by using thermostat.
The incubator is made up of double walled
chamber adjusted to a desired temperature. It
is done by using an external knob controlling
the thermostat system. The gap between two
walls is insulated to check heat condition. A
thermometer is inserted from the top for
Figure 1.5 recording the temperature.

Microbiology Laboratory Manual 10

 Temperature greatly influences the microbial growth. Therefore, instrument is
generally designed that can allow the desired microorganism to grow at a particular
temperature.
It is operated to allow the microbial growth on a suitable medium under proper
temperature. In an incubator, the variation in temperature should not be more than
one degree.
Small square type incubators are better than large ones. If a lower temperature than
the room is required, the water is circulated around the chamber to pass through an
ice chest.

Precautions: the door of the incubator should be opened only when necessary. If the
tubes are to be incubated for a long time or at higher temperature, the medium may
become too dry due to excessive evaporation. In such cases cotton plug should be
pushed inside the neck of the tube. The tube should be covered by a rubber cap so as to
cover the plug. If the petriplates are to be incubated for a long time, they may be placed
in moist chamber with a damp sterile cotton wool at the bottom.

6. Colony Counter
It is a device used for counting small or closely
growing colonies on the surface of media.
For accuracy, the lens fitted or mounted in it
helps to see the colonies.
The lens is movable on the box and can be
adjusted to see the colonies.
The petriplate is kept on a slanting platform
meant for it and illuminated with the help of
light source from beneath.
The numbers of colonies are read out with the
support of digital reading meter.
Figure 1.6

Microbiology Laboratory Manual 11

LABORATORY 2

The
Microscope
 THE MICROSCOPE
A good microscope is an essential tool for any microbiology laboratory. There are many
kinds of microscopes but the type most useful in diagnostic work is the compound
microscope. By means of a series of lenses and a source of bright light, it magnifies and
illuminates minute objects such as bacteria and other microorganisms that would
otherwise be invisible to the eye. This type of microscope will be used throughout your
laboratory course. As you gain experience using it, you will realize how precise it is and
how valuable for studying microorganisms present in clinical specimens and in cultures.
Even though you may not use a microscope in your profession, a firsthand knowledge of
how to use it is important. Your laboratory experience with the microscope will give you
a lasting impression of living forms that are too small to be seen unless they are highly
magnified. As you learn about these “invisible” microorganisms, you should be better
able to understand their role in transmission of infection.

USES OF MICROSCOPE
IN PARASITOLOGY
The microscope is used in Urine Analysis to check for the presence of
various types of crystals, and white blood cells, which could indicate a bladder
infection. The microscope is used in Parasitology to identify different types of
parasitic organisms intracellular and extracellular.

IN MICRBIOLOGY
The microscope is essential to the microbiology lab: most microorganisms
cannot be seen without the aid of a microscope, save some fungi. And, of course,
some microbes cannot be seen even with a microscope, unless it is an electron
microscope, such as viruses.

IN LABORATORY
It produces clear, high-quality images, whether an optical microscope,
which uses light to generate the image, scanning or transmission electron
microscope (using electrons), or a scanning probe microscope (using a probe). It is
also used as an instrument for viewing objects that are too small for the naked eye
to see. It allows us to make research, study some microorganisms, cells, and their
structures, make diagnostics, and so on.

Microbiology Laboratory Manual 13

 TYPES OF SIMPLE MICROSCOPE

MICROSCOPE
COMPOUND MICROSCOPE
ELECTRON MICROSCOPE

A. DEFINITION
A simple microscope is a magnifying glass that has a double convex lens with a short
focal length. The examples of this kind of instrument include the hand lens and reading
lens. When an object is kept near the lens, then its principal focus with an image is
produced, which is erect and bigger than the original object. The formed image is virtual
and cannot be projected on a screen like a real image.

FIGURE 2.1 SIMPLE MICROSCOPE PARTS AND FUNCTIONS

Eyepiece: It is the lens that is used to study the
samples and is placed at the top. It has a
magnification of 10X to 15X.
Base: This provides support to the microscope.
Tube: This is used to connect the eyepiece to the
objective lenses.
Objective lenses: These are found with the
magnification of 10X, 40X and 100X and are colour
coded. The lower power lenses are the shortest
lens and the highest power lenses are the longest
lens.
Revolving nose-piece: This is also known as the
turret. It is used for holding of other objective lens
and can be rotated while viewing the samples.
Diaphragm: It is used to control the amount of
light that passes through the stage.
Stage: It is the platform used for placing the slides
with samples.
Stage clip: These are used to hold the slides in the
proper place.
Coarse adjustment knob: It is used to focus on
scanning.
Fine adjustment knob: It is used to focus on oil.
Arm: It is used to support the tube and connects to

SIMPLE the base of the microscope.

Power switch: The main power switch used to turn
on or off the microscope.

MICROSCOPE Condenser: It is used to focus the light on the

sample and 400X power lenses are used.

Microbiology Laboratory Manual 14

 A. DEFINITION
A compound microscope is defined as a microscope with a high resolution and uses
two sets of lenses providing a 2-dimensional image of the sample. The term compound
refers to the usage of more than one lens in the microscope. Also, the compound microscope
is one of the types of optical microscopes. The other type of optical microscope is a simple
microscope. The difference between simple and compound microscope is that a simple
microscope uses only one lens while the compound microscope uses more than one lens.
The compound microscope is mainly used for studying the structural details of cell,
tissue, or sections of organs. The parts of a compound microscope can be classified into two:
Non-optical parts and Optical parts.

A. NON-OPTICAL PARTS

• Base- also known as the foot which is either U or horseshoe-shaped. It is a metallic

structure that supports the entire microscope.
• Pillar- the connection between the base and the arm is possible through the pillar.
• Arm- arm is also known as the limb which is a metallic handle forming the connection
between the arm to the inclined joint. The stage and the body tube are supported by the
arm.
• Inclination Joint- if the observation has to be done in a sitting position, then the
microscope can be tilted using the inclination joint.
• Stage- it is the metallic platform that is fitted to the lower part of the arm with a hole in
the center. The microscopic slides are placed on the stage either by using side clips or by
mechanical stage clips.
• Body Tube- the main purpose of the body tube is to hold the objective and ocular lenses
at the two ends. The end where the ocular lens is present is known as the head while the
end where the objective lens is placed is known as the nose piece. For the passage of light
rays through the body tube, there is a pathway.
• Draw Tube- the upper end of the body tube has a small fixed tube which is known as the
drawtube. The main function of the drawtube is to hold the ocular lens.
• Rack and Pinion- to bring the object under focus, the rack and pinion are either attached
to the body tube or the stage.
• Adjustment Screws- these are two pairs of adjusting screws that are used either for a
coarse adjustment or for fine adjustment. When a fine adjustment is made, the body tube
or the stage moves extremely short distances while in coarse adjustment, the body tube
and stage move up. Through fine adjustment, a sharp image can be obtained.
• Automatic Stop- the rack and pinion have a small screw that is used for stopping the
downward sliding of the body tube. This prevents damage to the objective lens.

Microbiology Laboratory Manual 15

B. OPICAL PARTS

Diaphragm- The amount of light falling on the object can be controlled through the
diaphragm. It is present below the stage. The disc and iris are the two types of
diaphragm.

Condenser- It is present below the diaphragm. The focusing of light can be done by
adjusting the condenser by moving it either up or down.

Reflector- A reflector is a mirror that is attached above the base. One side of the mirror
has a plane mirror while the other side has a concave mirror. When the light is strong,
the plane mirror side is used and when the light is weak, the concave mirror side is used.
The light on the object is directed with the help of the reflector through the diaphragm
and condenser.

Objective Lenses- These lenses are present over the nose piece. There are two to three
types of objective lenses: Low power, High power, and Oil immersion. The objective lens
is a compound lens that forms a real inverted image of the image inside the body tube.

Ocular Lens- The ocular lens is also known as the eyepiece. The image of microscopic
objects can be viewed through these lenses. There are four types of magnification that
can take place in the ocular lens: 5X,10X,15X, and 20X. The binocular head is the device
that uses two eyepieces and has many mirrors and prisms, which makes the passage of
light easier.

FIGURE 2.2
COMPOUND MICROSCOPE

COMPOUND
MICROSCOPE
Microbiology Laboratory Manual 16
 DEFINITION
ELECTRON
An electron microscope is a microscope
MICROSCOPE that uses a beam of accelerated electrons as
a source of illumination. It is a special type of
microscope having a high resolution of
PARTS AND FUNCTIONS
images, able to magnify objects in
nanometers, which are formed by controlled
Electron gun- use of electrons in a vacuum captured on a
The electron gun is a heated tungsten phosphorescent screen. Ernst Ruska (1906-
filament, which generates electrons. 1988), a German engineer and academic
Electromagnetic lenses professor, built the first Electron Microscope
The condenser lens focuses the electron in 1931, and the same principles behind his
beam on the specimen. A second prototype still govern modern EMs.
condenser lens forms the electrons into
FIGURE 2.3
a thin tight beam. TRANSMISSION ELECTRON MICROSCOPY
The electron beam coming out of the
specimen passes down the second of
magnetic coils called the objective lens,
which has high power and forms the
intermediate magnified image.
The third set of magnetic lenses called
projector (ocular) lenses produce the final
further magnified image.
Each of these lenses acts as an image
magnifier all the while maintaining an
incredible level of detail and resolution. FIGURE 2.4
SCANNING ELECTRON MICROSCOPY
Specimen Holder
The specimen holder is an extremely thin
film of carbon or collodion held by a
metal grid.
Image viewing and Recording
System
The final image is projected on a
fluorescent screen.
Below the fluorescent screen is a camera
for recording the image.

Microbiology Laboratory Manual 17

HOW TO USE A MICROSCOPE

1. OBSERVE THAT A FLAT PLATFORM, OR STAGE AS IT IS CALLED, EXTENDS BETWEEN THE UPPER
LENS SYSTEM AND THE LOWER SET OF DEVICES FOR PROVIDING LIGHT. THE STAGE HAS A HOLE
IN THE CENTER THAT PERMITS LIGHT FROM BELOW TO PASS UPWARD INTO THE LENSES ABOVE.
THE OBJECT TO BE VIEWED IS POSITIONED ON THE STAGE OVER THIS OPENING SO THAT IT IS
BRIGHTLY ILLUMINATED FROM BELOW. NOTE THE ADJUSTMENT KNOBS AT THE SIDE OF THE
STAGE, WHICH ARE USED TO MOVE THE SLIDE IN VERTICAL AND HORIZONTAL DIRECTIONS ON
THE STAGE. THIS TYPE OF STAGE IS REFERRED TO AS A MECHANICAL STAGE.

2. A BUILT-IN ILLUMINATOR AT THE BASE IS THE SOURCE OF LIGHT. LIGHT IS DIRECTED UPWARD
THROUGH THE ABBE CONDENSER. THE CONDENSER CONTAINS LENSES THAT COLLECT AND
CONCENTRATE THE LIGHT, DIRECTING IT UPWARD THROUGH ANY OBJECT ON THE STAGE. IT
ALSO HAS A SHUTTER OR IRIS DIAPHRAGM WHICH CAN BE USED TO ADJUST THE AMOUNT OF
LIGHT ADMITTED. A LEVER IS PROVIDED ON THE CONDENSER FOR OPERATING THE DIAPHRAGM.

3. THE CONDENSER CAN BE LOWERED OR RAISED BY AN ADJUSTMENT KNOB. LOWERING THE

CONDENSER DECREASES THE AMOUNT OF LIGHT THAT REACHES THE OBJECT. THIS IS USUALLY A
DISADVANTAGE IN MICROBIOLOGICAL WORK. IT IS BEST TO KEEP THE CONDENSER FULLY
RAISED AND TO ADJUST LIGHT INTENSITY WITH THE IRIS DIAPHRAGM.

4. ABOVE THE STAGE, ATTACHED TO THE ARM, A TUBE HOLDS THE MAGNIFYING LENSES THROUGH
WHICH THE OBJECT IS VIEWED. THE LOWER END OF THE TUBE IS FITTED WITH A ROTATING
NOSEPIECE HOLDING THREE OR FOUR OBJECTIVE LENSES. AS THE NOSEPIECE IS ROTATED ANY
ONE OF THE OBJECTIVES CAN BE BROUGHT INTO POSITION ABOVE THE STAGE OPENING. THE
UPPER END OF THE TUBE HOLDS THE OCULAR LENS, OR EYEPIECE (A MONOCULAR SCOPE HAS
ONE; A BINOCULAR SCOPE PERMITS VIEWING WITH BOTH EYES THROUGH TWO OCULARS).

5. DEPENDING ON THE BRAND OF MICROSCOPE USED, EITHER THE ROTATING NOSEPIECE OR THE
STAGE CAN BE RAISED OR LOWERED BY COARSE AND FINE ADJUSTMENT KNOBS. THESE ARE
LOCATED EITHER ABOVE OR BELOW THE STAGE. ON SOME MICROSCOPES THEY ARE MOUNTED
AS TWO SEPARATE KNOBS; ON OTHERS THEY MAY BE PLACED IN TANDEM WITH THE SMALLER
FINE ADJUSTMENT EXTENDING FROM THE LARGER COARSE WHEEL. LOCATE THE COARSE
ADJUSTMENT ON YOUR MICROSCOPE AND ROTATE IT GENTLY, NOTING THE UPWARD OR
DOWNWARD MOVEMENT OF THE NOSEPIECE OR STAGE. THE COARSE ADJUSTMENT IS USED TO
BRING THE OBJECTIVE DOWN INTO POSITION OVER ANY OBJECT ON THE STAGE, WHILE LOOKING
AT IT FROM THE SIDE TO AVOID STRIKING THE OBJECT AND THUS DAMAGING THE EXPENSIVE
OBJECTIVE LENS. THE FINE ADJUSTMENT KNOB MOVES THE TUBE TO SUCH A SLIGHT DEGREE
THAT MOVEMENT CANNOT BE OBSERVED FROM THE SIDE. IT IS USED WHEN ONE IS VIEWING
THE OBJECT THROUGH THE LENSES TO MAKE THE SMALL ADJUSTMENTS NECESSARY FOR A
SHARP, CLEAR IMAGE.

Microbiology Laboratory Manual 18

 M I C R O S C O P E :

Proper Care and Handling

1.
Always use both hands to carry the microscope, one
holding the arm, other under the base.

2. 3.
Before each use, examine the Keep the oculars, objectives,
microscope carefully and and condenser lens clean. Use
report any unusual condition dry lens paper only.
or damage.

4. 5.
At the end of each laboratory Replace the dust cover, if
period in which the microscope available, and return the
is used, remove the slide from microscope to its box
the stage, wipe away the oil on
the oil-immersion objective, and
place the low-power objective in
vertical position.

Handling and Examining Cultures

Microscopic examination of microorganisms provides important information about their
morphology but does not tell us much about their biological characteristics. To obtain
such information, we need to observe microorganisms in culture. If we are to cultivate
them successfully in the laboratory, we must provide them with suitable nutrients, such
as protein components, carbohydrates, minerals, vitamins, and moisture in the right
composition. This mixture is called a culture medium. It may be prepared in liquid form,
as a broth, or solidified with agar, a nonnutritive solidifying agent extracted from
seaweed. Agar media may be used in tubes as a solid column or as slants, which have a
greater surface area. They are also commonly used in petri dishes, or plates.

Solid media are essential for isolating and separating bacteria growing together in a
specimen collected from a patient, for example, urine or sputum. When a mixture of
bacteria is streaked across the surface of an agar plate, it is diluted out so that single
bacterial cells are deposited at certain areas on the plate. These single cells multiply at
those sites until a visible aggregate called a colony is formed. Each colony represents the
growth of one bacterial species. A single, separated colony can be transferred to
another medium, where it will grow as a pure culture.

Microbiology Laboratory Manual 19

Colonies of several different species are regularly present on the same agar plate when
certain patient specimens are inoculated onto them. Working with pure cultures permits
the microbiologist to study the properties of individual species without interference from
other species.
The appearance of colonial growth on agar media can be very distinctive for individual
species. Observation of the noticeable, gross features of colonies, that is, of their colonial
morphology, is therefore very important. The colour, density, consistency, surface
texture, shape, and size of colonies all should be observed, for these features can
provide clues as to the identity of an organism, although final identification cannot be
made by morphology alone.
In liquid media, some bacteria grow diffusely, producing uniform clouding, whereas
others look very granular. Layering of growth at the top, center, or bottom of a broth
tube reveals something of the organisms’ oxygen requirements. Sometimes colonial
aggregates are formed and the bacterial growth appears as small puff balls floating in
the broth. Observation of such features can also be helpful in recognizing types of
organisms
Disposal of Laboratory Wastes and Cultures

During laboratory practices, it has been noticed that the untreated waste is generally
disposed off by the laboratory staff. It happens due to their unskilled work culture. Most
of the laboratories situated in the rural area used discarded hospital material for land
filling purposes. Any material which contains microorganisms should be treated first and
thereafter, with the proper treatment should be thrown properly. The treatment is
necessary due to the reasons:
a) If it contains pathogenic microorganisms, the disease may transmit or spread to the
healthy persons.
b) It may contaminate soil and causes soil, water and air-pollution. Hence, to check
from such hazards, proper treatment is required to kill microorganisms.

The infected material is generally the solid or liquid culture media used for cultivation of
microorganisms, or it may also contain cotton plugs, paper, cotton or cotton swabs,
gloves, pins, PCR tubes, gel material etc.
Some of the materials such as cotton plugs, paper, napkins, swabs etc. should be
autoclaved first and then it is incinerated. But the microbial contaminants containing
materials should be treated with some disinfectant and thereafter autoclaved by putting
them in suitable containers. The molten material should be discarded.
Sometimes, HCl is also added to hydrolyze the agar, if present in the medium. This is
added before their safe disposal. All such laboratory materials should be disposed of
after autoclaving.

Microbiology Laboratory Manual 20

 EXAMPLES OF MICROORGANISMS
UNDER THE MICROSCOPE

FIGURE 2.5 SAMPLE

UNDER MICROSCOPE

FIGURE 2.6 SAMPLE

UNDER
MICROSCOPE

Microbiology Laboratory Manual 21

LABORATORY 3

Methods in
Microbiology
 DIFFERENT METHODS
IN MICROBIOLOGY
1 STERELIZATION
Sterilization is the process of destroying or physically removing all forms of microbial
life including vegetative cells, spores and viruses from a surface, a medium or an
article. The principal reasons for controlling microorganisms are:
1. To prevent transmission of disease and infection
2. To prevent contamination by undesirable microorganisms
3. To prevent deterioration and spoilage of materials by microorganisms .
The methods of sterilization employed depend on the purpose for which sterilization is
carried out, the material which has to be sterilized and the nature of the
microorganisms that are to be removed or destroyed. The various agents used in
sterilization can be grouped into physical and chemical agents

PHYSICAL AGENTS a
(PHYSICAL METHODS)
Sunlight Direct sunlight has an active germicidal effect due to the combined effect
of the ultraviolet radiation and heat. This is a natural method of sterilization.
Drying Moisture is essential for the growth of bacteria. Drying in air has therefore a
deleterious effect on many bacteria. But spores are unaffected by drying. Hence
this is very unreliable method.
Heat Heat has a killing effect on microorganisms and is one of the most popular
reliable methods to destroy. Microorganisms has a minimum, optimum and
maximum growth temperatures. Temperature below the minimum usually
produces static (inhibition of metabolism).

Microbiology Laboratory Manual 23

PHYSICAL AGENTS (PHYSICAL METHODS)
Temperature above the maximum, generally kill microorganisms. This is because
biochemical changes in the cells organic molecules result in its death. These
changes arise from alterations in enzyme molecules or chemical break down of
structural molecules especially in the cell membranes. Heat also drives off water
and this loss of water may be lethal to the organisms.
The killing rate of heat may be expressed as a function of time and temperature.
Each microbial species has a Thermal Death Time (TDT). It’s the minimum time
required to kill a population of microorganism in a microbial suspension at a given
temperature and under defined condition. Each species also has a Thermal Death
Point (TDP), the temperature at which it dies in a given time. In determining the
time and temperature for microbial destruction with heat, the following factors
are considered.
1. Type of organism to be killed
2. Type of material to the treated
3. Presence of organic matter
4. Acidic or basic nature of the material

NATURE OF HEAT:
a) DRY HEAT
Many objects are best sterilized in the absence of water by dry heat sterilization;
killing by dry heat is due to protein denaturation, oxidative damage and toxic
effect of elevated levels of electrolytes.
Methods of Sterilization by Dry Heat
1. Flaming
Inoculating loops and points of
forceps may be heated in the Bunsen
flame, till they are red-hot. Articles
such as mouth of the culture tubes,
cotton wool plugs, glass slides etc.
are passed over the flame without
allowing it to become red hot.

2. Incineration
This is an excellent method for
rapidly destroying, animal carcasses,
FIGURE 3.1 HOT
pathological material and
AIR OVEN
disposables.

Microbiology Laboratory Manual 24

3. Hot Air Oven
This is the most widely adopted method of sterilization by dry heat. The hot air oven
utilizes radiating dry heat for sterilization. This type of energy does not penetrate
materials easily and thus, long periods of exposure to high temperature are
necessary. For example, at a temperature of 160°C, a period of two hours is required
for the destruction of bacterial spores. Hot air oven is used to sterilize glassware,
forceps, scissors, scalpels, glass syringes, liquid paraffin, dusting powder etc. A
holding period of 160°C for an hour is used. The oven is usually heated by electricity,
with heating elements in the wall of the chamber and it must be filled with a fan to
ensure even distribution of hot air and elimination of air pockets. The materials
should be arranged in a manner which allows free circulation of hot air in between
the objects. It should not be over-loaded. Glass wares should be perfectly dry before
being placed in the oven. Test tubes, flasks etc. should be wrapped in craft paper.
Oven must be allowed to cool slowly for about 2 hours before the door is opened,
since the glasswares may get cracked by sudden or uneven cooling.

STERILIZATION CONTROL

a) MOIST HEAT
Moist heat kills microorganisms by coagulating their proteins and is much more rapid
and effective than dry heat because water molecules conduct heat better than air.
Lower temperature and less time of exposure are therefore required than for dry
heat. Moist heat readily kills viruses, bacteria, fungi etc.
A. TEMPERATURE BELOW 100°C
For pasteurization of milk, there are two methods:
Holding Method or Low Temperature Holding Method (LTH)
In this method, the milk is exposed to a temperature of 63°C (145°F) for 30
minutes in an appropriately designed equipment. This is followed by sudden
cooling to 13°C or below.
Flash Process or High Temperature Short Time (HTST)
In this method, the milk is exposed to a temperature of 72°C for 15 seconds
in the equipment. This is followed by sudden cooling to 13°C or below. The
finished product should be stored at a low temperature to retard growth of
microorganisms and pasteurization removes the pathogenic bacteria in milk. By
these processes all

The spores of a non – toxigenic strain of Clostridium tetani are used as a

microbiological test of dry heat efficiency. Paper stripes impregnated with 106
spores are placed in envelop and inserted into suitable packs. After sterilization is
over, the strips are removed and inoculated into thioglycollate or cooked meat media
and incubated for sterility test under strict anaerobic conditions for five days at 37°C.

Microbiology Laboratory Manual 25

non-sporing pathogens such as mycobacteria, salmonellae and brucella are
destroyed ‘Coxiella bumetic’ is relatively heat resistant and may survive the holder
method.
ii. Vaccine bath It’s used for killing non-sporing bacteria which may be present in
vaccine. In vaccine bath, the vaccine is treated with moist heat for one hour at 60°C.
iii. Serum containing coagulable proteins can be sterilized by heating for one hour at
56°C in a water bath for several successive days.

b) TEMPERATURE at 100°C
i. Boiling
Most of the vegetative forms of bacteria, fungi etc. are killed almost immediately at
90-100°C, but sporing bacteria required considerable periods of boiling. Boiling
water is not considered as a sterilizing agent because destruction of bacterial spores
and inactivation of viruses cannot always be assured. Under ordinary circumstances,
most species of microbes can be killed within 10 minutes. However, spores of
bacteria and fungi, protozoa cysts and large concentrations of Hepatitis A viruses
requires up to 30 minutes exposure or more because inadequate information exists
on the heat tolerance of many microorganisms, boiling water is not reliable for
sterilization purpose especially the sterilization of instruments and for surgical
procedures. In cases where boiling is considered adequate, the material should be
immersed in water and boiled for a period of 10-30 minutes. The lid of the sterilizer
should not be opened during that period. Addition of little acid, alkali or washing
soda will increase the efficiency of the process.
ii. Steam under atmospheric pressure (100°C)
Steam under atmospheric pressure is used to sterilize culture media which may
decompose if subjected to higher temperature. A Koch or Arnold sterilizer is an
instrument that generates free floating steam. The container and the medium are
simultaneously sterilized and evaporation from the medium is prevented one
exposure of 90 minutes usually ensures complete sterilization of the medium. This is
an inexpensive method.
iii.Sterilization above 100°C (steam under pressure)
Heat in the form of saturated steam under pressure is the most practical and
dependable agent for sterilization. Steam under pressure provides temperature
above those obtainable by boiling. Moreover, it has advantages of rapid heating,
penetration and moisture in abundance, which facilitates the coagulation of the
protein of microorganisms, resulting in complete destruction of all forms of microbial
life, including bacterial endospores. It is important to note that the sterilizing agent is
the moist heat not the pressure. The laboratory apparatus designed to use steam
under regulated pressure is called an autoclave. It is essentially a double jacketed
steam chamber equipped with devices which permit the chamber to be filled with
saturated steam and maintained at a designed temperature and pressure for any

Microbiology Laboratory Manual 26

period of time. The articles to be sterilized are placed in the sterilizing chamber
and steam is maintained in the steam jacket into the sterilizing chamber, cool air
is forced out and a special valve increases the pressure to 15 pounds/square inch
above normal atmospheric pressure. The temperature rises to 121.5°C and the
superheated water molecules rapidly conduct heat into microorganisms and will
be killed. The time for destruction of the most resistant bacterial spore is reduced
to 15 minutes. For denser objects, up to 30 minutes of exposure may be required.
Autoclave is an essential equipment in every microbiology laboratory. It’s used to
sterilize many media, solutions, discarded cultures, glass wares, metal wares etc.

FIGURE 3.2
AUTOCLAVE
FILTIRATION
Filtration is the process of removal of microorganisms from liquid or gases
using a mechanical device known as filter. This is an excellent way to reduce the
microbial population in solution of thermo labile materials such as sera, antibiotic
solutions, intravenous solutions, carbohydrates solutions used in the preparation
of culture media etc. As fluid passes through the filter, microorganisms are
trapped in the pores of the filtering material. The solution that drips through the
filter is collected in a previously sterilized container. Porosity, electric charges of
the filter, electric charge carried by the organisms, nature of the fluid being
filtered etc. can influence efficiency of filtration.
Types of filters: Seltz Filter, Berkefeld Filter, Membrane Filter, High Efficiency
Particulate Air (HEPA) filter
IRRADATION
The process of exposing organisms to anyone of the radiation such as UV-
rays, X-rays, gamma rays etc. is known as irradiation. Irradiation is an effective
method of sterilization. Two types of radiations are used for sterilization.

Microbiology Laboratory Manual 27

 a) Non ionizing radiation
 UV radiation
 Infrared radiation
b) Ionizing radiation
 X rays
 Gamma rays

b CHEMICAL METHODS
The physical agents for controlling microorganisms are generally intended to achieve
sterilization. Instead, they are expected only to destroy the pathogenic organisms on or
in or removal of pathogenic microorganisms is called disinfection. An agent, usually a
chemical that kills the pathogenic microorganisms on/in animate objects is known as a
disinfectant. A disinfectant does not necessarily sterilize an object because a few
microorganisms and viable spores may remain. The chemical agents that are applied to
the body to prevent infection or species are called antiseptic. The antiseptic prevents
the growth or action of microorganisms either by destroying them or by inhibiting their
growth and metabolism.
An antimicrobial agent is generally called as a germicide. A disinfectant or antiseptic
can be particularly effective against a specific group called as bactericides, fungicides
or algaecides. Some chemicals do not kill microorganisms, but they temporarily
prevent from multiplication. Many different chemicals are available for use as
disinfectants and each has its own advantages and disadvantages.

Characteristics of a desirable disinfectant

The disinfectant must be effective against a wide variety of infections/agents,

at high dilutions and in the presence of organic matter. The chemical must be
toxic for infection agents but it should not be toxic to people or corrosive for
common upon storage, colourless with a pleasant odors, soluble in water and
lipids for penetration into microbes and have a low surface tension so that it
can cracks in surface. If possible, the disinfectant should be relatively
inexpensive.

Microbiology Laboratory Manual 28

The factor influencing the effectiveness of chemical disinfectants:
1. Size of the microbial population
2. Nature of microbes present
3. Concentration and nature of the disinfectant
4. Duration of exposure
5. Temperature
6. Local environment

The main modes of action of disinfectant:

1. Protein coagulation
2. Disruption of cell membrane, thus resulting in exposure of the contents of the
cell to the adverse environment and loss of the constituents of the cell and
changes in the composition of the contained cytoplasm. These cause death of
cell.
3. Removal of free sulfhydryl groups which are essential for the functioning of the
enzymes and thus the life of the cells.
4. Inhibition of respiration of catabolic/anabolic reactions.
5. Loss of membrane integrity resulting in leakage of essential constituents such
as potassium cations, inorganic phosphate, pentose, nucleotides and nucleosides
and proteins.

Major Group of Chemical Agents: Phenol and Phenol compounds, Alcohols, Heavy metals and
their compounds, Dyes, Soaps and Detergents, Aldehydes, Gaseous agents

Microbiology Laboratory Manual 29

 LABORATORY 4

Cultural
Characteristics of
Microorganisms
CULTURAL CHARACTERISTICS
OF MICROORGANISMS
AIM:
To determine the cultural characteristics of microorganisms
in identifying and classifying organisms into taxonomic
groups.

PRINCIPLE:
When grown on a variety of media, microorganisms will
exhibit the difference in the microscopic appearance of their
broth. These differences are called cultural characteristics
and are used as a basis for separating microorganism into
taxonomic groups. The cultural characteristics for all taxon
microorganisms are contained in Bergey’s Manual of
systematic bacteriology.

THE FOLLOWING CHARACTERS

OF COLONY ARE NOTED:

1. Size: in millimeter
2. Shape: circular / irregular
FIGURE 4.1: GROWTH ON SOLID MEDIA
3. Surface : smooth, rough,
granular
4. Elevation : flat, low convex,
high convex, raised, umbonate,
umbulate
5. Edge: entire, undulate, lobate,
crenated, fimbricate, ciliate
6. Opacity : opaque, translucent,
transparent
7. Colour of colony
8. Consistency : mucoid, friable
9. Other properties : hemolysis,
pigmentation, swarming

Microbiology Laboratory Manual 31

MORPHOLOGY ON NUTRIENT AGAR SLANTS

The isolated bacteria can be identified based on their

colony characteristics in the following manner.
1. Degree of growth : scanty, moderate, abundant
2. Surface : smooth, rough, granular
3. Elevation : convex, flat, raised
4. Edge : entire, undulate, crenate
5. Opacity : opaque, translucent, transparent
6. Consistency : firm, butyrosis, powdery, mucoid, membranous
7. Colour of Colony : creamy white, lemon yellow, bluish green
8. Form : filiform, echinulated, beaded, effuse, rhizoid
9. Changes in Medium : changes in colour, pitting of agar TURBIDITY

FIGURE 4.2: GROWTH ON LIQUID MEDIA

FIGURE 4.3: GROWTH ON NUTRIENT AGAR SLANT

MORPHOLOGY ON NUTRIENT AGAR PLATES

These demonstrate well isolated colonies and are evaluated in the

following manner.
1. Size: pinpoint, moderate, small or large d) serrate: tooth like appearance
2. Colour of the colony e) filamentous: thread like, spreading edge
a) Form: the shape of the colony: 4. Elevation: the degree to which the colony
b) circular: unbroken, peripheral edge growth is raised on the surface is described as
c) irregular: intended, peripheral edge follows:
d) rhizoid: root like, spreading growth a) flat: elevation not discernible
3. Margin: The appearance of the outer edge of b) raised: slightly elevated
the colony is described as follows c) convex: dome shaped elevation
a) entire: sharp d) umbonate : raised with elevated convex
b) lobate: marked indentations central region
c) undulate: wavy indentations

Microbiology Laboratory Manual 32

GROWTH IN LIQUID MEDIA
The liquid medium (nutrient broth, peptone water and other liquid media) the following
characteristics are noted:
1) the degree of growth : scanty, moderate, abundant
2) presence of turbidity and its nature (uniform turbidity)
3) presence of deposits, pellicle formation on surface & its quality

MORPHOLOGY ON NUTRIENT BROTH CULTURES

These are evaluated for the distribution & appearance of the growth as follows:

1) uniform time turbidity : finely dispersed throughout

2) flocculent: flank aggregates, dispersed throughout
3) pellicle: thick, pad like growth on the surface
4) sediment: concentration of growth at the bottom of broth cultures may be granules

FIGURE 4.4: GROWTH ON PLATES

Microbiology Laboratory Manual 33

 EIGHT KINGDOM CLASSIFICATION
ACCORDING TO THOMAS CAVALIER-SMITH

I. E U B A C T E R I A
Eubacteria are prokaryotic microorganisms consisting of a single cell lacking a
nucleus and containing DNA is a single circular chromosome. Eubacteria can be either
gram-negative or gram-positive, they have economic, agricultural, and medical
importance. They include E. coli, Lactobacilli, and Azospirillum.
Eubacteria (sometimes called simply as “bacteria”) are small organisms that
cannot be seen by naked eyes; thus, microscopes are used to visualize and study their
morphology. To do so, bacteria are stained. Staining is an essential microbiological
technique as it helps in highlighting the whole bacterial structure and cellular shape.
Bacteria are classified according to Gram staining.
Its reproduction usually includes dividing the parent cell into two daughter
cells after the replication of genetic material in a process called binary fission. Some
bacteria have the ability to form a spore in unfavorable conditions such as deficiency
of nutrients, exposure to chemicals, or radiation.

FIGURE 4.5: GRAM+ ROD SHAPED LACTOBACILLUS

LACTOBACILLUS species are

probiotics ("good" bacteria) normally
found in human digestive and
urinary tracts. They can be
consumed for diarrhea and "gut
health." "Good" bacteria such as
Lactobacillus can help the body
break down food, absorb nutrients,
and fight off "bad" organisms that
might cause diseases.

Microbiology Laboratory Manual 34

II. A R C H A E B A C T E R I A
Archaebacteria are a group of microorganisms considered to be an ancient
form of life that evolved separately from the bacteria and blue-green algae, and they
are sometimes classified as a kingdom.
These high-temperature-loving bacteria represent life at the known upper
temperature limit. Within the marine environment, hyperthermophilic archaebacteria
have been found in shallow-water hydrothermal fields as well as in deep hot
sediments and vents (see Stetter, 1982, 1986; Neuner et al., 1990). Archaea bacteria
can also be found in some parts of the human body such as the colon, mouth, and
skin. Archaea bacteria are not usually pathogenic.
Archaebacteria are obligate or facultative anaerobes, i.e., they flourish in the
absence of oxygen and that is why only they can undergo methanogenesis. The
major types of Archaebacteria are Crenarchaeota, Euryarchaeota, Korarchaeota,
Thaumarchaeota, and Nanoarchaeota.

FIGURE 4.6: LOKIARCHAEOTA

LOKIARCHAEOTA were first discovered off the coast of Norway, 15 kilometers

from deep sea vents known as Loki’s castle. Lokiarchaeota are considered to be
important because they provide the best evidence to date for the evolutionary link
between simple single-celled organisms, called prokaryotes, and organisms with
complex cells called eukaryotes like multi-cellular animals and plants

Microbiology Laboratory Manual 35

III. A R C H E Z O A
The taxon Archezoa was proposed to unite a group of very odd eukaryotes
that lack many of the characteristics classically associated with nucleated cells, in
particular the mitochondrion. The hypothesis was that these cells diverged from other
eukaryotes before these characters ever evolved, and therefore they represent
ancient and primitive eukaryotic lineages. The kingdom comprised four groups:
Metamonada, Microsporidia, Parabasalia, and Archamoebae.
Until recently, molecular work supported their primitive status, as they
consistently branched deeply in eukaryotic phylogenetic trees. However, evidence has
now emerged that many Archezoa contain genes derived from the mitochondrial
symbiont, revealing that they actually evolved after the mitochondrial symbiosis. In
addition, some Archezoa have now been shown to have evolved more recently than
previously believed, especially the Microsporidia for which considerable evidence now
indicates a relationship with fungi. In summary, the mitochondrial symbiosis now
appears to predate all Archezoa and perhaps all presently known eukaryotes.

FIGURE 4.7: ARCHAMOEBA

The Class Archamoeba includes the genera, Entamoeba, Endolimax,

Mastigamoeba, Mastigella, Mastigina, Pelomyxa, and Phreatamoeba. Entamoeba
histolytica is an important representative of this group since it is a human
pathogen, causing amoebic dysentery (bloody diarrhea). Endolimax species are also
found as parasites in the intestines of various animals.

Microbiology Laboratory Manual 36

IV. P R O T O Z O A
Protozoa are unicellular, eukaryotic, heterotrophic organisms. They are either
free-living or parasites. There are around 65000 species of protozoans categorized in
different groups. They lack a cell wall. There are many different cell organelles, that
perform various tasks performed by different organs in higher animals, e.g. mouth,
anus, intestinal tract, etc. There are many protozoa, that cause various diseases in
animals and humans, e.g. Plasmodium (malarial parasite), Trypanosoma (sleeping
sickness), Trichomonas (trichomoniasis), etc. The protozoa have many stages in their
life cycle. Some of the stages of the life cycle are infectious.
Protozoa are found in the aquatic environment. They live in freshwater or
oceans. Some are free-living and some are parasitic in plants and animals. Mostly
they are aerobic but some are anaerobic and present in the rumen or human
intestine. Some of the species are found in extreme environments like hot springs.
Some of them form resting cyst to overcome dry environments.

FIGURE 4.8: AMOEBA

AMOEBA, also spelled ameba, plural amoebas or amoebae, any of the

microscopic unicellular protozoans of the rhizopodan order Amoebida. The well-
known type species, Amoeba proteus, is found on decaying bottom vegetation of
freshwater streams and ponds. There are numerous parasitic amoebas.

Microbiology Laboratory Manual 37

V. C H R O M I S T A
The name Chromista means "colored", and although some chromists, like
mildews, are colorless, most are photosynthetic. Even though they are photosynthetic,
chromists are not at all closely related to plants, or even to other algae. Unlike plants,
the Chromista have chlorophyll c, and do not store their energy in the form of starch.
Also, photosynthetic chromists often carry various pigments in addition to chlorophyll,
which are not found in plants. It is these pigments which give them their characteristic
brown or golden color.
Photosynthetic chromists are some of the most important organisms in aquatic
ecosystems. The cool and temperate coasts of continents are lined with kelp forests,
where many commercially important fish and shellfish feed and reproduce, and
diatoms are frequently the primary source of food for both marine and fresh-water
organisms.
In additional to their roles as producers for marine animals, chromists provide
many products for industry. Alginates are viscous chemicals extracted from kelp;
these are used in paper production, toothpaste, and in ice cream, where the alginate
helps to improve texture and ensure uniform freezing and melting. Ancient chromists,
like coccolithophorids, are responsible for deposits of limestone and other rock
formations. The skeletons of dead chromists accumulate on the floor of lakes and
oceans, where they may become thick deposits of silica or calcium carbonate. These
deposits are useful for interpreting ancient climate, and in searching for oil.

FIGURE 4.9: GOLDEN ALGAE

GOLDEN ALGAE (Prymnesium

parvum) is a microscopic, single-
celled species of algae. See family
tree. It occurs worldwide, and can live
in a wide range of water
temperatures and salinities. It has a
complex life cycle and can form
resting cysts under unfavorable
conditions.

Microbiology Laboratory Manual 38

VI. P L A N T A E
Plantae is a taxonomic group that includes land plants and green algae.
Kingdom Plantae includes multicellular, (mostly) autotrophic eukaryotes that (usually)
conduct photosynthesis. Kingdom is formerly the highest taxonomic rank or the most
general taxon used in classifying organisms. Further, the kingdom Plantae has been
classified into several subgroups based on the plant body, vascular system, and seed
development. These groups are Thallophyta, Bryophyta, Pteridophyta, Angiosperms,
and Gymnosperms.
Thallophyta includes plants with primitive and simple body structure. The plant
body is thallus, they may be filamentous, colonial, branched or unbranched. Examples
include green algae, red algae and brown algae.
Bryophytes do not have vascular tissues. The plant body has root-like, stem-
like and leaf-like structures. Bryophytes are terrestrial plants but known as
“amphibians of the plant kingdom” as they require water for sexual reproduction.
They are present in moist and shady places. Bryophyta includes mosses, hornworts
and liverworts.
Pteridophytes have a well-differentiated plant body into root, stem and leaves.
They have a vascular system for conduction of water and other substances.
Gymnosperms have a well-differentiated plant body and vascular tissues. They
bear naked seeds, i.e. seeds are not enclosed within a fruit.
Angiosperms are seed-bearing vascular plants with a well-differentiated plant
body. The seeds of angiosperms are enclosed within the fruits. Angiosperms are
widely distributed and vary greatly in size.

FIGURE 4.10: EPHEDRA

EPHEDRA, (genus Ephedra), genus

of 65 species of gymnosperm shrubs
of the family Ephedraceae. Ephedra is
an evolutionally isolated group and is
the only genus in the order
Ephedrales (division Gnetophyta).
Species are distributed in dry regions
in both the Eastern and Western
hemispheres.

Microbiology Laboratory Manual 39

VII. F U N G I
Fungi are eukaryotic organisms that include microorganisms such as yeasts,
moulds and mushrooms. The organisms found in Kingdom fungi contain a cell wall
and are omnipresent. They are classified as heterotrophs among the living organisms.
To name a few – the appearance of black spots on bread left outside for some days,
the mushrooms and the yeast cells, which are commonly used for the production of
beer and bread are also fungi. They are also found in most of the skin infections and
other fungal diseases. Thus, we can say that fungi usually grow in places which are
moist and warm enough to support them. Following are the important characteristics
of fungi:
1. Fungi are eukaryotic, non-vascular, non-motile and heterotrophic organisms.
2. They may be unicellular or filamentous.
3. They reproduce by means of spores.
4. Fungi exhibit the phenomenon of alternation of generation.
5. Fungi lack chlorophyll and hence cannot perform photosynthesis.
6. Fungi store their food in the form of starch.
7. Biosynthesis of chitin occurs in fungi.
8. The nuclei of the fungi are very small.
9. The fungi have no embryonic stage. They develop from the spores.
10. The mode of reproduction is sexual or asexual.
11. Some fungi are parasitic and can infect the host.
12. Fungi produce a chemical called pheromone which leads to sexual reproduction in
fungi.

FIGURE 4.11: MUSHROOM

MUSHROOM, the conspicuous

umbrella-shaped fruiting body
(sporophore) of certain fungi, typically
of the order Agaricales in the phylum
Basidiomycota but also of some other
groups. Popularly, the term mushroom
is used to identify the edible
sporophores; the term toadstool is
often reserved for inedible or
poisonous sporophores.

Microbiology Laboratory Manual 40

VIII. A N I M A L I A
Kingdom Animalia constitutes all animals. Amongst the five kingdoms, the
largest kingdom is the animal kingdom. Animals are multicellular eukaryotes.
However, like plants, they do not possess chlorophyll or a cell wall. Therefore,
members of the animal kingdom exhibit a heterotrophic mode of nutrition. Kingdom
Animalia has been classified into ten different subphyla based on their body design or
differentiation.
Animals are eukaryotic, multicellular, species belonging to the Kingdom
Animalia. Every animal has its own unique characteristics. They obtain their energy
either by feeding on plants or on other animals. There are millions of species which
have been identified, few share similar characteristics while others differ drastically.
Animals are classified based on their characteristics.
They are eminent from algae, plants, and fungus where rigid cell walls are
absent. Some are also heterotrophic, in general, they digest their food within the
internal chambers which again distinguish them from algae and plants. Another elite
character of these species is that they are motile, except in certain life stages.

FIGURE 4.12: BLACK-FOOTED CAT

With a hunt success rate of

60%, the highest of all cats, the
black-footed cat (felis nigripes)
is the ultimate feline predator.
Being the most successful of
hunters in the felid species,
successfully hunting most of
their prey, where the lion only
has a hunt success rate of 20%.

Microbiology Laboratory Manual 41

LABORATORY 5

Culture
Media
 CULTURE MEDIUM
The food materials on which the organism is grown is known as culture medium
and the growth of organism is known as culture. Different microorganisms
require different nutrient materials. Thus, culture media vary in form and
composition, depending upon the species to be cultivated. It must contain all the
ingredients required by the organism and in certain proportions. Basically there
should be an energy source, various macro and micronutrients, vitamins etc. it
must have a suitable pH. Moreover, it must be sterile so that the organism
cultivated may form a pure culture.
A culture is an in vitro technique of growing or cultivating microorganisms or only
other cells in a suitable nutrients medium called culture medium in the laboratory.
The primary aim of constructing a culture medium for any microorganism is to
provide a balanced mixture of required nutrients, at concentrations that will
permit good growth. Culture media give artificial environment simulating natural
conditions necessary for growth.

CHARACTERISTICS OF AN BASIC REQUIREMENTS OF

IDEAL CULTURE MEDIUM CULTURE MEDIUM
 Satisfactory growth for small  Energy source
inoculum – even for single cell.  Carbon source
 Rapid growth  Nitrogen source
 Easy to prepare  Salts
 Cheap  pH
 Easily producible  Adequate oxidation
 Demonstrate all the characteristics  Growth factors
in which we are interested

Microbiology Laboratory Manual 43

Common Ingredients of Culture Media
1. Water It is essential for existence of living cells. They act as source of
hydrogen and oxygen.

2. Peptone Golden granular hygroscopic powder which are obtained

from meat, casein fibrin or soya bean flour. Function: nitrogen source,
carbon source, buffers

3. Meat Extract It contains protein degradation products,

carbohydrates, inorganic salts, enzymes, excites and growth factors
that are rich in vitamin B complex. Function: Source of growth factors,
inorganic salts etc.

4. Yeast Extract It contains proteins, amino acids, growth factors

(Vitamin B), Carbohydrates and inorganic salts like potassium and
phosphates.
Functions: Source of growth factors and hence excellent stimulators of
growth. It can be used as suitable for meat extract.

5. Electrolyte Mainly used are sodium chloride or other electrolytes.

Functions: Essential to maintain the osmotic pressure.

6. Agar Dried mucilaginous substance obtained from gelidium species

and other algae available as long shield or in powder form; contains
mainly long chain polysaccharides, protein like material and inorganic
salts. Functions: it melts at 98°C and solidifies at 42°C, hence used as
solidifying agent.

7. Fermentable Compounds Mainly used are sugars, alcohols etc.

Function: Act as source of energy, fermentation reactions are helpful in
the identification and classification of organisms.

8. Buffers Carbonates and phosphates are used as buffer. Function: To

resist change in pH of the medium.

Microbiology Laboratory Manual 44

TYPES OF MEDIA

a) LIQUID MEDIA OR BROTH

No solidifying agents (eg: agar) is added while preparing the medium. The most
commonly used non-synthetic liquid media are nutrient broth, peptone solution,
milk, blood, serum etc. broth is a clear transparent straw colored fluid prepared
from meat extract or peptone. Beef infusions are rich in minerals, organic
micronutrients, protein, protein derivatives and carbohydrates. They are often
supplemented with 1% peptone or yeast extract culture fluids made from beef
infusion are commonly called infusion broth, where as those made from beef
extract are called extract broth.
Advantages of Liquid Media
For obtaining bacterial growth from blood or water when large volumes have
to be tested.
For preparing bulk cultures for preparation of antigens or vaccines.
It’s used to study growth rate and the sedimentation rate of bacterial cells.
Disadvantages of Liquid Media
It’s difficult to isolate different types of bacteria from mixed population.
It’s difficult to study colony characteristics.
FIGURE 5.1:

LIQUID CULTURE MEDIA include media for sterility testing, food and
water quality monitoring as well as cell culture media. Media are
available in different volumes and packaging, ready to use, with a
variety of antibiotics, additives, and dropout components.

Microbiology Laboratory Manual 45

b) SEMI-SOLID MEDIA
The semi-solid medium remains in the semi-solid condition. It is prepared by
adding small amount of agar (0.5%) or gelatin. Agar is a complex carbohydrates
prepared from algae like gelidium and gracillaria. Agar forms a colloidal solution
in hot water and sets in the form of a jelly when cool. Gelatin is an animal extract
prepared by hydrolysis of collagen with boiling water. When dissolved in water,
it’s in the form of a liquid when warm and sets in the form as it cools. Many
bacteria, when grown on a gelatin medium, produce a digestive enzyme
gelatinase, which liquefies gelatin. This feature is important in the identification
and classification of bacteria.
The semi-solid medium may be selective which promotes the growth of one
organism and retards the growth of another organism. This type of medium can
be used to study bacterial motility (semisolid media are useful for cultivation of
microaerophlic bacteria).

FIGURE 5.2:

SEMI-SOLID MEDIA are prepared with agar at concentrations of

0.5% or less. They have soft custard like consistency and are
useful for the cultivation of microaerophilic bacteria or for
determination of bacterial motility.

Microbiology Laboratory Manual 46

c) SOLID MEDIA
The solid medium is solid in consistency. It is prepared by adding 2% or 1%
gelatin; agar or silica gel is sometimes an inorganic solidifying agent for
autotrophic bacteria. It’s used for colony characterization, colony identification,
etc.
Based on composition, culture media can be classified into:
Simple Media
Complex Media
Synthetic or defined Media
Semi Solid Media
Special Media

FIGURE 5.3: EXAMPLE OF SOLID MEDIA- BLOOD AGAR

1) Simple Media
It’s also called basal media. eg: Nutrient Broth. It consists of meat extract, peptone,
Sodium Chloride and water. Nutrient agar made by adding 2% agar to nutrient broth is
the simplest and commonest medium in routine diagnostic laboratories.
2) Complex Media
These have added ingredients for special purpose or for bringing out certain
characteristics or providing special nutrient required for the growth of certain organisms.
3) Synthetic or defined Media
These media are prepared from pure chemical substances and the exact
composition of the medium is known. They are used for research purpose.
4) Semi Solid Media
The nutritional requirements of some microorganisms include some additional
ingredients of unknown chemical composition such as peptone, meat extract, yeast
extract, etc. Chemical composition is not fully known. They are called semi solid media.
5) Special Media
No single medium or set of conditions can support the growth of all the different
types of organisms that occur in nature. To cultivate, recognize, enumerate and isolate
certain types of microorganisms many special purpose media are needed on the basis of
their application or function, these special media can be classified into different types.
Enriched Media Differential Media
Enrichment Media Selective Media
Selective Media Sugar Media
Indicator Media Transport Media

Microbiology Laboratory Manual 47

i. Enriched Media
In these media, substances like blood, serum or egg are added to a basal medium
eg. Blood Agar, Chocolate Agar, Egg Media etc
ii. Enrichment Media
Some substances are added to liquid media with the result that wanted organism
grows more in number than unwanted organism. Such media are used in mixture
cultures eg. Tetrathionate broth (inhibit coliforms and allow typhoid paratyphoid bacilli
to grow freely)
iii. Selective Media
It favors the growth of particular microorganism. This is like enrichment media with
the difference that inhibiting substance is added to solid medium. eg. Desoxycholate
citrate medium for dysentery bacilli
iv. Indicator Media
These media contain an indicator which changes colour when a bacterium grows
in them. eg. Incorporation of sulphite in Wilson and Blair medium Salmonella typhi
reduces sulphite to sulphide in Wilson and Blair medium and the colonies of S. typhi
have a black metallic sheen.
v. Differential Media
Media that distinguish between different groups of bacteria and even permit to
identification of microorganisms based on their biological characteristics. A medium
which has substances incorporated into it, enabling it to bring out differing
characteristics of bacteria and thus helping to distinguish between them. Such media
are called differential media eg. Mac conkey medium consists of peptone, lactose, agar,
neutral red and taurocholate. It shows lactose fermenters, are colourless or pale (this
may also be termed indicator medium). Blood agar is an enriched medium, but also
differentiates between hemolytic organisms and non- hemolytic organisms. So it also
acts as a differential medium.
vi. Sugar Media
The usual sugar media consist of 1% sugar concerned. In peptone water along
with appropriate indicator, a small tube (Durham’s tube) is kept inverted in sugar tube to
detect gas production.
vii. Transport Media
Delicate organisms like gonococci which may not survive the time taken which
may not survive the time taken for transporting the specimen to the laboratory or may
be overgrown by non-pathogens (dysentery or cholera organisms) require a special
medium called transport medium. eg. Stuart Medium for gonococci.
viii. Anaerobic Media
These media are used to grow anaerobic organisms. eg. Robertson’s Cooked Meat
Media

Microbiology Laboratory Manual 48

LABORATORY 6

Media
Preparation
 MEDIA PREPARATION
Growth medium or culture medium is a gel or liquid designed to support the
growth of microorganisms or cells. There are different types of media for growing
different types of organisms or cells. One commonly used type of media is
nutrient broth or agar. Some organisms, termed fastidious organisms, require
more specialized types of media.
Microbiological Media Preparation Overview Growth medium or culture medium is
a gel or liquid designed to support the growth of microorganisms or cells. There
are different types of media for growing different types of organisms or cells. One
commonly used type of media is nutrient broth or agar.

18 MEDIA PREPARATION

1. PEPTONE BROTH
Peptone : 10g
NaCl : 5g
Distilled water : 1000ml

FIGURE 6.1

2. NUTRIENT AGAR
Peptone : 5g
NaCl : 5g
Beef extracts : 3g
Agar : 20g
Distilled water : 1000ml
The ingredients are dissolved in
warm water and pH adjusted to
7.2-7.6. Autoclaved at 121°C, 15 lbs
for 15 minutes.

FIGURE 6.2

Microbiology Laboratory Manual 50

3. NUTRIENT BROTH
Peptone : 5g
NaCl : 5g
Beef extracts : 3g
Distilled water : 1000ml
The ingredients are dissolved
in warm water and pH
adjusted to 7.2-7.6.
Autoclaved at 121°C, 15 lbs
for 15 minutes. FIGURE 6.3

4. MAC CONKEY AGAR

Peptone : 20g
NaCl : 5g
Bile salt : 1.5g
Lactose : 10g
Neutral red solution : 10ml
Crystal violet : 0.001g
Agar : 13.5g
Distilled water : 1000ml
FIGURE 6.4

5. SABOURAUD'S DEXTROSE
AGAR (SDA)
Peptone : 10g
Dextrose : 40g
Chloramphenicol : 0.05g
Agar : 15g
Distilled water : 1000ml

FIGURE 6.5

Microbiology Laboratory Manual 51

6. SABOURAUD'S DEXTROSE
BROTH
Peptone : 10g
Dextrose : 40g
Chloramphenicol : 0.05g
Distilled water : 1000ml

FIGURE 6.6

7. MUELLER-HINTON AGAR
Beef infusion form : 300g
Acid hydrolysate of casein : 17.5g
Agar : 17g
Starch : 1.5g

FIGURE 6.7

8. LACTOSE BROTH
Peptone : 5g
Beef extract : 3g
Lactose : 5g
Distilled water : 1000ml

FIGURE 6.8

Microbiology Laboratory Manual 52

9. EMB (EOSIN METHYLENE
BLUE) AGAR
Peptone : 10g
Lactose : 5g
Sucrose : 5g
Dipotassium hydrogen
phosphate : 2g
Eosin Y : 0.40g
Methylene blue : 0.065g
Agar : 13.50g
Distilled water : 1000ml
FIGURE 6.9

10. METHYLENE BLUE

SOLUTION (1:25,000)
Methylene blue dye : 1mg
Distilled water : 25ml
Dissolved the methylene blue in
distilled water and was dispensed
into regular staining bottles.
FIGURE 6.10

11. CARBOHYDRATE
FERMENTATION
Peptone : 1g
Carbohydrates : 10g
NaCl : 5g
Phenol red indicator : 10ml (0.1g in
10ml ethanol)
Distilled water : 1000ml
Mix all the ingredients, except phenol red
indicator. Adjust pH to 7. Then add phenol red
indicator. Dispense the medium in 8ml test FIGURE 6.11
tubes containing the Durham’s tubes. Sterilize
the medium at 10lbs for 20 minutes.

Microbiology Laboratory Manual 53

12. OXIDATION-
FERMENTATION
Peptone : 20g
Dipotassium hydrogen phosphate :
2g
NaCl : 5g
Bromothymol blue : 3ml (1%
aqueous solution)
Agar : 13.50g
Distilled water : 1000ml
Mix all the ingredients, expect
Bromothymol blue indicator. Adjust pH
to 7.1. Then add Bromothymol blue
indicator. The medium is poured into FIGURE 6.12
the tube to a depth of about 4cm.
sterilized by autoclaving at 121°C for
20 minutes at 10 lbs. it was then
allowed to set.

13. VOGES -PROSKAUER

Reagents: Barrett’s A
α – naphthol : 5g
Ethanol : 100ml
Dissolve α – naphthol in small amount
of alcohol and then add remaining
alcohol to 100ml. Store in brown bottle
at 4°C.
Barrett’s B
Potassium hydroxide : 40g
Distilled water : 100ml
Cool the volumetric flask in cold water
with 80ml water, add KOH crystals,
FIGURE 6.13
dissolve and make up to100ml. Store
in polyethene bottles at 4°C.

Microbiology Laboratory Manual 54

14. CITRATE UTILIZATION
MgSo4 : 0.2g
Ammonium dihydrogen
phosphate : 1g
Dipotassium phosphate : 1g
Sodium citrate : 2g
Sodium chloride : 0.5g
Bromothymol blue : 0.08g
Agar : 15g
Distilled water : 1000ml

Dissolve the ingredients in 1000ml

distilled water. Dispense in tubes
and sterilize by autoclaving at
FIGURE 6.14
121°C for 20 minutes at 10 lbs.

15.NITRATE BROTH

Beef extract : 3g
Peptone : 5g
Potassium Nitrate : 1g
Distilled water : 1000ml
Dissolve all the ingredients and
sterilize by autoclaving at 121°C for
20 minutes at 15 lbs.
Reagents: Sulphanlic acid
Dissolve 8g of sulphanlic acid in 1 l
of acetic acid.
α- Naphthol amines
Dissolve 5g of α-Naphthol amines in
1 l of acetic acid. Immediately before
use, mix equal volumes of Sulphanlic
acid and α- Naphthol amines to give
the test reagent. FIGURE 6.15

Microbiology Laboratory Manual 55

16. UREASE TEST
Peptone : 1g
Phenol red : 0.012g
Dextrose : 1g
NaCl : 5g
Disodium phosphate : 1.2g
Mono potassium phosphate : 0.8g
Agar : 15g
Distilled water : 1000ml
Dissolve ingredients in 950ml
distilled water. Sterilize by
autoclaving at 10lbs for 20 minutes.
Cool to 58°C and aseptically add
50ml of 40% urea. Sterilize the urea
solution by autoclaving at 10lbs for
15 minutes, mix well and add the
Phenol red indicator. Dispense into
sterilized test tubes and allow to set FIGURE 6.16
in a slanting position.

17. MANNITOL MOTILITY

TEST
Peptone : 20g
NaCl : 5g
Potassium Nitrate : 2g
Mannitol : 64g
Agar : 6g
Distilled water : 1000ml
Phenol red : 4ml (1g in 100ml
ethanol)
Mix all the ingredients, expect
phenol red indicator. Adjust pH to 7.
Then add phenol red indicator.
Dispense in tubes. Sterilize the FIGURE 6.17
medium at 10lbs for 20 minutes.

Microbiology Laboratory Manual 56

 18. TRIPLE SUGAR IRON
AGAR TEST

Peptone : 20g
Yeast extract : 3g
Beef extract : 3g
Lactose : 10g
Sucrose : 10g
Glucose : 10g
Sodium chloride : 5g
Ferrous sulphates : 0.2g
Sodium thiosulphate : 0.3g
Agar : 12g
Distilled water : 1000ml Phenol red :
0.024g Mix all the ingredients,
expect phenol red indicator. Adjust
pH to 7. Then add phenol red
indicator. Sterilize by autoclaving at
121°C for 20 minutes. Allow the
medium to set in slopped form with
a butt about 1 inch long. The
medium is used in the form a butt FIGURE 6.18
and slant.

1. Sterilization
Materials:

2.8g of Agar
1000ml of distilled water
Weighing box
Aluminum foil

Microbiology Laboratory Manual 57

 PREPARATION | MEDIA IN PLATES
1. CALCULATE THE TOTAL AMOUNT OF MEDIUM NEEDED.

2. WEIGHT THE TOTAL AMOUNT OF POWDERED MEDIUM CALCULATED.

3. TRANSFER POWDERED MEDIA INTO THE WEIGHING CONTAINER.

4. TRANSFER POWDER INTO A FLASK OR BEAKER.

5. ADD REQUIRED AMOUNT OF DISTILLED WATER.

6. TRANSFER INTO A FLASK, THEN SEAL WITH A COTTON PLUG AND SOME FOIL.
PLACE IN AN AUTOCLAVABLE PLASTIC BAG AND STERILIZE USING AN AUTOCLAVE.

7. ALLOW THE MEDIUM TO COOL INSIDE A BIOSAFETY CABINET.

8. ASEPTICALLY POUR MEDIUM INTO THE PLATES.

9. INVERT THE PLATES AND STORE IN THE INCUBATOR FOR 24 HOURS BEFORE
USING.

PREPARATION | BROTH AND AGAR SLANTS

1. CALCULATE THE TOTAL AMOUNT OF MEDIUM NEEDED.

2. PREPARE A WEIGHING BOX OR A PIECE OF FOIL LARGE ENOUGH TO HOLD THE

POWDERED MEDIUM NEEDED.

3. TRANSFER POWDERED MEDIA INTO THE WEIGHING CONTAINER UNTIL THE

DESIRED AMOUNT IS OBTAINED.

4. TRANSFER THE POWDER INTO A FLASK OR BEAKER, OR IMMEDIATELY COVER THE

WEIGHING CONTAINER AND THEN LABEL FOR LATER USE.

5. ADD THE REQUIRED VOLUME OF DISTILLED WATER.

6. MELT THE AGAR IN A MICROWAVE OVEN UNTIL THE LIQUID IS CLEAR AND
HOMOGENOUS.
7. TO MAKE SLANTS, DISPENSE ABOUT 8ML OF MELTED AGAR INTO REGULAR TEST
TUBES. FOR MAKING BROTH, DISPENSE ABOUT 8ML OF BROTH INTO SCREWCAP
TUBES.

Microbiology Laboratory Manual 58

8. PLACE THE TUBES IN A BEAKER OR TEST TUBE RACK AND COVER WITH
ALUMINUM FOIL. PLACE THE BEAKER WITH THE TUBES IN AN AUTOCLAVABLE
PLASTIC BAG.

9. STERILIZE IN AN AUTOCLAVE IMMEDIATELY.

10. TO MAKE SLANTS, LAY THE TUBES CONTAINING THE AGAR ON A SLANTING
BOARD, ALLOW TO SOLIDIFY AND COOL. LET THE BROTH TUBES COOL
COMPLETELY.

11. : LABEL AND REFRIGERATE BOTH SLANTS AMD BROTH FOR FUTURE USE.

Microbiology Laboratory Manual 59

LABORATORY 7

Plating
Techniques and
Methods of
Isolation Pure
Culture
 PLATING STREAK PLATE METHOD
SPREAD PLATE METHOD
TECHNIQUES POUR PLATE METHOD

DEFINITION

The common plating techniques employed in microbiology are Streak

Plate Method, Spread Plate Method and Pour Plate Method

FIGURE 7.1
STREAK PLATE METHOD STEP BY STEP PROCEDURE

Microbiology Laboratory Manual 61

 A. STREAK PLATE METHOD
This method was developed by two bacteriologists, Leoffler and Gaffkey in the laboratory of
Robert Koch. This method is routinely employed for the isolation of bacteria in pure culture.
In this method a sterilized inoculating loop or transfer needle is dipped into a suitable diluted
suspension of microorganisms which is then streaked on the surface of an already solidified
agar plate to make a series of parallel, non-overlapping streaks. The process is known as
streaking and the plate so prepared is called a streak plate. The main objective of the streak
plate method is to produce well separated colonies of bacteria from concentrated
suspensions of cells.
A sterilized inoculating needle with a loop made up of either platinum or nichrome wire is
used for streaking. One loopful of specimen is transferred onto the surface of the agar plate
in a sterile petridish and streaked across the surface in the form of a zig-zag line. This
process is repeated thrice to streak out the bacteria on the agar plate so that some
individual bacteria are separated from each other. The first streak will contain more
organisms than the second and the second more than the third and so on. The last streaks
should thin so on. The last streaks should thin out the culture sufficiently to give isolate
colonies. The successful isolation depends on spatial separation of single cells. Each colony
usually represents the growth from a single organism when such a plate is incubated
colonies will appear on the surface of the medium. Because of the high concentration of
water in agar, some water of condensation forms in petriplate during incubation. Moisture is
likely to drip from the cover to the surface of the agar and spread out, resulting in a
confluent mass of growth and running individual colony formation. To avoid this, petriplates
are routinely incubated bottom side up. Pure colonies can be obtained from well isolated
colonies by transferring a small portion of each to separate culture media.

Microbiology Laboratory Manual 62

 B. SPREAD PLATE METHOD
The spread plate technique is used for the separation of a dilute, mixed
population of the microorganisms so that individual colonies can be isolated. In this
technique, a small volume of dilute microbial mixture is transferred to the center of
an agar plate and spread evenly over the surface with a sterile L-shaped bent glass
rod, while the petridish is spun, at some stage, single cells will be deposited with
the bent glass rod on the agar surface. Incubate the agar plate at 37ºC for 24 hours,
in the inverted position. The dispersed cells will develop into isolated colonies.
Because the number of colonies will be equal to the number of viable organisms in
the sample spread plates can be used to count the microbial population.

FIGURE 7.2
SPREAD PLATE METHOD STEP BY STEP PROCEDURE

Microbiology Laboratory Manual 63

 C. POUR PLATE METHOD
In pour plate method, successive dilutions of the inoculum (serially diluting
the original specimen) are added into sterile petriplate to which is poured melted
and cooled (42ºC - 45ºC) agar medium and thoroughly mixed by rotating the plates
which is then allowed to solidify. After incubation, the plates are examined for the
presence of individual colonies. The pure colonies may be isolated and transferred
into test tube culture media for making pure cultures. This technique is employed to
estimate the viable bacterial count in a suspension.

FIGURE 7.3
POUR PLATE METHOD STEP BY STEP PROCEDURE

Microbiology Laboratory Manual 64

 METHODS OF ISOLATION
OF PURE CULTURE
A culture that contains only one kind of microorganisms is called a pure
culture. A culture which contains more than one kind of microorganisms is called mixed
culture. Most of the cultures obtained in nature are mixed cultures. Pure cultures are
essential to study the cultural, morphological and physiological characters of an
individual species. There are different methods for obtaining pure cultures from mixed
cultures.
Streak Plate Method
Spread Plate Method
Pour Plate Method

Micromanipulator Method

 In this technique, a microscope is  Microorganisms can also be violated by

used to pick out a single bacterial cell controlling physical environment especially
with the help of a device known as temperature. Bacteria with different optimum
micromanipulator. A single viable cell growth temperature can be separated by
may be transferred on the culture incubating at different temperature. Only
medium to develop turbidity. thermophiles bacteria grow to 60ºC. A
 Enrichment, selective and indicator mixture containing vegetative and spore
media are widely used for the forming bacteria can be separated by heating
isolation of pathogens from at 80ºC. In this method, the bacteria in the
specimens such as faeces with varied vegetative state will be eliminated. This
flora. method is useful for the isolation of tetanus
bacilli from dust and similar sources.
 Pure culture may be obtained by
pre-treatment of specimens with  Separation between motile and non-motile
appropriate bactericidal substances bacteria can be effected using Craigie’s tube.
which destroy the unwanted bacteria. This consists of a tube of semisolid agar with
This method is the standard practice a narrow tube open at both ends placed in the
center of the medium in such a way that it
for the isolation of tubercle bacilli from
sputum and other clinical specimen. projects above the level of the medium. The
mixture is inoculated into the central tube, the
 Obligate aerobes and anaerobes motile bacteria alone transverse the agar and
may be separated by cultivation appear at the top of the medium outside the
under aerobic or anaerobic conditions. central tube.

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LABORATORY 8

CULTURE
PRESERVATION
TECHNIQUE

 CULTURE PRESERVATION
TECHNIQUES
Microbiologist or laboratories concerned with microbial studies preserve cultures
for a short period or many years conserving all the characteristics of the
organisms. These preserved cultures may be made available in future for various
purposes such as:
Use in the laboratory classes
Research work
Use as test agents for particular procedure
Some methods used for culture preservation include refrigeration, deep freezing,
freezing under liquid nitrogen and lyophilization.

1. Refrigeration
Live cultures on a culture medium can be successfully stored in refrigerators or
cold rooms maintained at 4ºC. Generally, the metabolic activities of the
microorganisms will be greatly slowed down at this temperature. Storing cultures
in a refrigerator at a temperature of 4ºC, slows growing protects from damage
due to evaporation of medium and preserve the culture. Thus growth will occur
slowly, nutrients will be utilized and waste products produced, which will
eventually kill the microorganisms. So subculturing of refrigerated cultures is to
be carried out at regular intervals. In the case of bacteria, subculturing should be
done at intervals of 2-3 weeks. In the case of fungi, regular subculturing is
necessary at intervals of 3-4 months.

FIGURE 8.1 REFRIGERATOR

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2. Deep Freezing
Cultures can be preserved for several years in glycerol at 40ºC in a deep freezer.
In this method approximately 2 ml of the glycerol solution is added onto the agar
slope culture by shaking. The culture suspension is transferred into each ampoule
which is placed in a mixture of industrial methylated spirit and CO2 and is
freezed rapidly to -70ºC. Ampoules are removed from the mixture and placed
directly into a deep freezer at 40ºC. During transfer from these stock cultures,
tubes are placed to water bath at 45ºC for a few seconds or until the suspensions
melt and are aseptically streaked onto agar plates.

FIGURE 8.2 DEEP FREEZER

3. Freezing Under Liquid Nitrogen

Freezing in liquid nitrogen at temperature of -196ºC
also suspends metabolism of cells and these survive
unchanged for long periods. In this method, cell
suspension in the presence of a stabilizing agent
such as glycerol or dimethyl sulfoxide, that prevents
the formation of ice crystals which may kill frozen
cells, is sealed into small ampoules and stored in
liquid nitrogen refrigerator. Most species of bacteria
can remain viable for 10-30 years or even more
without undergoing change in their characteristics.
The liquid nitrogen method has been successful with
many species that cannot be preserved by
lyophilization.

FIGURE 8.3 FREEZING USING LIQUID NITROGEN

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4. Lyophilization
Lyophilization or freeze drying is the rapid dehydration of organisms while they
are in a frozen state. Most of the microbes are protected from the damage
caused with water loss by this method. Because metabolism requires water, the
organisms are in a dormant state and can retain viability for over 30 years
unchanged in their characteristics. In this technique, the culture is rapidly frozen
at -70ºC and then dehydrated by vacuum and the tubes containing freeze dried
cultures are sealed and stored in the dark at 4ºC in refrigerators. It is the most
satisfactory method of long term preservation of microorganisms. It’s universally
used for the preservation of bacteria, viruses, fungi etc. Lyophilized cultures are
revived by opening the vials adding liquid medium and transferring the culture to
a suitable growth medium.

FIGURE 8.4 LYOPHILIZER

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 LABORATORY 9

BACTERIAL
IDENTIFICATION
and
BIOCHEMICAL TESTS
FOR
IDENTIFICATION OF
BACTERIA
 BACTERIAL
SIMPLE STAINING
DIFFERENTIAL STAINING

IDENTIFICATION
GRAM STAINING
Hanging-Drop and Wet-Mount
Preparations

SIMPLE STAINING

AIM
To compare the morphological shapes and arrangements of bacterial
cells.

Principle
In simple staining, bacterial smear is stained with a single reagent,
which produces a distinctive contrast between the organism and its
background. A simple stain that stains the bacteria is the direct stain. The
purpose of simple staining technique is to determine cell shape, size
arrangement of bacterial cells. Simple staining is performed by using
basic stains which have different exposure time (Crystal Violet 20-60 s,
Carbol fuschin 15-30 s and Methylene blue 1-2 miutes).

Procedure
1. Clean glass slide was taken and was washed and dried.
2. Bacterial smears were prepared from the bacterial cultures.
3. The slide was kept on the staining tray and 5 drops of stain was added
for a designed period.
4. The extra stain was poured off and the smear was washed gently
under slow running tap water.
5. The slide was then blot dried using blotting paper.
6. The slide was then examined under 10X, 45X and oil immersions
objects respectively.

Observation
On the basis of microscopic observation, bacteria appeared blue,
violet and red respectively depending on the stain taken.

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DIFFERENTIAL STAINING

Differential staining requires the use of at least 3 chemical reagents

that are applied sequentially to a heat fixed smear. Its function is to impart
its colour to all cells. In order to establish a colour contrast, the second
reagent used is the decolorizing agent. Based on the chemical
composition of cellular components the decolorizing agent may or may
not remove the primary stain from the entire cell or from any cell
structure. The final reagent is the counter stain. Following discoloration, if
the primary stain is not washed out, the counter stain cannot be absorbed
and neither the cell nor its components will retain the colour of the
primary stain. If the primary stain is removed, the decolorized cellular
components will accept and assume the contrasting colour of the counter
stain. In this way, cell type or their structure can be distinguished from
each other. On the basis of the stain that is retained the most important
differential stain used in bacteriology is the Gram stain.

DIFFERENTIAL STAINING
AIM
To differentiate two principal groups of bacteria.
Principle
The Gram stain, a differential stain was developed by Hans Christian
Gram, a Danish physician, in 1884. Gram staining classifies bacteria into 2
major groups, Gram positive and Gram negative bacteria. The Gram stain
reaction is based on the difference in the chemical and physical
composition of bacterial cell wall. Gram positive cells have a thick
peptidoglycan layer, whereas peptidoglycan layer in Gram negative cells
is much thinner and surrounded by outer lipid containing layer.
In Gram negative, the higher amount of lipid in the formation of
large pores thus facilitating the leakage of crystal violet-iodine complex
and resulting in the decolonization of the bacterium which later takes this
complex counter stain. In contrast, the Gram positive cell wall are thick
and composed mainly of proteins and cross linked mucopeptide, when
treated with alcohol it causes dehydration and closure of the cell wall
pores thereby not allowing the loss of complex and cell retains primary
stain. The bacteria which retain the primary stain appear dark blue or vio-

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let and not decolorized when stained with Gram’s method are called
Gram positive, where as those that lose the crystal violet used counter
stain, saffranin appear red are called as Gram negative.
The Gram stain uses different reagents in the order, crystal violet,
iodine solution, alcohol and saffranin.
Procedure
1. Thin smear was prepared of the given bacterial species on a clean
glass slide. 
2. Let the smear dry.
3. Heat fixed smear.
4. Hold the smear using the slide rack.
5. Covered each smear with crystal violet for 1 minute.
6. Washed each slide with distilled water for few seconds using wash
bottles.
7. Covered each smear with Gram’s iodine solution for 1 minute.
8. Gently washed with distilled water.
9. Decolorized with 95%.
10. Washed the slide with distilled water and drained.
11. Counter stain was applied saffranin for 30 seconds.
12. Washed with distilled water and blot dried with absorbent paper.
13. The stained slides were air dried and observed under the microscope.
Observation
1. Examined the slides microscopically using oil immersion objective.
2. Identified the Gram reaction of the given cultures and classified it and
described the morphology and arrangement of cells.
Those bacteria that appear blue are referred to as Gram positive and these appearing
pink are described as Gram negative.

FIGURE 9.1 GRAM STAINING

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HANGING-DROP AND WET-MOUNT PREPARATIONS

Procedure
1. Take a cover glass and clean it thoroughly, making certain it is free of
grease (the drop to be placed on it will not hang from a greasy
surface). It may be dipped in alcohol and polished dry with tissue, or
washed in soap and water, rinsed completely and wiped dry.
2. Take one hollow-ground slide and clean the well with a piece of dry
tissue. Place a thin film of petroleum jelly around (not in) the concave
well on the slide.
3. Gently shake the broth culture of Proteus until it is evenly suspended.
Using good aseptic technique, sterilize the wire loop, remove the cap
of the tube, and take up a loopful of culture. Be certain the loop has
cooled to room temperature before inserting it into the broth or it may
cause the broth to “sputter” and create a dangerous aerosol. Close and
return the tube to the rack.
4. Place the loopful of culture in the center of the cover glass (do not
spread it around). Sterilize the loop and put it down.
5. Hold the hollow-ground slide inverted with the well down over the
cover glass, and then press it down gently so that the petroleum jelly
adheres to the cover glass. Now turn the slide over. You should have a
sealed wet mount, with the drop of culture hanging in the well.
6. Place the slide on the microscope stage, cover glass up. Start your
examination with the low-power objective to find the focus. It is
helpful to focus first on one edge of the drop, which will appear as a
dark line. The light should be reduced with the iris diaphragm and, if
necessary, by lowering the condenser. You should be able to focus
easily on the yeast cells in the suspension. If you have trouble with the
focus, ask the instructor for help.
7. Continue your examination with the high-dry and oil-immersion
objectives (be very careful not to break the cover glass with the latter).
Although the yeast cells will be obvious because of their larger size,
look around them to observe the bacterial cells.
8. Make a hanging-drop preparation of the Staphylococcus culture
following the same procedures just described.
9. Record your observations of the size, shape, cell groups, and motility
of the two bacterial organisms in comparison to the yeast cells.
10. Discard your slides in a container with disinfectant solution.

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HANGING-DROP AND WET-MOUNT PREPARATIONS

FIGURE 9.2 Hanging Drop Method

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SOME SPECIAL FEATURES OF COMMON
MICROORGANISMS IN LABORATORY

1. Escherichia coli

Section : Facultative, Gram negative rods

Family : Enterobacteriaceace
Genus : Escherichia
Species : coli
Common coliform bacterium used in laboratory practices
The bacterium is rod shaped generally 1.3 x 2.5 µm in size
Gram negative, facultatively anaerobe, motile having peritrichous
flagella
It causes diarrhea due to the presence of enterotoxins
It is catalase positive and oxidase negative

Colony Characteristics
a) On Nutrient Agar
Small, regular, circular, translucent colonies
b) On Mac conkey Agar
Small, regular, circular, lactose fermenting colonies

Biochemical Tests

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2. Klebsiella

Section : Facultative, Gram negative rods

Family : Enterobacteriaceace
Genus : Klebsiella
No specific growth requirements and grow well on standard
laboratory media 
Grows best between 35 and 37ºC and at pH 7.2 
The bacterium is rod shaped, non-motile 
Gram negative, facultatively anaerobe 
It is catalase positive and oxidase negative

Colony Characteristics
a) On Nutrient Agar
Large, regular, convex, opaque, mucoid colonies
b) On Mac conkey Agar
Large, regular, convex, opaque, mucoid, lactose fermenting
colonies

Biochemical Tests

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3. Pseudomonas

Section : Facultative, Gram negative rods

Family : Enterobacteriaceace
Genus : Pseudomonas
The cells are straight or slightly curved of 1.5 – 5.0 x 0.5-1.0 µm in size
Aerobic and motile 
Some species are pathogenic to human, animals and plants 
They are catalase and oxidase positive

Colony Characteristics
a) On Nutrient Agar
Medium, regular, flat, translucent colonies with greenish
pigmentation
b) On Mac conkey Agar
Small, irregular, flat, translucent, non-lactose fermenting
colonies

Biochemical Tests

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3. Pseudomonas

Section : Facultative, gram positive cocci

Family : Staphylococcaceae
Genus : Staphylococcus
Species: aureus
They appear round (cocci) and form in grape-like clusters. 
Non-Motile 
It is a common cause of skin infections, respiratory disease and food
poisoning. 
It is catalase and coagulase positive

Colony Characteristics
a) On Nutrient Agar
Small, regular, circular, entire, smooth, convex, opaque,
golden yellow colonies
b) On Mac conkey Agar
Small, regular, circular, entire, smooth, convex, opaque,
lactose fermenting colonies

Biochemical Tests

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 BIOCHEMICAL TESTS FOR
IDENTIFICATION OF BACTERIA
1) CARBOHYDRATE FERMENTATION
AIM
To determine the ability of microorganisms to degrade and ferment
carbohydrate with the production of acid and gas.

Principle
Most microorganisms use carbohydrate differently depending on
their enzymes components. In fermentation, substrate and alcohols
undergo anaerobic dissimilation and produce an organic acid (For
example lactic acid, formic acid or acetic acid). The pH indicator Phenol
Red is used to detect the production of acid, which is red at a neutral pH 7
and changes to yellow at a slightly acidic pH of 6.8. This indicates a
positive reaction.

In some cases, acid production is accompanied by the evaluation of

gas such as Hydrogen or Carbon dioxide. To detect the presence of gas
produced or Durham’s tube (an inverted inner vial) is placed in the
fermentation broth, in which the evaluation of gas will be visible as a
bubble.
Cultures that are not capable of fermenting any carbohydrate and not producing
concomitant evolution of gas are noted. This is a negative reaction.

MATERIALS REQUIRED
8 ml Test Tube, Durham’s Tube, Phenol Red Indicator, Sugar (Glucose,
Lactose, Sucrose)

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 Procedure
1. Using sterile technique, culture was inoculated into its appropriately
labeled medium by means of loop inoculation. 
2. Care was taken during this step not to shake the fermentation tube. 
3. 1 tube of each fermentation broth was kept uninoculated as a
comparative control. 
4. All the tubes were incubated at 37°C for 24 hours and the reaction was
observed.

Observation
All carbohydrate broth cultures were observed for colour and
presence or absence of gas bubble by comparing with the uninoculated
tube (control).

Note
A- Only acid is formed; the
broth has turned yellow
AG- Acid & Gas formed, the
broth turned Yellow and gas
bubble is trapped
-ve- No change
FIGURE 9.3 Carbohydrate Fermentation Test

Sugar fermentation test

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2) OXIDATION – FERMENTATION TEST
AIM
To determine the oxidation fermentation characteristics of
microorganisms.

Principle
This method depends upon the use of semisolid tube medium
containing the carbohydrate (Glucose) together with a pH indicator. The
acid is produced only at the surface of medium where conditions are
aerobic the attack on the medium where conditions are aerobic the
attack on the sugar is oxidative. If acid is produced throughout the
medium including lower layers and where the conditions are aerobic
breakdown is fermentative.
Fermenting organism (Enterobacteriaceace, Vibrio) produce an
acidic reaction throughout the medium in the covered (anaerobic) as well
as open (aerobic) tube. Oxidizing organisms (Pseudomonas) produce an
acidic reaction only in the open tube. Organisms that cannot breakdown
carbohydrate aerobically/anaerobically (alkali genes faecalis) produce an
alkaline reaction in the open tube and no change in the covered tube. This
medium may be used for detecting gas production and motility.

MATERIALS REQUIRED
Bacterial broth culture, D-F medium, liquid paraffinaol

Procedure
1. Using sterile technique, two
tubes of medium were
inoculated by stabbing with
sterile urine.
2. Two inoculated tubes were used
as control.
3. Liquid paraffin was poured over
the medium to form a layer
about 1cm in depth into one of
the tube of each pair.
4. The tubes were incubated at
FIGURE 9.4 Oxidation Fermentation Test 37°C for 24-48hrs was observed.

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 Observation
The tubes were observed for the colour of the medium and the type
of metabolism was recorded.

3) INDOLE PRODUCTION TEST

AIM
To determine the ability of microorganisms to decompose the amino
acid tryptophan to indole.

Principle
Tryptophan an essential amino acid oxidized by some bacteria by the
enzyme tryptophanase resulting in the formation of indole, pyruvic acid
and ammonia. In this experiment, the medium contains the substrate
tryptophan which is utilized by the microorganisms.

This ability to hydrolyse tryptophan with the production of indole is

not a characteristic of all microorganisms and therefore serves as a
biochemical mask. The presence of indole is detected by adding Kovac’s
reagent, which produces a cherry red reagent layers. This colour is
produced by the reagent which is composed of Paradimethyl
aminobenzaldehyde yielding the cherry red colour.

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 Indole Reaction with Kovac’s Reagent
Culture producing a red reagent layers following addition of the
Kovac’s reagent are indole positive. The absence of red colouration
demonstrates that the substrate tryptophan was not hydrolyzed and
indicating indole negative reaction.
Another reagent used is Ehrlisch’s reagent. It’s believed to be more
sensitive than Kovac’s reagent and is recommend for the detection of
indole production by anaerobic and non-fermentative Gram negative
organism Kovac’s reagent was used usually initially to classify the
members of Enterobacteriaceace family.

MATERIALS REQUIRED
15 ml test tubes, bacterial culture, peptone water, Kovac’s reagent
Procedure
1. The peptone water tubes were
inoculated with bacterial broth
culture using sterile needle
technique.
2. An uninoculated tube was kept
as control.
3. Both tubes were incubated at
37°C for 24-48 hours.

FIGURE 9.5 Indole Production Test

4. After proper incubation, 1 ml of Kovac’s reagent was added to both

tubes including the control.
5. The tubes were shaken gently after an interval for 10 – 15 minutes.

Observation
The tubes were observed for the colour in the top reagent layer.
note:
Development of cherry red colour in the top layer of the tube is a
positive test. Absence of red colouration is indole negative.
Examples
Positive: E. coli, Proteus vulgaris
Negative: Klebsiella sp., Proteus mirabilis

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4) METHYL RED TEST
AIM
To determine the ability of microorganism to oxidize glucose with the
production and stabilization of high concentrations of acid end products

Principle
All enteric organisms oxidize glucose for energy production and the
end products of this process will vary depending on the specific
enzymatic pathway present in the bacteria. In this test, the pH indicator
methyl red detects the presence of large concentrations of acidic red
detects the presence of large concentrations of acidic products. The test
can be used in differentiating Escherichia coli and Enterobacter
aerogenes (both coliform bacteria) that are used as indicator of the
sanitary quality of water, foods etc.

Both of these organisms initially produce organic acid end products

during the early incubation period. The low acid end products produce
acidic pH (4) which is stabilized and maintained by E. coli at the end of
incubation. During the later incubation period Enterobacter aerogenes
enzymatically converts these acids into nonacid end products such as 2,3
butanedial and acetyl methyl carbinol (pH 6).

At a pH of 4, Methyl red indicator will turn red throughout the tube,

which is indicating of a positive test. At pH 6, still indicating the presence
of acid but with a lower hydrogen ion concentration, the indicators turn
Yellow, which is indicating the negative test.

MATERIALS REQUIRED
MR broth, 24 hours broth cultures, Methyl red indicator, inoculating loop

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 Procedure
1. Using sterile technique experimental organisms were inoculated into
appropriately labeled tubes containing MR broth by means of loop
inoculation.
2. Uninoculated tube was kept as control.
3. Both tubes were incubated at 37°C for 24-48 hours.
4. After proper incubation 5 drops of MR indicator was added to both
tubes including control.
5. It was mixed well and colour was observed.

Observation
The tubes were observed for changes in the colour of Methyl Red.

interpretation
The colour of MR reagents
remaining red is a positive test
and the colour turning to
yellow is negative.
Examples
MR positive – E. coli, Proteus sp;
Salmonella sp.
MR negative – Klebsiella,
FIGURE 9.6 Methyl Red Test Enterobacter sp.

5) VOGES – PROSKAUER TEST

AIM
To determine the ability of many microorganisms to produce
acetone (acetyl methyl carbinol) during fermentation of glucose.

Principle
This determines the ability of many bacteria to ferment
carbohydrates with the production of non- acidic / neutral end products,
acetyl methyl carbinol or its reduction product, acetyl methyl carbinol or
its reduction products, acetyl- methyl carbinol or its reduction product 2,3
Butylene glycol from the organic acids.

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 The reagent used in this test, Barrett’s reagent, consists of a mixture of
alcoholic α- naphthol and 40% potassium hydroxide solution. Detection of
the acetyl methyl carbinol requires this end product to be oxidized to a
diacetyl compound. This reaction will occur in the presence of α- naphthol
catalyst and a guanidine group that is present in the peptone. At a result,
a pink complex a guanidine group that is present in the peptone. As a
result, a pink complex is complex is formed imparting a rose colour to the
medium. Acetyl Methyl Carbinol reaction with Barrett’s reagent .

Development of deep rose colour in culture with in a minute following

the addition of Barrett’s reagent is indicative of presence of the acetyl
methyl carbinol and represents a positive result. The absence of rose
colouration is a negative result.

Procedure
1. Using sterile technique, the experimental
organism was inoculated into VP broth
by means of loop inoculation.
2. One tube is kept uninoculated as control.
3. The tube will be incubated at 37°C for 24-
48 hours.
4. After proper incubation, about 3 ml of
Barrett’s reagent A & 1 ml of Barrett’s
reagent B was added into both tubes
including control. FIGURE 9.7 Voges Proskauer Test

5. The tubes were shaken gently for 30 seconds with the caps off to
expose the media to oxygen.
6. The reaction was allowed to complete in 15 – 30 minutes and tubes were
observed.
Observation
The tubes were observed for the development of crimson red colour.
Note: the colour may be more intense at the surface.
Interpretation: Red colour formation indicates a positive test and colour change
is negative.
eg. Positive – Klebsiella sp., Enterobacter Negative – E. coli, Proteus sp.

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6) CITRATE UTILIZATION TEST
AIM
To determine the ability of a microorganism to utilize citrate as the
sole source of carbon and as energy source for the growth and
ammonium salt as a sole source of nitrogen.
Principle
Citrate test is used to differentiate among enteric bacteria on the
basis of their ability to utilize / ferment citrate as the sole carbon source. In
the absence of glucose or lactose some microorganisms utilize citrate as a
carbon source. This ability depends on the presence of citrase enzyme
that facilitates the transport of citrate in the cell. Citrate, the first major
intermediate in Krebs’s cycle is produced by the condensation of active
acetyl CoA with oxalo acetic acid and acetate. These products are then
enzymatically converted to pyruvic acid and carbon dioxide. During this
reaction the medium becomes alkaline; CO2 combines with sodium and
water to form carbonate, an alkaline product. This changes the
bromothymol blue indicator in the medium from green to Prussian blue.

Citrate test is preferred / performed by

inoculating the microorganisms in to an
organic synthetic medium. Simmons citrate
agar (solid) or Koser’s citrate medium
(liquid) in which sodium citrate is the only
source of carbon and energy.
Bromothymol blue is green when
acidic (pH 6.8 and below). When alkaline
(pH 7.6 and above). Formation of blue
colour constitutes a positive test. Citrate
FIGURE 9.8 Citrate Utilization negative culture will show no growth and
the medium will remain green.

MATERIALS REQUIRED
Bacterial broth, Simmons Citrate Agar Slants, Inoculation Loop

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 Procedure
1. Using sterile technique Simmons citrate agar slant was inoculated
with the test organism by means of a stab and streak inoculation.
2. An uninoculated tube was kept as control.
3. Both tubes were incubated at 37°C for 24 – 48 hours & was observed

Observation
The tubes were observed for growth and colouration of the medium.

Interpretation

Colour of the medium if turned blue, a positive result is indicated. Colour of

the medium remains as green, indicates a negative result.

7) NITRATE REDUCTION TEST

AIM
To determine the ability of bacteria to produce an enzyme nitrate
reductase .

Principle
The reduction of nitrate by some aerobic and facultative anaerobic
microorganisms occur in the absence of molecular oxygen an anaerobic
process whereby the cell uses in organic substances such as nitrates or
sulphates to supply oxygen that is subsequently utilized as a final
hydrogen acceptor during energy formation. The biochemical
transformation may be utilized as follows:

Some organisms possess the enzymatic capacity to act further on

nitrates to reduce them to ammonia or molecular nitrogen. These
reactions may be described as follows:

Nitrate reduction can be determined by cultivating organisms a

nitrate broth medium. The medium is basically a nutrient broth
supplemented with 0.1% potassium nitrate (KNO3) as the nitrate
substrate.

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In addition, the medium is made into a semisolid by the additional of 0.1%
agar. The semisolid impedes the diffusion of oxygen in to the medium,
there by favoring the anaerobic requirement necessary for nitrate
reduction. An organisms ability to reduce nitrate to nitrite is determined
by the addition of two reagent solution A, which is sulphanlic acid
followed by solution B, which is α-napthylamine followed reduction, the
addition of solution A and B will produce an immediate cherry red colour.

Cultures not producing a colour change suggest one of two possibilities

Nitrates were not reduced by the organism
The organism possessed such potent nitrate reductase enzymes that
nitrate were rapidly reduced beyond nitrates to ammonia or even
molecular nitrogen.
This test determines the production of an enzyme called nitrate
reductase, resulting in the reduction of nitrate (NO3). With this enzyme,
nitrate is reduced to nitrite (NO2). It then forms nitrous acid that reacts
with the first reagent sulphanlic acid, and that reacts with the other
reagent α-napthylamine to form a red colour. The development of red
colour, therefore, verifies that nitrates were not reduced to nitrites by the
organism. If nitrites were reduced a negative nitrate reduction had
occurred. If the addition of zinc does not produce colour change, the
nitrates in the medium were reduced beyond nitrites to ammonia or
nitrogen gas. This is a positive reaction or result. Reduction of nitrate is
generally an anaerobic respiration in which an organism derives its
oxygen from nitrate.
MATERIALS REQUIRED
Bacterial broth, Nitrate broth, Nitrate reagent and inoculation loop.

Procedure
1. Using sterile technique the test organism was inoculated in to nitrate
broth by means of loop inoculation.
2. An uninoculated broth was kept as control.
3. Both tubes were incubated at 37°C for 24 – 48 hours.
4. After proper incubation equal amounts of nitrate reagent (solution A &
B) were added to nitrate broth Cultures and to the control tube and
the reaction was observed

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 observation
The tubes were observed to see a red colour has been developed or
not. A minute quantity of zinc was added to cultures in which no red
colour was developed and it was observed to see if red colour has been
developed or not.

Interpretation

Development of red colour indicates nitrate positive and no colour change

indicates a negative test.
Eg:
Positive: all members of Enterobacteriaceace
Negative: Haemophilus duceryi.

8) UREASE TEST
AIM
To determine the ability of microorganism to degrade urea by means
of the enzyme urease.

Principle
Urease is a hydrolytic enzyme that attacks nitrogen and carbon bond
in amide compounds such as urea and forms the alkaline end products
ammonia. The presence of urease is detectable when the organisms are
grown in a urea broth medium containing the pH indicator phenol red. As
the substrate urea is split into its products, the phenol red to turn to a
deep pink. This is a positive reaction for the presence of urease. Failure of
deep pink colour to develop is evidence of negative reaction.

MATERIALS REQUIRED
Bacterial broth cultures, Christener’s urea agar slant and the inoculation
loop

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 Procedure
1. Using sterile technique, the test organism was inoculated the media
by means of loop of inoculation.
2. An uninoculated tube was kept as control.
3. The tubes were incubated at 37°C for 24-24 hours and the reaction
was observed.
Observation
The tubes were observed to see if pink colour has developed or not.
Interpretation

Development of pink colour indicator a positive test and no colour change

shows a negative test,
Eg Urease Positive – Klebsiella sp., Proteus sp.
Urease Negative – E. coli, Salmonella sp.

FIGURE 9.9 Urease Test

9) MANNITOL MOTILITY TEST

AIM
To detect whether the given organism is motile and also mannitol is
fermenting or not.

Principle
Mannitol motility test medium is an example of semisolid agar
media; motile bacteria swarm and give a diffused spreading growth that
is easily recognized by the naked eye. The final sterile medium should be
quite clear and transparent. After incubating the stabbed culture, non-
motile bacteria generally give growth that are confined to stab line and
have sharply defined margins leaving the surrounding medium clearly
transparent. Motile bacteria typically give diffused, hazy growth that
spreads throughout the medium rendering it slightly opaque. This test
also helps to identify whether the microorganisms ferment Mannitol or
not. It produces acidic end products which in turn change the red colour
of phenol red indicator to yellow.

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 materials required
Bacterial culture broth, mannitol fermentation media (semisolid) and
inoculation loop.

procedure
1. Using sterile technique the test organism was inoculated in to the
medium using stab inoculation method.
2. An uninoculated tube was kept as control.
3. Both tubes were incubated at 37°C for 24-48 hours and the reaction
was observed.
observation
The tubes were observed for motility
and also for colour changes from red to
yellow.

Interpretation

Diffused growth – Motile bacteria eg:

Pseudomonas sp.
Growth at stab line only – Non-motile
FIGURE 9.10 Mannitol Motility Test bacteria eg: Staphylococcus aureus only
Red colour – Mannitol nonfermenting eg: Bacillus cereus
Yellow colour - Mannitol fermenting eg: E. coli

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10) TRIPLE SUGAR IRON AGAR TEST
AIM
To identify the microorganisms based on the ability to ferment the
carbohydrates (Glucose, Sucrose and Lactose).

Principle
The triple sugar- iron agar test is designed to differentiate among the
different groups or genera of the Enterobacteriaceace, which are all Gram
negative bacilli capable of fermenting glucose with the production of acid
and to distinguish them from other gram negative intestinal bacilli. This
differentiation is based on the differences in carbohydrate fermentation
patterns and hydrogen sulfide production by the various groups of
intestinal organisms. Carbohydrate fermentation is indicated by the
presence of gas and a visible colour change of the pH indicator, phenol
red. The production of hydrogen sulphide in the medium is indicated by
the formation of a black precipitate that will blacken the medium in the
butt of the tube.
To facilitate the observation of
carbohydrate utilization patterns, TSI
Agar contains three fermentative sugars,
lactose and sucrose in 1% concentrations
and glucose in 0.1% concentration. Due to
the production of acid during
fermentation, the pH falls. The acid base
indicator Phenol red is incorporated for
detecting carbohydrate fermentation
that is indicated by the change in colour
FIGURE 9.11 Triple Sugar Iron Agar Test of the carbohydrate medium from
orange red to yellow in the presence of acids. In case of oxidative
decarboxylation of peptone, alkaline products are produced and the pH
rises. This is indicated by the change in colour of the medium from orange
red to deep red. Sodium thiosulfate and ferrous ammonium sulfate
present in the medium detects the production of hydrogen sulfide and is
indicated by the black colour in the butt of the tube.
Carbohydrate fermentation is indicated by the production of gas
and a change in the colour of the pH indicator from red to yellow. To
facilitate the detection of organisms that only ferment glucose, the glu-

Microbiology Laboratory Manual 94

cose concentration is one-tenth the concentration of lactose or sucrose.
The amount of acid production in the slant of the tube during glucose
fermentation oxidizes rapidly, causing the medium to remain orange red
or revert to an alkaline pH. In contrast, the acid reaction (yellow) is
maintained in the butt of the tube since it is under lower oxygen tension.
After depletion of the limited glucose, organisms able to do so will
begin to utilize the lactose or sucrose. To enhance the alkaline condition
of the slant, free exchange of air must be permitted by closing the tube
cap loosely. If the tube is tightly closed, an acid reaction (caused solely by
glucose fermentation) will also involve the slant.

MATERIALS REQUIRED
Bacterial broth cultures, TSI agar slants, Inoculation Loop.

Procedure
1. Using sterile technique, the test organism was inoculated into the
media by means of stab and streak inoculation.
2. An uninoculated broth was kept as control.
3. Both tubes were incubated at 37°C for 24 hours and the reaction was
observed.
observation
The tubes were observed for the colour of both the butt and slant
and also gas production by means of cracks or bubble or blackness of
butt.

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 Interpretation

A/A: ferments glucose and either sucrose, lactose, or both. 

K/A: does not ferment lactose or sucrose; does ferment glucose. 
K/K: a non-fermenter. 
Black precipitate in stab: produces H2S (and ferments glucose).

11) CATALASE TEST

FIGURE 9.12 Catalase Test

AIM
To demonstrate the presence of catalase in an organism.
Principle
Catalase is an enzyme, which is produced by microorganisms that
live in oxygenated environments to neutralize toxic forms of oxygen
metabolites and H2O2. The catalase enzyme neutralizes the bactericidal
effects of hydrogen peroxide and protects them. Anaerobes generally lack
the catalase enzyme. Catalase mediates the breakdown of hydrogen
peroxide H2O2 into oxygen and water. To find out if a particular bacterial
isolate is able to produce catalase enzyme, small inoculums of bacterial
isolate is mixed into hydrogen peroxide solution (3%) and the rapid
elaboration of oxygen bubbles occurs. The lack of catalase is evident by a
lack of or weak bubble production. Catalase-positive bacteria include
strict aerobes as well as facultative anaerobes. They all have the ability to
respire using oxygen as a terminal electron acceptor. Catalase-negative
bacteria may be anaerobes, or they may be facultative anaerobes that
only ferment and do not respire using oxygen as a terminal electron
acceptor (ie. Streptococci).

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uses
The catalase test is primarily used to distinguish among Gram-positive
cocci: Member of the genus Staphylococcus is catalase-positive, and
members of the genera Streptococcus and Enterococcus are catalase-
negative.
Catalase test is used to differentiate aero tolerant strains of
Clostridium, which are catalase negative, from Bacillus species, which
are positive.
Semi quantitative catalase test is used for the identification of
Mycobacterium tuberculosis.
Catalase test can be used as an aid to the identification of
Enterobacteriaceace. Members of Enterobacteriaceace family are
Catalase positive.

MATERIALS REQUIRED
24 hours old bacterial culture, glass slide, petridish, 3% H2O2, applicator
sticks
Procedure
1. Transfer a small amount of bacterial colony to a surface of clean, dry
glass slide using a loop or sterile wooden stick.
2. Place a drop of 3% H2O2 on to the slide and mix. 
3. A positive result is the rapid evolution of oxygen (within 5-10 s) as
evidenced by bubbling.
4. A negative result is no bubbles or only a few scattered bubbles.
5. Dispose of your slide in the biohazard glass disposal container.

precautions
Do not use a metal loop or needle with H2O2; it will give a false positive
and degrade the metal.
If using colonies from a blood agar plate, be very careful not to scrape
up any of the blood agar as blood cells are catalase positive and any
contaminating agar could give a false positive.

observation
The release of bubbles was observed and compared with control.

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 Interpretation
Bubble Formation : Catalase Positive
No Bubble Formation: Catalase Negative
Examples
Catalase Positive : Staphylococcus aureus
Catalase Negative: Streptococcus pyogenes
Note
Care must be taken while performing catalase test of growth from blood agar
plate because blood (RBC) contains RBC catalase.

12) OXIDASE TEST

AIM
To test the production of
oxidase bacteria.

Principle
The oxidase test is a key test
to differentiate between the
families of Pseudomonadaceae
(ox +) and Enterobacteriaceace
(ox-), and is useful for speciation FIGURE 9.13 Oxidase Test

and identification of many other bacteria those that have to use oxygen as
the final electron acceptor in aerobic respiration. The enzyme cytochrome
oxidase is involved with the reduction of oxygen at the end of the electron
transport chain. There may be different types of oxidase enzymes
produced by bacteria. The colorless redox reagent, tetra methyl-
pphenylenediamine dihydrochloride (or dimethyl) used in the test will
detect the presence of the enzyme oxidase and reacting with oxygen,
turn a colour. The oxidase reagent contains a chromogenic reducing
agent, a compound that changes color when it becomes oxidized, so it
acts as an artificial electron acceptor for the enzyme oxidase. The oxidized
reagent forms the coloured compound indophenol blue.

MATERIALS REQUIRED
Oxidase disc, 24 hours old test organism, applicator stick or glass rod.

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 Procedure
1. The test organisms was rubbed over the reagent impregnated, filter
paper disc using sterile applicator sticks or glass rod.
2. Controls were also kept along with the test and the reaction was
observed within 10 seconds.
observation
The colour changes to purple were observed with the prescribed
time.

important
Acidity inhibits oxidase enzymes activity therefore the oxidase test
must not be performed on colonies that produce fermentation on
carbohydrates containing media like Mac Conkey Agar.
Interpretation
Formation of purple colour indicates a positive test. No colour changes show a
negative test.
eg. Oxidase Positive: Pseudomonas sp., Vibrio sp.
Oxidase Negative: E. coli, Klebsiella

precautions
The test reagent is to be freshly prepared.
Nichrome wire is not used to take bacterial growth.
Cultures should not be very cold.
Culture from selective media should not be used.
The colour changes should be observed within the prescribed time.

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13) COAGULASE TEST

AIM
To distinguish coagulase producing
Staphylococcus aureus from other
species of Staphylococcus.

Principle
Staphylococcus aureus is known to
produce coagulase, which can clot
plasma into gel in tube or agglutinate
cocci in slide. This test is useful in
differentiating S. aureus from other FIGURE 9.14 Coagulase Test
coagulase-negative staphylococci.
Most strains of S.aureus produce two types of coagulase, free coagulase
and bound coagulase. While free coagulase is an enzyme that is secreted
extracellular, bound coagulase is a cell wall associated protein. Free
coagulase is detected in tube coagulase test and bound coagulase is
detected in slide coagulase test. Slide coagulase test may be used to
screen isolates of S. aureus and tube coagulase may be used for
confirmation. While there are seven antigenic types of free coagulase,
only one antigenic type of bound coagulase exists. Free coagulase is heat
labile while bound coagulase is heat stable.

Slide Coagulase Test: The bound coagulase is also known as clumping

factor. It cross-links α and β chain of fibrinogen in plasma to form fibrin
clot that deposits on the cell wall. As a result, individual coccus sticks to
each other and clumping is observed.
Tube Coagulase Test: The free coagulases secreted by S. aureus act with
coagulase reacting factor (CRF) in plasma to form a complex, which is
thrombin. This converts fibrinogen to fibrin resulting in clotting of
plasma.

MATERIALS REQUIRED
EDTA anticoagulant human plasma, clean glass slide, test tubes, pipettes,
distilled water and inoculation loop.

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 Procedure
Slide Coagulase Test: Dense suspensions of Staphylococci from culture
are made on two ends of clean glass slide. One should be labeled as “test”
and the other as “control”. The control suspension serves to rule out false
positivity due to auto agglutination. The test suspension is treated with a
drop of citrated plasma and mixed well. Agglutination or clumping of
cocci within 5-10 seconds is taken as positive. Some strains of S. aureus
may not produce bound coagulase, and such strains must be identified by
tube coagulase test.

observation
The slides were observed for clumping or not within prescribed time.

Interpretation
Clumping formation - Positive reaction
No clumping formation – Negative reaction

Tube Coagulase Test

Three test tubes are taken and labeled “test”, “negative control” and
“positive control”. Each tube is filled with 0.5 ml of 1 in 10 diluted rabbit
plasma. To the tube labeled test, 0.1 ml of overnight broth culture of test
bacteria is added. To the tube labeled positive control, 0.1 ml of overnight
broth culture of known [[S. aureus is added and to the tube labeled
negative control, 0.1 ml of sterile broth is added. All the tubes are
incubated at 37°C and observed up to four hours. Positive result is
indicated by gelling of the plasma, which remains in place even after
inverting the tube. If the test remains negative until four hours at 37°C,
the tube is kept at room temperature for overnight incubation.

observation
The tubes were observed for clotting in the prescribed time.
Interpretation
Clumping formation - Positive reaction
No clumping formation – Negative reaction
Examples
Coagulase Positive: Staphylococcus aureus
Coagulase Negative: E. coli

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 LABORATORY 10

Fungi and
Identification of
Fungal
Contaminants in
Plant Tissue
Culture
 FUNGI
Fungi are unicellular or multi cellular organisms which live either as saprophytes
or parasites. They are the major contaminating organisms in the tissue cultures
because of their simple rapid reproductive processes through asexual and sexual
spores. Elimination of contaminants is crucial for the successful tissue culture
production as the fungi species during their rapid growth, utilize the culture media
and destroy the explants.

TECHNIQUES

1. Lacto Phenol Cotton Blue

Lacto Phenol Cotton Blue (LPCB)
of tear mount staining technique was
employed for identification. Two
methods are employed for LPCB
staining, namely, Tear mount method
and slide culture method. In LPCB
staining, cotton blue stain gives dark
blue colour to the fungal structure
against light blue background. The
FIGURE 10.1
cytoplasm will also be in light blue
colour. Phenol acts as fungicide and lactic acid acts as clearing agent. To
perform tear mount method, one or two drops of Lacto phenol cotton
blue stain was added to a clean slide. Using a flame sterilized needle a few
fungal mycelia was placed on the stain and the mycelia was gently teased
and spread using a sterile needle. Cover glass was carefully placed taking
extra caution to avoid air bubbles. Excess stain was removed using tissue
paper and observed under 10X and 45X objective of microscope.

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 2. Slide Culture Techniques

Contaminations were also

studied using slide culture method
for a double confirmation. Slides
were arranged over the V- shaped
tube in a petriplate. 1 cm X 1 cm
square block of Sabouraud’s
dextrose agar (SDA) was carefully
placed on the center of the glass
slide block. Cover slip was placed
with sterile forceps and moistened
cotton in petriplate was kept for
FIGURE 10.2
promoting the fungal growth.
After two to three days incubation, agar block was carefully placed on a
glass slide containing Lacto phenol cotton blue staining. Block was later
observed under 10X and 45X magnification of microscope.

3. Germ Tube Test

It is used for the observation of
germ tubes in Candida sp. Using
micropipette, dispense 3drops of fresh
pooled human serum into test tubes
with a sterile wooden applicator
sticker/ needle touch a yeast
(suspected sample) place the stick into
serum. Incubated the sample 35°C for
2-3hours. By placing on clean glass
slide, examine it with microscope. FIGURE 10.3

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 STUDY OF CHARACTERISTICS OF
SOME COMMON FUNGI

1) Aspergillus species
Colony Morphology:
Colonies are wooly at first, white to
yellow, then turning dark brown to
black. Reverse is white to yellow.

Microscopic Morphology:
Conidiophores are smooth and
colourless and turned dark toward
vesicles. The vesicle was globes and
bearing phialides mycelium septate.
FIGURE 10.4

2) Penicillium species
Colony Morphology:
Colony appeared as bluish green
mycelium was septate and ridged

Microscopic Morphology:
Penicillium has brush like appearance
formed of chains of spores extending
from the ends of phialides borne on
short branches of conidiophores on the
hyphae.
FIGURE 10.5

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3) Mucor species
Colony Morphology:
Colony was rapidly growing, filling the
test tube or petridish in 5-7 days with a
fluffy asexual mycelia ie, at first white
but later become gray to brown.
Microscopic Morphology:
Mycelium was broad, non-septate, and
colourless without rhizoids shows few
irregular cross wall, sporangiophores
FIGURE 10.5
arose singly from them the mycelium
forming a thick fluff.
They were either unbranched with terminal sporangia or branched with
spherical multispored sporangia on cell at the end of the hyphae.

4) Rhizopus species
Colony Morphology:
Colonies are columnar, fast growing
and cover an agar surface with a dense
cottony growth that is at first white
becoming grey or yellowish brown
with sporulation.
Microscopic Morphology:
Sporangiophores up to 1500 µm in
length and 18 µm in width, smooth
walled, non - septate, simple or
branched, arising from stolons
opposite rhizoids usually in groups of 3 FIGURE 10.6
or more. Sporangia are globose, often
with a flattened base, grayish black,
powdery in appearance, up to 175 µm
in diameter and many spored.

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5) Fusarium species
Colony Morphology:
Colony was fluffy to cottony, owing to
extensive mycelium some diffusible
pigment produced on reverse side
(orange).
Microscopic Morphology:
Conidiophores singly or grouped,
septate, micro conidia are one walled
and often numerous in chain or balls.
FIGURE 10.7
Macroconidia were elongate and
cylindrical.

6) Candida species
Colony Morphology:
Colonies were creamy white, pasty,
smooth, dull with foul odour and yeast-
like in appearance.

Microscopic Morphology:
All Candida species produce
blastoconidia singly or in small clusters.
Blastoconidia may be round or
elongate. Most species produce
pseudohyphae which may be long,
branched or curved. True hyphae and
FIGURE 10.8
chlamydospores are produced by
strains of some Candida spp.

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7) Phytophthora species
Colony Morphology:
White cottony colonies, media colour
changed into red

Microscopic Morphology:
Highly branched, septate and
nucleated

FIGURE 10.9

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 IDENTIFICATION OF FUNGAL CONTAMINANTS
IN PLANT TISSUE CULTURE LAB
Objective
To identify various fungal contaminants in plant tissue culture lab

Technical Programme
Lacto Phenol Cotton Blue (LPCB) of tear mount staining technique was
employed for identification. Two methods are employed for LPCB staining,
namely, tear mount method and slide culture method. In LPCB staining blue
colour gives to cytoplasm against light blue background walls of hyphae can be
visualized easily. Phenol act as fungicide and lactic acid act as clearing agent. To
perform tear mount method, one or two drops of Lacto phenol cotton blue stain
was added to a clean slide. Using a flame sterilized needle a few fungal mycelia
was placed on the stain and the mycelia was gently teased and spread using a
sterile needle. Cover glass was carefully placed taking extra caution to avoid air
bubbles. Excess stain was removed using tissue paper and observed under 10X
and 45X objectives of microscope. Various fungal smears were identified based
on their morphological characteristics from the banana, pineapple and passion
fruit tissue culture bottles.
Contaminations were also studied using slide culture method for a double
confirmation. Slides were arranged over the V- shaped tube in a petriplate. 1 cm
X 1 cm square block of Sabouraud’s dextrose agar (SDA) was carefully placed on
the center of the glass slide block. Cover slip was placed with sterile forceps and
moistened cotton in petriplate was kept for promoting the fungal growth. After
two to three days incubation, agar block was carefully placed on a glass slide
containing Lacto phenol cotton blue staining. Block was later observed under 10X
and 45X magnification of microscope.

Observation

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Result
A wide range of microorganisms cause contamination in tissue culture
laboratory, fungi, yeast, molds and bacteria were the predominant microbes.
Among them fungi were the major contaminants, 73.135% of consisting of fungal
contamination and of bacteria were 26.87%.

FIGURE 10.10
MACROSCOPIC AND MICROSCOPIC OBSERVATIONS
OF SOME FUNGAL CONTAMINANTS

Microbiology Laboratory Manual 110

 LABORATORY 11

IDENTIFICATION OF
DISEASE CAUSING
FUNGAL PATHOGEN OF
PASSION FRUIT NURSERY
PLANTS
AND
IDENTIFICATION OF
DISEASE CAUSING
PATHOGENS IN PASSION
FRUIT PLANTS OF PRS
FIELD
 IDENTIFICATION OF DISEASE
CAUSING FUNGAL PATHOGEN OF
PASSION FRUIT NURSERY PLANTS
OBJECTIVE
To identify the infective agent on passion fruit seedling from
roof top nursery.

TECHNICAL PROGRAMME

The soil and plant samples were collected from the roof top
nursery and weighed 1g of sample and suspended in 9 ml sterile
distilled water in tubes (10ˉ¹). Arranged 5 sets of tubes, each set
contained 9 ml of sterile distilled water. Shaked and homogenized
the first and transferred 1 ml from it to the second. Similarly, 1ml
sample was serially transferred 10ˉ² dilutions into third tube
containing 9ml of sterile water to get a final dilution of 10ˉ³.
Repeated the procedure for 10ˉ⁴, 10ˉ5 , 10ˉ6 dilutions. The same
procedure was followed in plant samples. Aseptically poured 1 ml soil
suspension from 10-1 , 10-2 , 10-3 , 10-4 , 10-5 into sterile petriplate
mixed with 15 ml of SDA at 45-50°C and mixed well. Incubate the
plates at room temperature for 4-5 days. After proper incubation
stained the colonies using LPCB stain method.

RESULTS

Microbiology Laboratory Manual 112

RESULT

Fusarium sp. was found to be in large number in both plant

and soil samples studied. This is the reason for the damping off
disease of seedlings in passion fruit planted in roof top nursery.

FIGURE 11.1 Fusarium sp. (45X)

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 IDENTIFICATION OF DISEASE
CAUSING PATHOGENS IN PASSION
FRUIT PLANTS OF PRS FIELD
AIM
To identify the disease causing pathogens for infected plants 55-
8, 55-9,Vazhakulam purple variety plants 12, 10, 9, 8, 7 and 6, 86-7, 134-
4, 55-5 and 125.

MATERIALS REQUIRED
SDA, Nutrient Agar (NA) plates, sterile distilled water, test tubes
and routine lab equipment

TECHNICAL PROGRAMME
As per the information from the field, that the passion fruit
plants started to wilt, plant pathologist visited the field and collected
the soil samples, stem samples etc. Aseptically collected the samples
and packed well. Checked the symptoms of the disease.

SYMPTOMS
Sample varieties showed wilting, root rot, stem rot, and stem
discoloration. During that time leaves were falling from the plant.
When removed the soil nearby the root system had shown rotting
except tap root.

Techniques Include

1. Surface sterilization of the sample 3. Inoculation of leaf, stem, root samples

2. Serial dilution of the soil sample on SDA plates
Preparation of SDA
Serial dilution
Inoculation of samples
Plating (Spread plate)
LPCB for fungal
Colony Counting
identification
Sub culturing (Streaking)
Gram’s Staining 4. Gram Staining
Biochemical tests (if 5. Hanging Drop Motility
applicable)

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 Surface Sterilization of the Samples
Washed the samples (root & stem samples collected from the diseased
plants) in running tap water for few minutes.
Washed again using sterile distilled water.
Rinsed the samples using 70% alcohol.
Washed again using distilled water for removing the alcohol.
Rinsed the samples using 0.1% Mercuric chloride (HgCl2) for 1 minute.
Washed the samples with distilled water for removing the excess mercuric
chloride .
Serial Dilution

Weigh 1 g of soil sample and add it to 10 ml sterile distilled water

and mix well. The sample was serially diluted up to 10ˉ⁴ by transferring
1ml. first three dilutions spread on 3 Nutrient Agar (NA) and 3 SDA
plates by transferring 0.1ml of each dilution. Incubate NA plates in
incubator at 37°C for 24 hrs and also incubate SDA plates at room
temperature for 3-4 days.

Sub cultured the colonies for getting pure culture by streak plate
method and performed Gram staining.

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 Gram Staining Result
Purple-rods were obtained by gram staining, Gram positive
bacteria. Both 55-8 and 55-9 have the same bacteria. It’s a common soil
microbe, which is not responsible for this infection.

Performed LPCB for the fungal growths obtained from leaf and
root samples on SDA plates. Macroscopic observations of the fungal
growth obtained from leaf and root samples on SDA plates are
discussed below:

Microbiology Laboratory Manual 116

 LPCB Results

The fungal pathogen was Phytophthora sp..

FIGURE 11.2 Microscopic view of Phytophthora &

Fusarium sp.

Microbiology Laboratory Manual 117

LABORATORY 12

MICRO-
BIOLOGICAL
EXAMINATION
OF MILK
 MICROBIOLOGICAL EXAMINATION
OF MILK
Methylene Blue Reductase Test

Principle
This reductase test is based on the oxidation-reduction activities
of the bacteria present in the milk sample. The indicator used in the
reaction is methylene blue which is color sensitive to oxygen
concentration. The indicator is blue in the oxidized state and leuco or
white in the reduced conditions. The speed of color disappearance of
methylene blue is proportional to the microbial load in the milk
sample. The more the bacteria present the faster will be the
reduction.
The classification of milk as per methylene blue reductase test is as
follows.
1. Class I- Excellent, not decolorized in 8 hours (<500 bacteria/ml)
2. Class II-Good, decolorized in less than 8 hours but not less than
6 hours (>500 bacteria/ml)
3. Class III-Fair, decolorized in less than 6 hours but not less than
2 hours (>40,00,000 bacteria/ml)
4. Class IV- Poor, decolorized in less than 2 hours. (> 2,00,00,000
bacteria/ml)

MATERIALS REQUIRED
Milk sample, methylene blue solution, Mc Cartney bottles, pipettes,
water bath set at 37oC, distilled water, Bunsen burner.

note
All the glass wares were sterilized before use.

Microbiology Laboratory Manual 119

procedure
1. Methylene blue solution was prepared by dissolving 1 mg
methylene blue powder aseptically in 25 ml of distilled water.
2. Transferred 10 ml of milk sample into sterile Mc Cartney bottle
using sterile pipettes.
3. Added 1 ml of methylene blue solution to the milk sample using a
separate sterile pipette.
4. The bottle was closed with the stopper.
5. The contents of the tube were mixed by gently inverting it 2-3
times.
6. Incubate the Mc Cartney bottle in a water bath at 37oC for 6
hours.
7. Controlled tubes containing 10 ml boiled milk and 1 ml of
methylene blue was also incubated.  Recorded the time for
discoloration.

Microbiology Laboratory Manual 120