Contents

Sr. No. Practical P. No.

1 Biosafety levels 2
2 Equipment and Laboratory Materials used in Microbiology 11
3 Microscope and Microscopy 14
4 Sterilization techniques (Physical/Heat method of sterilization) 17
5 Sterilization techniques (Chemical method of sterilization) 22
6 Sterilization techniques (Mechanical/Filtration methods) 24
7 Types of media and culturing 27
8 Culturing of bacteria in liquid medium 33
9 Culturing of bacteria on solid medium 35
10 Colony and cell morphology 40
11 Bacterial cell count 44
12 Gram staining of bacteria, 46
13 Testing sensitivity to antimicrobial substances 49
14 Biochemical tests 51
15 Growth Kinetics 64

1
 Practical No. 1
Biosafety levels

Biosafety
Safety from exposure to Infectious Agents
Biosafety Level:
A biosafety level is a set of biocontainment precautions required to isolate dangerous biological
agents in an enclosed laboratory facility.
Importance:
● Laboratorians recognize hazards of processing infectious agents
● Guidelines developed to protect workers in microbiological and medical labs through
engineering controls, management policies, work practices
● Precautions so people researching or trying to identify organisms do not become infected
● While handling or testing clinical specimens, workers could accidentally infect themselves or
coworkers
● Labs must adhere to very specific safety regulations to work with organisms that pose a
threat to human health
Biosafety levels (1-4):
There are four biosafety levels; protective practices increase with each
Biosafety Level 1 labs - work with least dangerous agents, require fewest precautions.
Biosafety Level 4 labs - have strictest methods because dealing with agents that are most
dangerous to human health
Primary barriers: Physical barriers or personal protective equipment between lab worker and
pathogen. For example: Gloves, masks, lab coat, goggle, shoes cover
Secondary barriers: Structural aspects of the laboratory that make working environment safer
against infection. For example: Sinks for hand washing, special containment areas, special air
ventilation patterns
BIOSAFETY LEVEL 1
● Work is generally conducted on open bench tops using standard microbiological practices.
● Special containment equipment or facility designs is not required nor generally used.

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● Laboratory personnel have specific training in the procedures conducted in the laboratory and
are supervised by a scientist with general training in microbiology.
Standard Microbiological Practices:
● Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted
in the work areas where there is reasonable likelihood of exposure to potentially infectious
materials.
● Food is stored outside the work area in cabinets or refrigerators designated and used for this
purpose only.
● Mouth pipetting is prohibited; mechanical pipetting devices are used.
● All procedures are performed carefully to minimize the creation of splashes or aerosols.
● Work surfaces are decontaminated at least once a day and after any spill of viable material.
● All cultures, stocks, and other regulated wastes are decontaminated before disposal by an
approved decontamination method.
● Materials to be decontaminated outside of the immediate laboratory are to be placed in a
durable, leak-proof container and closed for transport from the laboratory.
● Materials to be decontaminated at off-site from the laboratory are packaged in accordance
with applicable local, state, and federal regulations, before removal from the facility.
● Special containment devices or equipment such as a biological safety cabinet is generally not
required for manipulations of agents assigned to Biosafety Level 1.
● It is recommended that laboratory coats, gowns, or uniforms be worn to prevent
contamination.
● Gloves should be worn if the skin on the hands is broken or if a rash exists.
Laboratory Facilities:
● Each laboratory contains a sink for hand washing.
● The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not
appropriate, and should not be used because proper decontamination following a spill
extremely difficult to achieve.
● Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and
moderate heat.
● Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are
accessible for cleaning.

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● If the laboratory has windows that open, they are fitted with fly screens.
BIOSAFETY LEVEL 2
● Risk Group 2 infectious agents are pathogens that can cause human or animal disease but,
under normal circumstances, are unlikely to be a serious hazard to laboratory workers, the
community, livestock, or the environment.
● Infections by risk group 2 organisms are not considered to be a serious hazard. They are a
moderate individual risk and limited community risk.
● Laboratory exposures rarely cause infection leading to serious disease; effective treatment
and preventive measures are available and the risk of spread is limited.
● Examples of infectious agents in this risk level are E. coli, many influenza viruses, some
fungi like ringworm; human herpes simplex viruses; Transmissible Gastro-enteritis of swine;
Mouse Hepatitis Virus; and a few parasites.
● Primary hazards: accidental needle sticks, exposure to eyes and nose, ingestion of
infectious materials
● Agents do not cause lethal infections that are not transmissible via airborne route (do not
cause infection if tiny droplets become airborne and are inhaled, which might occur if the
material were spattered)
● Agents are pathogens for which immunization or antibiotic treatment is available
● Extreme care should be taken with contaminated needles and sharp lab instruments
Procedures required to contain risk group 2 organisms:
● The laboratory should be separated from all other activities.
● A biohazard sign should be present and visible. (policies to restrict access to lab)
● All the surfaces in the laboratory should be readily cleanable
● An autoclave should be present.
● A certified HEPA filtered class 1 or 2 biological cabinet should be available for the
manipulation of organisms.
Gloves and a laboratory coat should be worn at all times in the laboratory

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Fig 1.1: Biohazard Symbol
BIOSAFETY LEVEL 3
● Risk Group 3 infectious agents are pathogens that usually cause serious human or animal
disease, or which can result in serious economic consequences, but do not ordinarily spread
by casual contact from one individual to another (high individual risk, low community risk),
or that can be treated by antimicrobial or antiparasitic agents.
● Risk Group 3 pathogens include bacteria such as anthrax, Q Fever, tuberculosis, and viruses
such as hanta viruses, Human immunodeficiency viruses (HIV - all isolates), eastern and
western equine encephalitis viruses.
Procedures required to contain risk group 3 organisms:
● Laboratory staff must be fully trained in the handling of pathogenic and other hazardous
material, in the use of safety equipment, disposal techniques, handling of contaminated
waste, and emergency response.
● Standard operating procedures must be pasted in visible spot.
● Equipment must include an autoclave and a certified HEPA filtered class II biological safety
cabinet
● All activities involving infectious materials to be conducted in biological safety cabinets or
other appropriate combinations of personal protective and physical containment devices.
BIOSAFETY LEVEL 4
Designed for use with:
● Highly toxic/infectious agents
● Agents that are at a very high risk for forming infectious aerosols
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● Life threatening agents
Examples: Ebola virus, Marburg virus, Lassa virus
Special Practices:
❖ Only those that work in the BSL-4 lab will be allowed entry
❖ A logbook must be signed each time one comes in or out of the lab with the date and time
❖ The doors must have a universal hazard label with։
• Description of the infectious agent (s)
• Responsible director
❖ All lab personnel are trained specially for BSL-4 by lab director
❖ Lab personnel must be vaccinated for the agents:
• Present in the lab
• That have potential to be in the lab
❖ Baseline serum samples are collected and stored for serological surveillance
• Make sure the personnel have sufficient antibodies for the pathogens they are working with
❖ Supplies/materials are brought into the lab by a double door autoclave that։
• is secured so that materials can be removed from the interior door
• is decontaminated after each use
❖ Plastic is substituted for glass when possible
• Eliminate sharps such as capillaries, scalpels, needles and syringes
• Broken glass should be handled with a dust pan and broom
• Non-disposable and disposable sharps collected in a hard container to be transported to an
autoclave for decontamination; disposables are thrown out
Safety Equipment:
Primary barriers:
❖ Class III biological safety cabinets for handling infectious material or։
❖ BSC must be validated on a 12 month period
❖ The PPE required is a
• one-piece jumpsuit
• gloves
• closed toe shoes
• All will be removed in the inner room after showering

6
 Secondary barrier:
❖ BSL-4 labs are housed in separate buildings or isolated within a building with lower BSL labs
❖ Outer and inner change rooms
❖ Walls, floors and ceilings have sealed internal shells
• keep pests out
• Liquid and heat stabile for decontaminating purposes
❖ Drains have chemical disinfectant traps attached to liquid waste decontamination system
• All waste must be decontaminated including shower waste
❖ HEPA filters in vents for sterile air
• Filter air exhaust from the class III biological safety cabinets.

Fig 1.2: One-piece suit

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Fig 1.3: Biosafety Cabinet Class I Fig 1.4: Biosafety Cabinet Class II

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Fig 1.5: Biosafety Cabinet Class III

9
Table 1.1: Type of biosafety rules in details

10
 Practical No. 2
Equipment & Materials Used in Microbiology

1. Agar Plate — Petri dish containing solidified culture medium.

2. Agar Slant — Test lube containing solidified culture medium at a slope for preparing cultures
and preservation of pure cultures in the lab. .
3. Agar Stab — Test tube containing solid, culture medium for growth of anaerobic organisms
especially when sealed with oil or deep culture system.
4. Anaerobic Jar’— Closed chamber with controlled out-fits for air. It is used for anaerobic
cultivation of microorganisms, in the laboratory..
5. Autoclave — Used for sterilization. Normally a pressure of 1.09-kg cm -2 (15 lb. per square inch) is
used for 15 minutes or longer for moist, heal sterilization.
6. Broth — Liquid medium containing nutrients in which bacteria are grown.
7. Broth Culture — Growth of bacteria in liquid medium.
8. Canisters — For carrying pipettes and Petri dished in a safe manner.
9. Centrifuge machine — Used to spin ‘the suspension, blood, etc. for the separation of cells from
the fluid part. Available in ordinary (5000 rpm) and high speed (15,000,rpm) versions. The
ultracentrifuge (60.000rpm) is employed for sub-cellular particle separation.
10. Colony Counter —‘Illuminated stage with magnifying lens attachment used for counting
bacterial colonies on the surface of Petri dish ‘culture medium.
11. Cotton Swabs — Small slick with cotton wool for taking samples from ‘any surface such as
wounds, throat, skin, etc.
12. Culture Hood — A closed cabinet with UV light arrangement in order to maintain sterilized
environment for culture manipulation.
13. Culture Medium — Mixture of nutrients for the growth of microorganisms. Available in the form
of ready- to-use, as powdered or is prepared from separate ingredients. Commonly used culture
media are nutrient agar, nutrient broth, MacConkey agar, etc.
14. Desiccator- A closed air-tight jar containing suitable hygroscopic chemical for the removal of
moisture content from the substrate.
15. Dropping Bottles — Used for carrying staining solution in the lab. These deliver drop wise flow of
the staining solutions.

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16. Glass Cavity Slide — Glass slide having concavity in its center. It is used for determining motility
of microorganisms under microscope.
17. Glass Cover Slip — Rectangular or circular thin piece of glass (usually 1 cm 2 or diameter) used for
permanent mounting of smears or cell culture experiments.
18. Glass Flask5 — Made of usually Pyrex glass, available in varying sizes and capacity as round or
flat bottom. These are used for mixing purposes or making nutrient media. Flat-bottomed flasks
are preferred.
19. Glass Slide — Rectangular piece of glass sheet (3.0 x 1.5. cm) used for making bacterial or fungal
smears, blood smears, etc. for detailed microscopic study.
20. Hot Air Oven — For sterilizing glassware, metal-ware and oils at usually a temperature of 171 to
200°C. Also used for drying objects.
21. Incubator — For growing microbial cultures at specific and constant temperature. Ordinary
incubators work at 25—40°C, while the low temperature incubators are set at - 1 to 10°C.
Carbon dioxide incubators work at 25—40°C.
22. Inoculating Needles and Loops — These are used [or transferring microorganisms. They must be
flamed to red-hot before and after use. They should be cooled before being used to pick up
organisms. These are also helpful in preparing slide smears.
23. Laminar Air Flow Cabinet—Semi-closed cabinet with filtered airflow arrangement used for open
culture manipulation.
24. Lypholization apparatus — Meant for the preservation of cultures for extended period of lime.
25. Magnetic stirrer — Instrument provided with hot plate arrangement, used for proper
homogenous mixing of solutions, media, etc. under warm conditions.
26. Microdispenser — Micropipettes delivering minute amounts of solutions in microliters ( μL) for
making multiple dilutions, These are available in single, 4, 8 and 12 channels.
27. Micrometers — Stage and ocular micrometers, used for micrometrical study.
28. Microscope — Simple, compound or binocular used for detailed study of microbial (bacterial or
fungal) or blood smears, etc.
29. Petri Dish (named after its inventor) — Normally made of Pyrex glass and consists of a bottom
and a lid, It is used for growing microorganisms. It must be placed with lid down in the
incubator. It is labelled on the underside. The glass Petri dish is reusable. Petri dish is also
manufactured in plastic material as disposable. Different sizes such as small, medium, regular
and king size are available for different purposes.

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30. pH Meter — Calibrated instrument with buffers, used for determining the acidity or alkalinity
(pH) of, solutions, broths or culture media.
31. Pipettes —Used for transferring liquid materials. Pasteur pipettes are used for pouring small
amounts. while graduated pipettes are employed for transferring specific amount of material.
32. Plate Carriers — For incubation of agar plates and storage of used plates.
33. Refrigerator — Essential for keeping samples, cultures. etc. at 4_10oC.
34. Seitz Filters — Stainless steel assembly with asbestos filters for sterilization of liquid media or
serum, etc.
35. Staining Rack — Used to support slides during staining.
36. UV Lamp —Used for decontamination of laboratory environment.,
37. Vacuum Pump — Used for creating negative or positive pressure in filtration systems.
38. Water Bath — Used to maintain constant temperature of agar and other materials. Also useful
for boiling sugar solutions and heal inactivation of serum.
39. Water Still — Made of Pyrex glass. It is required for preparing distilled or redistilled water
needed for reconstitution or preparation of culture media.
40. Weighing Balance — Required for weighing ingredients for making solutions, media, etc.

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 Practical No. 3
Microscope & Microscopy

Microscope & Microscopy

Microscope is an instrument used to obtain an enlarged image of a small object. Microscopes are
classified as:
a. Light microscope — Simple and compound microscopes where the ordinary light source (visible
light spectrum) is utilized to make the final image of the object. These are the most commonly
used microscopes in the microbiological laboratories

b. Electron microscope — The transmission and the scanning electron microscopes are employed
Lo resolve very minute particles like viruses and peptides through the use of electron beam
under vacuum These are primarily used for detailed examination of sub-cellular particles in
research laboratories.

3.1 Light Microscope

Study the microscope and identify the following parts on it. Note also any accompanying information on
the parts:
1. The lenses: The ordinary light microscope is a compound microscope. II has two sets of lenses in
contrast to the simple magnifying glass that is a simple microscope The set of lenses nearer the eye
is the ocular or eyepiece The usual magnification of the ocular lenses is 20 x 2 5 x 6 x 10 x and 12 x The
set of lenses nearer the object is the objective The objectives are carried on a revolving nosepiece
The nose-piece usually carries three to four objectives:-

Objectives lens Usual magnification of Focal length

objective lens

Low power 10 x 18

Medium power 20 x 9 mm

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 High dry 40 x 4 mm

Oil immersion 100 x 1.2-1.6 mm

2. The mirror This transmits light from a light source which may be an electric lamp or natural daylight
When a condenser is present the flat side of the mirror is used If a condenser is absent concave side
of the mirror is employed In the modern microscopes mirror is absent and the lamp transmits light.
3. The sub stage condenser (also called the Abbe condenser after its discoverer) It focuses light on the
object. It should be flush with the stage. . . . .
4. The iris diaphragm: The amount of light entering the microscope is controlled with the iris
diaphragm. II should be adjusted to suit the nature i the specimen being observed, depending on
whether more or less light is required. It is fitted beneath the condenser.
5. The mechanical stage: This is the platform where the slide is placed. It is provided with. two
mechanical manipulator knobs (one for forward backward and the Other for side
movements)..These are usually located on or immediately below the stage. It helps in locating exact
position of the specimen or object under microscopic field. .. . .
6. The adjustment Knobs: These are three: the coarse adjustment knob, the fine adjustment knob and
the sub-stage adjustment knob (used to raise the condenser). All are working to get fine image of
the object.
7. Magnifying power of the microscope: It is the combined power of ocular x objective of the
microscope lens adjusted or available. . .

8. Resolving power of the microscope: It is the ability of microscope, to distinctly separate closely
related objects. It is calculated as RP = λ .NA where A =. wavelength o. visible light spectrum (4000
5000 Ao), NA=numerical aperture of thy lens (04rrd oil immersion lend (1.25)

3.2 Microscopy
Microscopy is the study of objects under the microscope. There are seven categories of light
microscopes intended for various kinds of studies. These are:
a. Bright field microscope — Commonly used for viewing stained bacteria and fungi for their
morphological and micrometrical studies.
b. Fluorescent microscope — Used for the identification of bacteria, fungi and specific antigens,
coaled with fluorescent dye, for much greater details.

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c. Dark field microscope — Very small objects including bacterial capsules may be viewed through
negative staining under dark field microscope.
d. Phase contrast microscope — Viable bacteria may be viewed directly without any staining
through phase contrast microscope.
e. Nomarski differential interference contrast microscope — A three dimensional view of the
object may be observed irrespective of the staining method adopted under this microscope.
f. Inverted microscope — The cell cultures and the fungi growing at the base of the Petri dish may
be viewed directly under the inverted microscope.
g. Ultraviolet light microscope — Very high resolving power of light microscope may be obtained
and detailed structures of the cells may be viewed in this microscope.

3.3 Procedure to view a specimen under compound microscope:

1. Connect the light microscope to a power source.

2. Turn the revolving nosepiece so the lowest objective lens is in position.

3. Mount your specimen onto the stage adequately protected by placing a coverslip.

4. Use the metal clips to keep your slide in place positioned in the center.

5. Look into the eyepiece and slowly rotate the coarse adjustment knob to bring your specimen to
focus.

6. Adjust the condenser for the maximum amount of light. Use the diaphragm under the stage to
adjust.

7. Now slowly rotate the fine adjustment knob until you obtain a clearer image of your specimen.

8. Examine your specimen.

9. After you're done viewing with the lowest power objective, switch to the medium power
objective and re-adjust the focus with the fine adjustment knob.

10. Proceed to the high power objective once you have it focused.

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17
 Practical No. 4
Sterilization techniques (Physical/Heat method of sterilization)

Introduction
Sterilization is defined as total destruction of all microorganisms (whether or not pathogenic) and
their spores, usually through the use of drastic methods such as concentrated toxic chemicals
(alcohols, chlorine, formaldehyde, glutar-aldehydes, etc.), very high temperature, or intense
radiation. A sterilized item cannot support life in any form.
Methods involved in sterilization

Fig 2.1: List of methods

This is the most common method of sterilization. The heat used kills the microbes in the
substance. The temperature of the heat and duration of heating are the factors that affect the
extent of sterilization. In heat sterilization process, the longer the exposure to heat the better is
the sterilization at a given temperature. As the temperature of heat raises the time span required
for sterilization decreases. Further, the sterilization time increases with a decrease in temperature
and vice-versa. But one needs to maintain minimum sterilization time or minimum contact time
for the heat to be in touch with microbes or bacteria and thereby kill them. The heat method of
sterilization is again of two types based on the type of heat used.
A) Moist heat methods. B) Dry heat methods.
A. Moist heat method of sterilization:
Here heat is applied in the form of steam or just boiling. This method includes techniques like
1. Boiling. 2. Pasteurization. 3. By use of steam (Autoclave).

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1. Boiling is preferred for metallic devices like surgical scissors, scalpels, needles, etc. Here
substances are boiled to sterilize them.
2. Pasteurization is the process of heating the milk at a temperature of 6o degrees or 72 degrees
3 to four times. Here alternative heating and cooling kills all the microbes and molds without
boiling the milk.
3. Using Steam (autoclaving): It is the most common method used for drugs as it is powerful
enough even to kill bacterial spores. Bacterial spores are the forms of bacteria which are inert.
They form a rigid cover over the cell wall during harsh climate. This cover prevents any damage
to cell and drying of the cell. By steam sterilization, these forms of bacteria are also killed as
steam destroys the cell wall.
Principle:
In this method sterilization is done by steam under pressure. Steaming at temperature higher than
100°C is used in autoclaving. The temperature of boiling depends on the surrounding
atmospheric pressure. A higher temperature of steaming is obtained by employing a higher
pressure. When the autoclave is closed and made air-tight, and water starts boiling, the inside
pressures increases and now the water boils above 100°C. At 15 ib per sq. inch pressure, 121°C
temperatures is obtained. This is kept for 15 minutes for sterilization to kill spores. It works like
a pressure cooker.
Construction of Autoclave
Autoclave is a metallic cylindrical vessel. On the lid, there are:
(1) A gauge for indicating the pressure,
(2) A safety valve, which can be set to blow off at any desired pressure, and
(3) A stopcock to release the pressure. It is provided with a perforated diaphragm. Water is
placed below the diaphragm and heated from below by electricity, gas or stove.
Working of Autoclave
(a) Place materials inside,
(b) Close the lid. Leave stopcock open,
(c) Set the safety valve at the desired pressure,
(d) Heat the autoclave. Air is forced out and eventually steam ensures out through the tap,
(e) Close the tap. The inside pressure now rises until it reaches the set level (i.e. 15 Win), when
the safety valve opens and the excess steam escapes,

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(f) Keep it for 15 minutes (holding time),
(g) Stop heating,
(h) Cool the autoclave below 100°C,
(i) Open the stopcock slowly to allow air to enter the autoclave.

Fig 2.2: Procedure of autoclave

Advantages – The penetrating nature of steam makes it a great solution to destroy proteins in
any microorganism after a certain amount of time. It is environmentally safe having no toxic
byproducts.
B. Dry heat methods
Here the substances are subjected to dry heat like
1. Flaming 2. Incineration 3. Hot air oven. 4. Radiation sterilization
1. Flaming

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It is the process of exposing metallic device like the needle, scalpels, and scissors to flame for
few minutes. The fire burns the microbes and other dust on the instrument directly.
2. Incineration
It is done especially for inoculating loops used in microbe cultures. The metallic end of the loop
is heated to red hot on the flame. This exposure kills all the germs.
3. Hot Air Oven
It is one of the most common methods used for sterilization. Glass wares, swab sticks, all glass
ware, syringes, powder and oily substances are sterilized in hot air oven. For sterilization, a
temperature of 160°C is maintained (holding) for one hour. Spores are killed at this temperature.
It leads to sterilization. Hot air oven is suitable for dry material like powders, metal devices,
glassware, etc.
Working and Principle
It is an apparatus with double metallic walls and a door. There is an air space between these
walls. The apparatus is heated by electricity or gas at the bottom. On heating, the air at the
bottom becomes hot and passes between the two walls from below upwards, and then passes in
the inner chamber through the holes of the apparatus. A thermostat is fitted to maintain a
constant temperature of 160°C.

Fig 2.3: Hot air oven

Advantages– Dry heat sterilization is preferred for heat stable products that are sensitive to
moisture.
4. Radiation

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This method involves exposing the packed materials to radiation for sterilization. There are two
types of radiations available for sterilization i.e. non-ionic and ionic radiation.
 Non-ionic radiations are safe to the operator of sterilization, and they are like Ultra Violet
radiations, they can be used even at the door entrances to prevent entry of live microbes through
the air.
 Ionizing radiation sterilization. They are powerful radiation and very useful for sterilization.
The operator needs to protect himself from exposure from these radiations by use of special
clothing. Ex: X-rays, γ-rays, etc.

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 Practical No. 5
Sterilization techniques (Chemical method of sterilization)

Here the articles are subjected to sterilization by using toxic gasses. The gas penetrates quickly
into the material like steam so, the sterilization is effective. But the chances of explosion and cost
factors are to be considered. The gasses used for sterilization are very poisonous. The commonly
used gas is ethylene oxide with a combination of carbon-dioxide. Carbon dioxide is added to
minimize the chances of an explosion.
Modes of action of chemicals:
● Protein coagulation
● Disruption of cell membrane resulting in exposure, damage or loss of the contents.
● Removal of free sulfhydryl groups essential for the functioning of the enzymes.
● Substrate competition – a compound resembling the essential substrate of the enzyme diverts
or misleads the enzymes necessary for the metabolism of the cell and causes cell death.
Alcohols
The types of alcohol used in disinfection are ethanol (80%), propanol (60%), and isopropanol
(70%). Alcohols are quite effective against bacteria and fungi, less so against viruses. They do
not kill bacterial spores. Due to their rapid action and good skin penetration, the main areas of
application of alcohols are surgical and hygienic disinfection of the skin and hands.
One disadvantage is that their effect is not long-lasting (no depot effect). Alcohols denature
proteins.
Aldehydes
Formaldehyde (HCHO) is the most important aldehyde. It can be used in a special apparatus for
gas sterilization. Its main use, however, is in disinfection. Formaldehyde is a water-soluble gas.
Formalin is a 35% solution of this gas in water. Formaldehyde irritates mucosa; skin contact may
result in inflammations or allergic eczemas. Formaldehyde is a broad-spectrum germicide for
bacteria, fungi, and viruses. At higher concentrations, spores are killed as well. The mechanism
of action of formaldehyde is based on protein denaturation.
Ethylene oxide
This highly reactive gas (C2H4O) is flammable, toxic, and a strong mucosal irritant. Ethylene
oxide can be used for sterilization at low temperatures (20–60 8C). The gas has a high

23
penetration capacity and can even get through some plastic foils. One drawback is that this gas
cannot kill dried microorganisms and requires a relative humidity level of 40–90% in the
sterilizing chamber. Ethylene oxide goes into solution in plastics, rubber, and similar materials,
therefore sterilized items must be allowed to stand for a longer period to ensure complete
desorption.
Chlorine compounds
Chlorine denatures proteins by binding to free amino groups. Generally, chlorine is used in the
form of sodium hypochlorite which is commonly known as household bleach. It is very useful
for sterilization purpose as it is very inexpensive and also effective against bacteria and viruses.
But it has some disadvantages as well; First of all high concentration of this agent corrodes
metals and also damages cloths. Secondly the strength of this solution decreases with time so
whenever you want to use it always prepare its fresh solution.
Oxidants
This group includes ozone, hydrogen peroxide, and peracetic acid. Their relevant chemical
activity is based on the splitting off of oxygen. Most are used as mild antiseptics to disinfect
mucosa, skin, or wounds.
Equipment:

● A spray bottle with 70% ethanol

● A contaminated surface (Bench top).
● Cotton

Procedure:

1. Spray the 70% ethanol evenly over the contaminated surface, making sure to cover all
areas. This helps in effectively killing or reducing microorganisms on the surface.
2. Dry the surface with Cotton Budd.

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 Practical No. 6
Sterilization techniques (Mechanical/Filtration methods)

This method is used for sterilizing thermolabile solutions, which will otherwise be degraded by
other conventional heating methods. The drug solutions are passed through the sterile bacteria
proof filter unit and subsequently transferring the product aseptically into the sterile containers
which are then sealed
Procedure:
The solutions to be sterilized is passed through the filter and collected in the sterile receiver by
the application of positive pressure to the non-sterile compartment or negative pressure to the
sterile side.
Membrane filters
● Membrane filters are made of cellulose-derivative (acetate or nitrate). They are very fine.
They are fixed in some suitable holders.
● Nominal pore size is 0. 22 ± 0. 02 µm or less is required.
● The membranes are brittle when dry. In this condition they can be stored for years together.
They become very tough when dipped in water.
● They are sterilized by autoclaving or by ethylene oxide gas. They cannot be sterilized by
dry heat as they decompose above 1200C.
● They are suitable for sterilizing aqueous and oily solutions but not for organic solvents
such as alcohol, chloroform etc.
● Membrane filters are generally blocked by dirt particles and organisms. Pre-filtration
(through glass-fibre paper prefilter) reduces the risks of blockage of the final filter.
Examples of membrane filters:
MF-Millipore – it is a mixture of cellulose esters

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Sintered (or fritted) glass filters
Borosilicate glass is finely powdered in a ball-mill and the particles of required size are
separated. This is packed into disc mounted and heated till the particles get fused. The discs thus
made are used for filtration. As these are made of glass and hence do not absorb liquids during
filtration. The disadvantage is that they are very brittle and break easily
They are cleaned with the help of sulfuric acid.
Seitz filter:
These are made of asbestos or other material. They are pad like and thicker than membrane
filters. They do not rupture during filtration. But the solution might get absorbed by the filter pad
itself

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Equipment:

● Sample to be filtered
● Sterile Syringe 5cc
● Syringe Filters 0.22µ
● Falcon tubes 15ml and 50ml

Procedure:

1. Syringe filters are available in a variety of pore sizes, with the most common pore
sizes being 0.22 microns that will exclude most microbial contaminants
2. Take sample in a sterile syringe.
3. Open the disposable syringe filter.
4. Attach syringe to the filter carefully.
5. Collect the filtrate in a sterile falcon tube.

Advantages of sterilization by filtration:

1. Thermolabile solutions can be sterilized.
2. It removes all the living microorganisms.
Disadvantages of sterilization by filtration:
1. Filters may break down suddenly or gradually on use.
2. Sterility testing is obligatory on the filtered solution.
3. Filter media may be absorbed on the filter surface.
4. Viruses are not removed by filtration.
5. Suspensions and oils cannot be sterilized by this method due to their heavy load of
particulate matters and viscosity.

27
 Practical No: 7
Types of media and culturing

Introduction
All micro-organisms require water, sources of energy, carbon, nitrogen, mineral element and
vitamin plus oxygen in their growth medium. On a small scale, it is simple to device a medium
containing pure compounds, but the resulting medium although satisfy the growth, may be
unsuitable for use in a large scale process.
The essential part of media designing is to be considering the stoichiometry of growth and
formation of desired product which is given below:
Carbon and energy source + Nitrogen source + O2 + Other requirements ----> Biomass +
Products + CO2 + H2O+ Heat Essential characteristics of formulated media
1. It should produce the maximum yield, maximal concentration with maximal rate of production
of desired biomass, of consistent quality which is always readily available.
2. It should also have minimal problems during media designing.
3. It should have minimal problems during recovery of desired product/ biomass especially
during aeration and agitation, extraction, purification and waste treatment.
Basic media constituents
1. Water
It is major component of all fermentation media. Following factors need to be considered about
water for media preparation
● pH
● Dissolve salt
● Effluent contamination
Mineral water content of water is very important in brewing, and most critical in the mashing
process, and historically influenced the siting of breweries and the type of beer produced.
2. Energy Sources
Energy for growth comes from either the oxidation of medium components or from light. Most
industrial micro-organisms are chemo-organotrophs, therefore the commonest sources of energy
will be the carbon source such as carbohydrates, lipids and proteins.
3. Carbon sources

28
Carbon requirement for the medium is normally provided by:
● Sucrose: Sugarcane, sugar beet molasses
● Glucose: Corn sugar, starch, cellulose
● Lactose: Milk whey
● Fats: Vegetable oil
● Starch: Maize grains, cereals, potatoes and cassava
● Hydrocarbons: Petroleum fractions
4. Nitrogen Sources
Microorganisms generally can use inorganic or organic N.
● Inorganic sources: ammonia, ammonium salts –
● Organic sources: amino acid, proteins and urea, Corn steep liquor, Yeast extract,
Peptones, Soya bean meal.
5. Oxygen sources
Oxygen is always provided in water.
Some organisms require molecular oxygen as terminal oxidizing agents to fulfill their energetic
needs through aerobic respiration. These organisms are obligatorily aerobic.
For obligate anaerobes molecular 02, is a toxic substance.
Some organisms are facultative anaerobes and can grow with or without molecular 02.
Types of media and culturing
Culture media contains nutrients and physical growth parameters necessary for microbial growth.
All microorganisms cannot grow in a single culture medium and in fact many can’t grow in any
known culture medium.
Organisms that cannot grow in artificial culture medium are known as obligate
parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate
parasites. Bacterial culture media can be distinguished on the basis of composition, consistency
and purpose.
Classification of culture media used in Microbiology laboratory on the basis of consistency
1. Solid medium
Solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert
solidifying agent. Solid medium has physical structure and allows bacteria to grow in

29
 physically informative or useful ways (e.g. as colonies or in streaks). Solid medium is useful
for isolating bacteria or for determining the colony characteristics of the isolate.
2. Semisolid media
They 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.
3. Liquid (Broth) medium
These media contains specific amounts of nutrients but don’t have trace of gelling agents
such as gelatin or agar. Broth medium serves various purposes such as propagation of large
number of organisms, fermentation studies, and various other tests. e.g. sugar fermentation
tests, MR-VR broth
Classification of culture media based on the basis of composition
1. Synthetic or chemically defined medium
A chemically defined medium is one prepared from purified ingredients and therefore whose
exact composition is known.
2. Non synthetic or chemically undefined medium
Non-synthetic medium contains at least one component that is neither purified nor completely
characterized nor even completely consistent from batch to batch. Often these are partially
digested proteins from various organism sources. Nutrient broth, for example, is derived from
cultures of yeasts.
Synthetic medium may be simple or complex depending up on the supplement incorporated in it.
A simple non-synthetic medium is capable of meeting the nutrient requirements of organisms
requiring relatively few growth factors whereas complex non-synthetic medium support the
growth of more fastidious microorganisms.
Classification of Bacterial Culture Media based on the basis of purpose/ functional use/
application
Many special purpose media are needed to facilitate recognition, enumeration, and isolation of
certain types of bacteria. To meet these needs, numerous media are available.
1. General purpose media/ Basic media

30
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone
water, nutrient broth and nutrient agar are considered as basal medium. These media are
generally used for the primary isolation of microorganisms.

Nutrient Agar
2. Enriched medium (Added growth factors):
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes
them enriched media. Enriched media are used to grow nutritionally exacting (fastidious)
bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media.
Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate
agar is also known as heated blood agar or lysed blood agar.

Blood Agar

31
3. Selective and enrichment media are designed to inhibit unwanted commensal or
contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective
media are agar based, enrichment media are liquid in consistency. Both these media serve the
same purpose. Any agar media can be made selective by addition of certain inhibitory agents that
don’t affect the pathogen of interest. Various approaches to make a medium selective
include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these.
a. Selective medium
Principle: Differential growth suppression
Selective medium is designed to suppress the growth of some microorganisms while allowing
the growth of others. Selective medium are agar based (solid) medium so that individual colonies
may be isolated.
Examples of selective media include:
1. Mannitol Salt Agar and Salt Milk Agar used to recover [Link] contains 10% NaCl.

Mannitol Salt Agar grows halophilic (salt-loving) bacteria. Pathogenic Staph is growing
on left (yellow side) and normal flora Staph growing on right (pink).
2. MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits
most gram positive bacteria.

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4. Differential/ indicator medium: differential appearance:
Certain media are designed in such a way that different bacteria can be recognized on the basis
of their colony colour. Various approaches include incorporation of dyes, metabolic substrates
etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media
are called differential media or indicator media. Differential media allow the growth of more
than one microorganism of interest but with morphologically distinguishable colonies.
Examples of differential media include:

Mannitol salts agar (mannitol fermentation = yellow)

Mac Conkey agar (lactose fermenters, pink colonies (E. coli) whereas non- lactose fermenter
produces pale or colorless colonies.

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 Practical No. 8
Culturing of bacteria in liquid medium

Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. However,
a liquid culture is capable of supporting a higher density of bacteria and is used to grow up
sufficient numbers of bacteria necessary for an experimental use. LB is the standard medium
used to grow bacteria, principally Escherichia Coli. Also known as Luria broth, or Luria-Bertani
medium or Lennox broth. Bertani was the inventor of LB and was a student with Max Delbruck
at Caltech. They were investigating phage lysis of E. coli. The following protocol is for
inoculating an overnight culture of liquid LB with bacteria.
Protocol:
1. Prepare liquid LB. For example, to make 1 liter of LB, weigh out the following into a
500mL glass bottle:
● 10g NaCl
● 10g Tryptone
● 5g Yeast Extract
● and dH2O to 1 liter
Note: If your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other
dry ingredients above.
1. Loosely close the cap on the bottle (do NOT close all the way or the bottle may explode!)
and then loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to
cool to room temperature. Now screw on the top of the bottle and store the LB at room
temperature.
2. When ready to grow your culture, add liquid LB to a tube or flask and add the appropriate
antibiotic to the correct concentration.
Note: If you intend to do a mini-prep you will usually want to start 2mL in a falcon tube, but for
larger preps you might want to use as much as a liter of LB in a 2L Erlenmeyer flask.
3. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
4. Drop the tip or toothpick into the liquid LB + antibiotic.
5. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
6. Incubate bacterial culture at 37°C for 12-18hr in a shaking incubator.

34
7. After incubation, check for growth, which is characterized by a cloudy haze in the media.
Note: Some protocols require bacteria to be in the log phase of growth. Check the instructions
for your specific protocol and conduct an OD600 to measure the density of your culture if needed.
Note: A good negative control is LB media + antibiotic without any bacteria inoculated. You
should see no growth in this culture after overnight incubation.
8. (Optional) For long term storage of the bacteria, you can proceed with Creating a Glycerol
Stock.

Antibiotic Concentrations
Commonly Used Antibiotics Recommended Concentration

Ampicillin 100 µg/mL

Bleocin 5 µg/mL

Carbenicillin 100 µg/mL

Chloramphenicol 25 µg/mL

Coumermycin 25 µg/mL

Gentamycin 10 µg/mL

Kanamycin 50 µg/mL

Spectinomycin 50 µg/mL

Tetracycline 10 µg/mL

35
 Practical No. 9
Culturing of bacteria on solid medium

Organisms can be grown in liquid media (broth) or on solid media. For example, Nutrient media
is referred to as Nutrient Broth when in the liquid form, and Nutrient Agar when in the solid
form. Agar, a galactan obtained from marine algae is used as the hardening agent in solid media.
Broth, with added agar powder, is heated to 121 oC in an autoclave, dissolving the agar and
sterilizing the medium. The molten medium may be poured into plates or tubes. The agar-media
will remain liquid at temperatures above 45oC. Below 45oC the agar will harden, and supply a
solid surface for the growth of bacteria. Agar plates and slants may be inoculated after they have
solidified. To avoid any condensation-droplets from falling onto agar surfaces and smearing the
inoculum, agar plates are inverted during incubation.
Batch makes about X plates
The below mentioned values is for 1L media preparation and at least 40 plates, you can minimize
it into what would you need.
Making the LB Agar
1. Add 250 mL of dH2O to a graduated cylinder.
2. Weigh out 8.50g of pre-mix LB Agar powder or:
5.0 g tryptone
2.5 g yeast extract
5.0 g NaCl
3.5 g agar
3. Mix powder well to bring into solution
4. Add dH2O to total volume of 500 mL and transfer to 1 L flask
5. Put on stirring hot plate and heat to boil for 1 min while stirring.
6. Transfer to 1 L pyrex jar and label with autoclave tape.
7. Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
8. Let agar cool to ~55oC (you should be able to pick up the jar without a glove)

36
Pouring the Plates
1. Make sure bench top has wiped down with bleach/EtOH.
2. Remove sterile Petri dishes from plastic bag (save the bag for storage).
3. Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not lift the cover
off excessively (you should be able to just open up enough to pour).
4. Swirl plate in a circular motion to distribute agar on bottom completely.
5. Let each plate cool until its solid (~20 minutes) then flip so as to avoid condensation on the
agar.
6. Store plates in plastic bags in fridge with: name, date and contents.
Streak Plate Method
For economy of materials and time, this method is best. It requires a certain amount of skill,
however, which is forthcoming with experience. A properly executed streak plate will give as
good isolation as is desired for most work. The important thing is to produce good spacing
between colonies.
Materials:
Electric hot plate (or tripod and wire gauze), Bunsen burner and beaker of water
Wire loop, thermometer, and china marking Pencil, LB agar pour and sterile Petri plate,
One mixed culture of Serratia marcescens, Escherichia coli, and Micrococcus luteus (or
Chromobacterium violaceum) etc etc.
1. Prepare your tabletop by disinfecting its surface with the disinfectant that is available in the
laboratory. Use a sponge to scrub it clean.
2. Label the bottom surface of a sterile Petri plate with your name and date. Use china marking
pencil.
3. Liquefy a tube of nutrient agar, cool to 50° C, and pour the medium into the bottom of the
plate. Be sure to flame the neck of the tube prior to pouring to destroy any bacteria around the
end of the tube. After pouring the medium into the plate, gently rotate the plate so that it
becomes evenly distributed, but do not splash any medium up over the sides.
Agar-agar, the solidifying agent in this medium becomes liquid when boiled and resolidifies at
around 42° C. Failure to cool it prior to pouring into the plate will result in condensation of
moisture on the cover. Any moisture on the cover is undesirable because if it drops down on the

37
colonies, the organisms of one colony can spread to other colonies, defeating the entire isolation
technique.
4. Streak the plate by one of the methods, see below figure.
5. Incubate the plate in an inverted position at 25° C for 24–48 hours. By incubating plates
upside down, the problem of moisture on the cover is minimized.

38
39
40
 Practical No. 10
Colony and cell morphology

Colony Morphology of Bacteria

Bacteria grow on solid media as colonies. A colony is defined as a visible mass of
microorganisms all originating from a single mother cell. Key features of these bacterial colonies
serve as important criteria for their identification.

41
Colony morphology can sometimes be useful in bacterial identification. Colonies are described
as to such properties as size, shape, texture, elevation, pigmentation, effect on growth
medium. In this blog post you will find common criteria that are used to characterize the
bacterial growth.
Colony Shape: It includes form, elevation and margin of the bacterial colony.
1. Form of the bacterial colony: – The form refers to the shape of the colony. These forms
represent the most common colony shapes you are likely to encounter. e.g. Circular,
Irregular, Filamentous, Rhizoid etc.
2. Elevation of bacterial colony: This describes the “side view” of a colony. These are the
most common. e.g. Flat, raised, umbonate (having a knobby protuberance), Crateriform,
Convex, Pulvinate (Cushion-shaped)
3. Margin of bacterial colony: The margin or edge of a colony may be an important
characteristic in identifying an organisms. Common examples are Entire (smooth),
irregular, Undulate (wavy), Lobate, Curled, Filiform etc.

42
 Colonies that are irregular in shape and/or have irregular margins are likely to be motile
organisms. Highly motile organism swarmed over the culture media. Such as Proteus spp.
4. Size of the bacterial colony: The size of the colony can be a useful characteristic for
identification. The diameter of a representative colony may be measured in millimeters or
described in relative terms such as pin point, small, medium, large. Colonies larger than
about 5 mm are likely to be motile organisms.
Appearance of the colony surface: Bacterial colonies are frequently shiny and smooth in
appearance. Other surface descriptions might be: dull (opposite of glistening), veined, rough,
wrinkled (or shriveled), glistening.

Mixed growth of mucoid Lactose fermenting colonies and NLF colonies in MacConkey Agar
Consistency/Texture: Several terms that may be appropriate for describing the texture or
consistency of bacterial growth are: dry, moist, viscid (sticks to loop, hard to get off),
brittle/friable (dry, breaks apart), mucoid (sticky, mucus-like)
Color of the colonies (pigmentation): Some bacteria produce pigment when they grow in the
medium e.g., green pigment produces by Pseudomonas aeruginosa, buff colored colonies
of Mycobacterium tuberculosis in L.J medium, red colored colonies of Serratia marcescens.
Opacity of the bacterial colony: Is the colony transparent (clear), opaque (not transparent or
clear), translucent (almost clear, but distorted vision–like looking through frosted glass),
iridescent (changing colors in reflected light).
Cell morphology of bacteria

43
Bacteria and Archaea are classified by direct examination with the light microscope according to
their morphology and arrangement.
The basic morphologies are spheres (coccus) and round-ended cylinders (bacillus). But there
may be others such as helically twisted cylinders (spirochetes), cylinders curved in one plane
(selenomonads) and unusual morphologies (such as the square, flat box-shaped cells of the
archaean genus Haloquadratum). Arrangements include pairs, tetrads, clusters, chains and
palisades.

Colony morphology of Yeasts

Yeast, a type of fungi (plural for fungus), is found in many places from nature, to research labs
and even everyday kitchens for baking. Yeast colonies generally look similar to bacterial
colonies. Some species, such as Candida, can grow as white patches with a glossy surface.

44
 Candida Albicans
Colony morphology of Molds
Molds are actually fungi, and they often appear whitish grey, with fuzzy edges. They usually turn
into a different color, from the center outwards. Two examples of molds are shown below:

Green Mold (Trichoderma harzianum) Black Mold (Aspergillus nidulaus)

Practical No. 11
Bacterial cell count

45
A viable cell count allows one to identify the number of actively growing/dividing cells in a
sample. The plate count method or spread plate relies on bacteria growing a colony on a nutrient
medium. The colony becomes visible to the naked eye and the number of colonies on a plate can
be counted. Colony-forming unit (CFU or cfu) is a measure of viable bacterial. CFU measures
only viable cells. For convenience the results are given as CFU/mL (colony-forming units per
milliliter) for liquids and CFU/g (colony-forming units per gram) for solids.
To quantify the number of cells in a culture, the cells can be simply plated on a petri dish with
growth medium. If the cells are efficiently distributed on the plate, it can be generally assumed
that each cell will give rise to a single colony or Colony Forming Unit (CFU). The colonies can
then be counted, and based on the known volume of culture that was spread on the plate; the cell
concentration can be calculated. As is with counting chambers, cultures usually need to be
heavily diluted prior to plating; otherwise, instead of obtaining single colonies that can be
counted, a so-called "lawn" will form: thousands of colonies lying over each other. Additionally,
plating is the slowest method of all: most microorganisms need at least 12 hours to form visible
colonies.
Although this method can be time consuming, it gives an accurate estimate of the number of
viable cells (because only they will be able to grow and form visible colonies). It is therefore
extensively used in experiments aiming to quantify the number of cells resisting drugs or other
external conditions. In addition, the enumeration of colonies on agar plates can be greatly
facilitated by using colony counters.
The materials needed to perform a plate count are:
⮚ Sterile 0.9% NaCl (sterile saline)
⮚ Sterile tubes, tips and spreaders
⮚ Agar plates (three per sample)
Procedure:
● Make a ten-fold dilution serial dilution of your bacterial culture (broth). Dilute the
suspension to a dilution factor of 10-6 (a million-fold dilution).
● Spread out aliquots using a sterile bacterial spreader (0.1 ml) of each dilution onto 3 agar
plates.
● Incubate the plates at 37oC for 24 hours.

46
 ● Count the number of bacterial colonies that appear on each of the plates that has between
30 and 200 colonies.
● Any plate which has more than 200 colonies is designated as "too numerous to count”.
Plates with fewer than 30 colonies do not have enough individuals to be statistically
acceptable.
● To compute the estimated number of bacteria on the surface that you tested, use the
following formula:
B = N/d
where: B = number of bacteria; N = average number of colonies counted on three plates; d =
dilution factor. Example: Plate 1: 56 CFU; Plate 2: 75 CFU; Plate 3: 63 CFU; Average (N) =
64.7; Dilution (d ) = 1/1,000; B = (64.7x1,000) = 64,700 bacteria in 0.1 ml, 647,000 bacteria per
ml.

Practical No. 12
Gram staining of bacteria

47
A technique that is used to define and examine different types of microbes
Types of staining techniques
1. Simple stains techniques
Simple staining is performed with basic dyes such as crystal violet or methylene blue.
2. Differential stains techniques
Differential stain technique distinguishes two kinds of organisms. There are two types of this
technique; one is Gram stain technique while other is acid fast technique.
Gram stain technique
A most commonly used technique for study of microbes like bacteria.
Principle
This technique separates bacteria into two groups, Gram‐positive bacteria and Gram‐negative
bacteria.
Materials:
⮚ Clean glass slides
⮚ Inoculating loop
⮚ Bunsen burner
⮚ Bibulous paper
⮚ Microscope
⮚ Lens paper and lens cleaner
⮚ Immersion oil
⮚ Distilled water
⮚ 18 to 24 hour cultures of organisms
Reagents:
1. Primary Stain - Crystal Violet
2. Mordant - Grams Iodine
3. Decolourizer - Ethyl Alcohol
4. Secondary Stain - Safranin

Procedure:
1. Smear preparation

48
A small sample of microorganisms is placed on a slide and permitted to air dry. The smear is
heat fixed by quickly passing it over a flame. Heat fixing kills the organisms, makes them adhere
to the slide, and permits them to accept.
2. Primary stain
Apply crystal violet as primary stain
3. Mordant
Use iodine as a mordant
4. Decolourizer
Wash with ethyl alcohol that acts as a decolourizing agent. Gram‐positive bacteria retain the
crystal‐violet iodine stain; however, the Gram‐negative bacteria lose the stain.
5. Secondary stain
The Gram‐negative bacteria subsequently stain with the Safranin dye, the counterstain. These
bacteria appear red under the oil‐immersion lens, while Gram‐positive bacteria appear blue or
purple, reflecting the crystal violet retained during the washing step.

49
The Gram staining procedure used for differentiating bacteria into two groups

50
 Practical No. 13
Testing sensitivity to antimicrobial substances

The agar diffusion method is widely used in industry for testing the sensitivity of micro-
organisms to antibiotics, antiseptics, toothpaste, mouthwashes, disinfectants, etc. The method
involves preparing a pour or spread plate of a test micro-organism, adding small amount of test
substance to either a well cut in the agar medium or (preferably) a paper disc which is then
placed on the agar surface. After incubation, an inhibitory effect on the test organism is indicated
by a clear zone (no growth) around the test substance; microbial growth is visible to the naked
eye in areas of the plate that are unaffected. This is a straightforward activity that tests several
practical skills and is relevant to other aspects of biology and to everyday life. In addition to
using laboratory reagents, e.g. stains, and antibiotic discs, many preparations with antimicrobial
activity are readily available in pharmacists and supermarkets. There is also the opportunity to
think of less obvious materials, e.g. plants and their products.
Materials
● Take a nutrient agar pour or spread/lawn plate of e.g. Bacillus subtilis, Micrococcus luteus,
Escherichia coli or Saccharomyces cerevisiae on malt agar.
● Sterile filter paper discs
● Sterile distilled/demineralised water (control)
● Samples to be tested, 3 (e.g. mouthwashes, selected for a range of active ingredients)
● Bunsen burner
● Forceps
● Alcohol (70 % IDA) in a small beaker covered in foil (Caution: flammable, should be kept
covered away from flames)
● Incubator at 25–30 °C (if available)
Procedure
Aseptic technique should be used throughout.
1. Mark and label four sections on the base of the Petri dish, for the three different samples and
control (sterile water).

51
2. Using sterile forceps (flamed with alcohol and cooled) remove one filter paper disc. Dip into
the first test sample, drain on the side of the container and place firmly onto the appropriate
section of the seeded agar plate.
3. Wash the forceps free of the sample.
4. Repeat for the remaining samples and the control (sterile water). Remember to rinse and
sterilise the forceps between each sample and to open the plate for the minimum possible
time.
5. Seal the lid to the base with tape.
Incubation of the plate
6. Invert the plate and incubate at 25–30 °C or at room temperature for 48 hours.
7. Examine the plate (without opening). Measure and record the size of any zones of inhibition
around the filter paper discs. Consider what factors might be affecting the size of the zones of
inhibition.

Fig 13.1: Antibiotic test

52
 Practical No. 14
Biochemical tests

Introduction
Biochemical tests are among the most important methods for microbial identification. Routine
biochemical tests include tests for carbohydrate fermentation, IMViC test, urease, oxidase and
catalase test.
1. Carbohydrate fermentation tests:
The carbohydrate fermentation test is used to determine whether or not bacteria can ferment a
specific carbohydrate. Carbohydrate fermentation patterns are useful in differentiating among
bacterial groups or species. The term fermentation is often used to describe the breaking down or
catabolism of a carbohydrate under anaerobic conditions. Therefore, bacteria capable of
fermenting a carbohydrate are usually facultative anaerobes. It tests for the presence of acid
and/or gas produced from carbohydrate fermentation. Basal medium containing a single
carbohydrate source such as Glucose, Lactose, Sucrose or any other carbohydrate is used for this
purpose. A pH indicator (such as Andrade’s solution, Bromcresol purple (BCP), Bromothymol
blue (BTB) or Phenol red) is also present in the medium; which will detect the lowering of the
pH of the medium due to acid production. Small inverted tubes called Durham tube is also
immersed in the medium to test for the production of the gas (hydrogen or carbondioxide).
Media
Carbohydrate broth medium
Peptone 10
Sodium chloride 5 gram
Phenol red/bromocresol purple 0.016 gram
Carbohydrate 10 gram
Distilled water 1000 ml
Procedure
1. Dissolve the media ingredients to distilled water and adjust the pH to 6.8 in case of
bromocresol purple or 7.4 if using phenol red.
2. Distribute the media in small test tubes and insert Durham tubes in inverted position to detect
gas production.

53
3. Place cotton plugs to tubes and autoclave them.
4. Alternatively, 90 ml of the basal medium (medium without the carbohydrate) is sterilized in
the autoclave and to this, after cooling, 10 ml of the carbohydrate solution (10%) sterilized
by membrane filtration is added. This is particularly essential for heat-sensitive
carbohydrates, which undergo degradation during heat sterilization.
5. After autoclaving, inoculate each tube with 18-24 h old culture of test microorganism.
Perform the test in duplicate or triplicate and incubate at 37ºC for 24 h. Unioculated media is
used as negative control.
6. Next day, check tubes for color change or gas production.
Acid production:
a. Positive: After incubation the liquid in the tube turns yellow (indicated by the change in the
color of the phenol red indicator). It indicates that there is drop in the pH because of the
production of the acid by the fermentation of the carbohydrate (sugar) present in the media.
NOTE:*If you are using other pH indicators please check corresponding colors in particular
pH.
b. Negative: The tube containing medium will remain red, indicating the bacteria cannot
ferment that particular carbohydrate source present in the media.
Gas Production
c. Positive: A bubble (small or big depending up the amount of gas produced) will be seen in
the inverted Durham tube.
d. Negative: There won’t be any bubble in the inverted Durham tube i.e. bacteria does not
produce gas from the fermentation of that particular carbohydrate present in the media i.e.
anaerogenic organism.
Interpretation:
● Colour of broth changes to yellow and gas accumulates in Durham tube: Fermentative for the
carbohydrate and acrogenic.
● Colour of broth changes to yellow, but no gas accumulates in Durham tube: Fermentative for
the carbohydrate and anaerogenic.
● Colour of broth does not change: Non-fermentative for the carbohydrate.

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 2. IMViC test
The IMViC tests are useful for differentiating the Enterobacteriaceae, especially when used
alongside the urease test. When used alone, the IMViC tests are particularly useful for
differentiating Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella
pneumoniae. Each of the letters in “IMViC” stands for one of these tests. “I” is for indole; “M” is
for methyl red; “V” is for Voges-Proskauer, and “C” is for citrate, lowercase “i” is added for the
ease of pronunciation. IMViC is an acronym that stands for four different tests
● Indole test
● Methyl red test
● Voges-Proskauer test
● Citrate utilization test
a. Indole test
Indole test is used to determine the ability of an organism to split amino acid tryptophan to form
the compound indole. Tryptophan is hydrolysed by tryptophanase to produce three possible end
products – one of which is indole. Indole production is detected by Kovac’s or Ehrlich’s reagent
which contains 4 (p)-dimethylamino benzaldehyde, this reacts with indole to produce a red

55
coloured compound. Indole test is a commonly used biochemical test (e.g. in IMVIC test, SIM
test etc). Indole test helps to differentiate Enterobacteriaceae and other genera.
Tryptophan medium Composition
Ingredients Gms / Litre
Meat peptone/ Casein enzymic hydrolysate 10.000
Sodium chloride 5.000
DL-Tryptophan 1.000
Final pH (at 25°C) 7.2±0.2
Procedure
1. Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test
organism in tryptophan broth.
2. Incubate at 37°C for 24-28 hours in ambient air.
3. After incubation, add 0.5 ml of Kovac’s reagent to the broth culture.

Expected results:
● Positive: Pink colored rink after addition of appropriate reagent
● Negative: No color change even after the addition of appropriate reagent.
e.g. Klebsiella pneumoniae

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b. Methyl Red / Voges-Proskauer (MR/VP)
This test is used to determine which fermentation pathway is used to utilize glucose. In the
mixed acid fermentation pathway, glucose is fermented and produces several organic acids
(lactic, acetic, succinic, and formic acids). The stable production of enough acid to overcome the
phosphate buffer will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an
aliquot of the culture broth and the pH is below 4.4, a red color will appear. If the MR turns
yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been utilized. The
2,3 butanediol fermentation pathway will ferment glucose and produce a 2,3 butanediol end
product instead of organic acids. In order to test this pathway, an aliquot of the MR/VP culture is
removed and a-naphthol and KOH are added. They are shaken together vigorously and set aside
for about one hour until the results can be read. The Voges-Proskauer test detects the presence of
acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-
red to pink”. If the culture is negative for acetoin, it will turn “brownish-green to yellow”. Note:
A culture will usually only be positive for one pathway: either MR+ or VP+. Escherichia coli is
MR+ and VP-. In contrast, Enterobacter aerogenes and Klebsiella pneumoniae are MR- and
VP+. Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR- and VP-.
Media and Reagents
Methyl red-Voges-Proskauer (MR/VP) broth (formulated by Clark and Lubs) is used in this test.
The composition of MR/VP broth is as follows:
Ingredient MR/VP broth (g/L)
Polypeptone 7g
Glucose 5g
Dipotassium phosphate 5g
Distilled water 1L
Final pH 6.9
Methyl Red pH Indicator
● Methyl red: 0.1 g in 300 mL of 95% ethyl alcohol.
● Distilled water: 200 mL
Procedure for Methyl Red (MR) Test
MR-VP broth is used for both MR Test and VP test. Only the addition of reagent differs, and
both tests are carried out consecutively.

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 1. Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms
under investigation.
2. Incubate at 35 °C for up to 4 days.
3. Add about 5 drops of the methyl red indicator solution to the first tube (for Voges-
Proskauer test, Barrit’s reagent is added to another tube).
4. A positive reaction is indicated, if the colour of the medium changes to red within a few
minutes.

c. Citrate test
Citrate utilization test is commonly employed as part of a group of tests, the IMViC (Indole,
Methyl Red, VP and Citrate) tests, that distinguish between members of the Enterobacteriaceae
family based on their metabolic by-products. Citrate utilization test is used to determine the
ability of bacteria to utilize sodium citrate as its only carbon source and inorganic ammonium
dihydrogen phosphate (NH4H2PO4) is the sole fixed nitrogen source.
Principle of citrate utilization test
When an organic acid such as citrate (remember Krebs cycle) is used as a carbon and energy
source, alkaline carbonates and bicarbonates are produced ultimately. In addition, ammonium
hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen
source. Utilization of exogenous citrate requires the presence of citrate transport proteins

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(permeases). Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and
acetate. The oxaloacetate is then metabolized to pyruvate and CO2.
Citrate = oxaloacetate + acetate
oxalacetate = pyruvate + CO2

Further metabolic breakdown is dependent upon the pH of the medium.

A. Under alkaline conditions, pyruvate is metabolized to acetate and formate.
pyruvate = acetate + formate
B. At pH 7.0 and below, lactate and acetoin are also produced.
pyruvate = acetate + lactate + CO2
pyruvate = acetoin + CO2

The carbon dioxide that is released will subsequently react with water and the sodium ion in the
medium to produce sodium carbonate, an alkaline compound that will raise the pH. In addition,
ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole
nitrogen source.
Growth usually results in the bromothymol blue indicator, turning from green to blue. The
bromothymol blue pH indicator is a deep forest green at neutral pH. With an increase in medium
pH to above 7.6, bromothymol blue changes to blue

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Citric acid media composition
Ingredient Simmons Citrate Agar (g/L)
Ammonium dihydrogen phosphate 1g
Dipotassium phosphate 1g
Sodium chloride 5g
Sodium citrate 2g
Magnesium sulfate 0.20 g
Agar 15 g
Bromothymol blue 0.08 g
Distilled water 1L
Final pH 6.9
Procedure of citrate utilization test:
1. Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a
colony that is 18 to 24 hours old.
2. Incubate at 35°C to 37°C for 18 to 24 hours. Some organisms may require up to 7 days of
incubation due to their limited rate of growth on citrate medium.
3. Observe the development of blue color; denoting alkalinization.
Expected results in citrate utilization test:
Citrate positive: growth will be visible on the slant surface and the medium will be an intense
Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate
catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change
from the original green color to blue .
Citrate negative: trace or no growth will be visible. No color change will occur; the medium
will remain the deep forest green color of the uninoculated agar. Only bacteria that can utilize
citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium,
thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant

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3. Catalase test
Introduction
Catalase is the enzyme that breaks hydrogen peroxide (H 2O2) into H2O and O2. Hydrogen
peroxide is often used as a topical disinfectant in wounds, and the bubbling that is seen is due to
the evolution of O2 gas. H2O2 is a potent oxidizing agent that can wreak havoc in a cell; because
of this, any cell that uses O2 or can live in the presence of O 2 must have a way to get rid of the
peroxide. One of those ways is to make catalase. The catalase reaction is used in the
identification of gram positive cocci (differentiate between Streptococcus and Staphylococcus
species) and some gram positive bacilli.
Reagent
Catalase Reagent (Hydrogen peroxide 3%)
Procedure
1. Place a small amount of growth from your culture on to a clean microscope slide. If using
colonies from a blood agar plate, be very careful not to scrape up any of the blood agar—blood
cells are catalase positive and any contaminating agar could give a false positive.
2. Add a drop of Catalase Reagent onto the smear.
3. A positive result is the rapid evolution of O2 as evidenced by bubbling.

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4. A negative result is no bubbles or only a few scattered bubbles.
5. Dispose of your slide in the biohazard glass disposal container.

4. Oxidase test
The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the
bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the
reagent (tetramethyl-p-phenylenediamine) to (indophenols) purple color end product. When the
enzyme is not present, the reagent remains reduced and is colorless.

All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron
acceptor in respiration. This does NOT mean that they are strict aerobes. Bacteria that are
oxidase-negative may be anaerobic, aerobic, or facultative; the oxidase negative result just means

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that these organisms do not have the cytochrome c oxidase that oxidizes the test reagent. They
may respire using other oxidases in electron transport.
Oxidase test is most helpful in screening colonies suspected of being one of
the Enterobacteriaceae (all negative) and in identifying colonies suspected of belonging to other
genera such as Aeromonas, Pseudomonas, Neisseria, Campylobacter, and Pasteurella (positive).
Test requirements for Oxidase test: Moist filter paper with the substrate (1% tetramethyl-p-
phenylenediamine dihydrochloride), or commercially prepared paper disk, wooden wire or
platinum wire.
Procedure of Oxidase test:
1. Take a filter paper soaked with the substrate tetramethyl-p-phenylenediamine
dihydrochloride
2. Moisten the paper with a sterile distilled water
3. Pick the colony to be tested with wooden or platinum loop and smear in the filter paper
4. Observe inoculated area of paper for a color change to deep blue or purple within 10-30
seconds
Expected results of Oxidase test
Positive: Development of dark purple color (indophenols) within 10 seconds
Negative: Absence of color

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5. Urease test
Urea is a diamide of carbonic acid. It is hydrolyzed with the release of ammonia and carbon
dioxide. Many organisms especially those that infect the urinary tract, have an urease enzyme
which is able to split urea in the presence of water to release ammonia and carbon dioxide.
The ammonia combines with carbon dioxide and water to form ammonium carbonate which
turns the medium alkaline, turning the indicator phenol red from its original orange yellow color
to bright pink.

Composition of Christensen’s Urea Agar

Ingredients Grams/liter
Peptone 1g
Dextrose 1g
Sodium chloride 5g
Potassium phosphate, monobasic 2g
Urea 20 g
Phenol red 0.012 g
Agar 15 to 20 g
Preparation of Christensen’s Urea Agar
1. Dissolve the first six ingredients in 100 ml of distilled water and filter sterilize (0.45 mm
pore size).
2. Suspend the agar in 900 ml of distilled water, boil to dissolve completely, and autoclave at
121°C and 15 psi for 15 minutes
3. Cool the agar to 50-55 °C
4. Aseptically add 100 ml of filter-sterilized urea based to the cooled agar solution and mix
thoroughly
5. Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes during cooling until
solidified (It is desirable to have a long slant and short butt).

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Procedure for urease test
1. Streak the entire slant surface with a heavy inoculum from an 18-24 hour pure culture (Do
not stab the butt as it will serve as a color control).
2. Incubate tubes with loosened caps at 35°C.
3. Observe the slant for a color change at 6 hours and 24 hours unless specified for longer
incubation.
Result and Interpretation
Organisms that hydrolyze urea rapidly (Proteus spp., Morganella morganii, and
some Providencia stuartii strains) will produce strong positive reactions within 1 or 6 hours of
incubation; delayed positive organisms (e.g. Klebsiella spp and Enterobacter species ) will
produce weak positive reactions in the slant in 6 hours of incubation which will be intense during
further incubation. The culture medium will remain a yellowish color if the organism is urease
negative e.g. Escherichia coli.
In routine diagnostic laboratories the urease test result is read within 24 hours.
▪ If organism produces urease enzyme, the color of the slant changes from light orange to
magenta.
▪ If organism do not produce urease the agar slant and butt remain light orange (medium
retains original color).

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 Practical No. 15
Growth Kinetics

Objectives: To establish growth curves for an unknown bacterial species and observe the
different phases of growth.
Introduction:
Bacteria normally reproduce by binary fission, forming two equal-sized progeny cells and thus
doubling their number with each division. This type of cell division is called exponential; i.e.,
one cell divides to form two, each of these cells divides so that four cells are obtained, and so
forth in a geometric progression. The unit of microbial growth is "generation time", which is the
time required to achieve a doubling of the population size and is designated as tgen. Generation
time can be estimated by determining cell numbers during the period of active cell division and
is expressed mathematically as:
tgen = t ln 2/ ln Xt − ln X0= t log 2 /log Xt − log X0
In the above equations, t is the elapsed time during which growth is measured, and Xo and Xt are
the number of bacteria at times zero and t, respectively. The presence of 2 in the numerator of the
equations is due to the fact that when doubling of the population number occurs, the quantity
Xt/Xo (called the doubling constant) will be equal to 2. The generation time of many bacteria is
usually several hours. However, under optimum conditions, E. coli has a generation time of
about 20 minutes. Thus it can be calculated that a single E. coli cell will produce about a
thousand progeny in 3.3 hours and over a million in 6.6 hours. If the generation time and the
elapsed time are known, then the number of generations can be estimated as follows:
Number of generations = t/ tgen

A bacterial population follows a characteristic growth curve which has four phases: the lag
phase, the log or exponential growth phase, the stationary phase, and the death phase.
1. Lag phase: During this phase, there is no increase in cell number; rather, bacteria are
preparing for reproduction and synthesizing DNA and various inducible enzymes needed
for cell division.

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 2. Log phase: This phase, also called the exponential phase, follows the lag phase and starts
with a rapid increase in bacterial number. At this stage, the logarithm of bacterial biomass
increases linearly with time so that numbers of bacterial cells in a given interval of time is
proportional to the biomass of bacteria present.
3. Stationary phase: The number of bacteria reaches a maximum in this phase and does not
increase further (the growth rate is exactly equal to the death rate). This phase is
sometimes called the plateau stage. A bacterial population may reach stationary growth
when a required nutrient is exhausted, when inhibitory end products accumulate, or when
physical conditions are inappropriate for growth. The duration of the stationary phase
varies, with some bacteria exhibiting very long stationary phases.
4. Death phase: Eventually, the number of viable bacterial cells begins to decline, signaling
the onset of the death phase. No further divisions occur in this phase. Death rate, in many
cases, follows the same kinetics as the exponential growth.
Principle:
Turbidity, which is a measure of the growth of microorganisms, is determined via an instrument
called a spectrophotometer. The basis for the use of this instrument is that less light passes
through a turbid solution than through a clear solution; i.e., the more turbid a solution (more
growth), the less the amount of light transmitted through the solution. Since the optical density of
the culture is proportional to the cell density, measuring the turbidity of the culture solution can
be used in estimating numbers of bacterial cells, if a growth curve for the conditions used has
already been established. This is the most common method used to rapidly estimate bacterial
numbers.
Laboratory Supplies: 10 ml culture of an unknown microorganism, 50 ml LB broth in 125-ml
flasks, Spectrophotometer, Un-inoculated LB broth in small test tube for blank.
Procedure:
1. Obtain a flask of LB and transfer 5 ml of broth to a small test tube. This is your blank. Adjust
the wavelength to 550 nm on the spectrophotometer and calibrate the machine to 0.0 O.D. or
100% transmittance with your blank. Take the blank out but do not discard it.
2. Inoculate your flask with 10 ml of the culture provided and swirl the flask well.
3. Work quickly to withdraw 5 ml of the sample into a clean small test tube, using a pipette.

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4. Place the culture flask immediately at the assigned incubating temperature and aeration
condition and record the time.
5. Measure the O.D. of your sample next. Record the 0 time absorbance. Discard the contents of
your sample test tube. This test tube can be re-used for your next sample without washing.
6. At 15-minute intervals for up to 90 minutes, shake your flask vigorously and transfer 5 ml of
the culture to your sample test tube.
7. Record the optical density at each time period. Use your blank each time to make sure your
readings are correct and other students have not altered the machine’s calibration. Your
instructor will ask all groups to write their results in a table on the blackboard. Copy these results
to your Results Sheet. If more than one group is doing a growth condition, get their average data.
8. Plot the optical density (y-axis) versus time (x-axis) for each growth condition on the same
regular graph paper.

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