Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 1

INDEX
Experiment Title of Experiment Page Marks Signature
Number Number of Teacher

1 TO Study principle and working

of microscope.

2 To study different laboratory

equipment used in
pharmaceutical microbiology.

3A To prepare and sterilize

nutrient broth, nutrient agar
slant ,stabs and plates.

3B To study different inoculation

techniques.

4A To isolate microorganisms from

given bacterial culture by Pour
Plate Method.

4B To isolate of pure culture of

micro-organisms by multiple
streak plate technique / other
techniques.

5A To identify given bacterial

culture by monochrome staining
(simple staining ) method.

5B To identify given bacterial

culture by negative staining
method.

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 5C To identify given bacterial
culture by Gram staining
method.

5D To identify given bacterial

culture by endospore staining
method.

6 To study motility of bacteria by

Hanging Drop Technique.

7 To perform bacteriological
analysis of water by most
probable number (MPN)
method.

8 To identify given microbial

culture using biochemical tests
(INDOLE)

9 To check sterility of given

marketed samples as per IP A)
Sterile water for injection (S)

B) Ophthalmic drops (O)

C) Tap water (T)

10A To perform the microbiological

assay of antibiotic (Penicillin)

10B To perform the microbiological

assay of antibiotic
(Streptomycin)

Date: 12/04/2021

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 INTRODUCTION TO PHARMACEUTICAL MICROBIOLOGY PRACTICALS

1. Always wear an apron, head cover and mask before entering the microbial laboratory to

protect from microbial contamination and laboratory hazards.

2. While entering the laboratory, check the following things required for the practical:

• Journal and Rough notebook .

• Clean apron, head cover and mask

• Match box and rubber gloves

• Inoculating wire loop

• Glass marker, Butter paper, Tissue paper

3. Take care to avoid contamination or fire hazards with respect to your clothes and body

parts.

4. Keep the doors and windows of the laboratory closed during practical sessions to avoid

contamination by air currents.

5. Keep your belongings like books, journals or bags away from the working platform,

6. Always wash your hands with soap/sanitizer before and after each practical session.

7. Clean your work bench with disinfectant before working.

8. Use rubber bulbs while pipetting out solutions.

9. Always use the dustbin for discarding solid waste material. Do not use wash basins for

disposing waste.

10. Avoid touching your eyes, ears, mouth during work as it may facilitate the chance of

infection by pathogenic micro-organisms.

11. Avoid eating, drinking, talking while working.

12. If there is any gas leakage/ light problem/ accident, immediately report to your laboratory

attendant or teacher.

13. Don’ t mishandle the chemical solutions, strains, instruments, and apparatus.

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 14. Always keep the burners at a safe distance from organic solvents. When not in use,

keep the burners off.

15. Always maintain aseptic conditions while working with micro-organisms.

16. Always use sterile inoculation needle or loop.

17. Never open culture tubes/ plates directly, inhale its contents.

18. Do not start your burner with an adjacent burner or transfer fire.

19. Open culture tubes/plates near the vicinity of flames of the burner.

20. After completion of work always label the cultures with short terms/ codes to keep your

work in observable conditions.

21. After completion of your work, clean the platform with a disinfectant.

Date: 19/04/2021

EXPERIMENT NO: 1

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AIM: TO STUDY PRINCIPLE AND WORKING OF MICROSCOPE

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

TERMS in Microscopy:
• Magnification
• It is the degree of enlargement of an image.
• Magnification can be enhanced by making the lens more convex or by bringing
the object closer to the lens.
• Total magnification of a specimen can be calculated by multiplying the
magnification of all lenses present in the microscope.
• Maximum magnification of compound microscope (1000X) = Magnifying Power of
oil immersion objective lens (100X) x magnifying power of oculars (10X).

• Resolution
• It is the ability of lenses to separate small objects that are close together for
distinguishing their fine and detail structure.
• Microscope with a resolution power of 0.2 nm indicates that it can distinguish
between two points only if they are minimum 0.2 nm distant apart.

• Working distance
Distance between the front surface of lens and the surface of cover slip / the
specimen to be observed.

• Numerical aperture
• It is the ability of lens to gather light.

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 • Defined by two components - refractive index of the medium in which the
lenses work (μ) and the angle of the cone in which the light enters an
objective.
• NA is calculated by the following formula-

Where, λ is the wavelength of light used.

CLASSIFICATION OF MICROSCOPES
1] Depending on number of lenses :-
Microscope

PARTS of Microscope: (Explain and write their roles)

OBJECTIVE LENS: It forms the initial image of specimen on the slide. It is the
optical element that gathers light from the object being observed and focuses the
light rays to produce a real image.
ROLE:
• Collect light rays coming from any point of object and unite them at a point of the
image.
• Real image formation and magnification.

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 EYEPIECE / OCULARS: Ultimate part of a compound microscope through which
the final image of the specimen can be visualized by human eye.
ROLE:
• To remagnify the image formed by objective lens and create a final virtual image.
• To rectify the remaining aberrations in the image missed by objective lens.

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 DIAGRAM OF COMPOUND MICROSCOPE

CONDENSERS: Series of lenses mounted in series under the stage. It is made

up of iris diaphragm and a shutter like apparatus mounted on the filter.
ROLE:

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 • It contracts light rays into a strong beam and pass through the opening of stage.
• Iris diaphragm, adjustable optical part that controls the light intensity after it is
screened by the filters.

ARM: It is the part of microscope that connects the tube to the base.
BASE: It supports the microscope and houses the illuminator.
NOSEPIECE: Upper part of a compound microscope that holds objective lens.
BODY TUBE: The body tube connects the eyepiece to the objective lens.
SPECIMEN STAGE: The place where specimen to be viewed is placed.

• Cedar wood oil is essential for observing slide under oil immersion lens.
Justify.

• For higher magnification and greater resolution,objective lens with higher NA is

recommended.
• The refractive index (μ) of air is 1.00 and cannot be greater than 1 (since
maximum value for θ cannot exceed 90° and sin 90° is 1.00).
• Thus no objective lens in air can have a NA greater than 1.00.
• So, replacing air with colorless immersion oil (cedar wood / microscopy oil)
possessing higher refractive index 1.5 (same as glass) will help to enhance NA
and thus,magnification as well as resolution of the image.

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 • Using oil immersion lens will prevent the detrimental reflection and refractive of
the light rays at the surfaces of slides or lenses.

Date: 19/04/2021

EXPERIMENT NO: 2
AIM: TO STUDY DIFFERENT LABORATORY EQUIPMENTS USED IN
PHARMACEUTICAL MICROBIOLOGY

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

THEORY:
Following equipments and instruments are used in pharmaceutical microbiology
practical:

AUTOCLAVE
PRINCIPLE
• The autoclave works on the principle of moist heat sterilization where steam
under pressure is used to sterilize the material present inside the chamber.
• The high pressure increases the boiling point of water and thus helps achieve a
higher temperature for sterilization.
• Water usually boils at 100°C under normal atmospheric pressure (760 mm of
Hg); however, the boiling point of water increases if the pressure is to be
increased.
• Similarly, the high pressure also facilitates the rapid penetration of heat into
deeper parts of the material for oxidation of cellular components. Moisture
present in the steam causes the coagulation of proteins leading to an irreversible
loss of function and activity of microbes.
• This principle is employed in an autoclave where the water boils at 121°C at the

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 pressure of 15 psi or 775 mm of Hg.

CONSTRUCTION
Following are the different parts of a laboratory bench autoclave-
a. Pressure Chamber
• The pressure chamber is the main component of a steam autoclave consisting of
an inner chamber and an outer jacket.
• The inner chamber is made up of stainless steel or gunmetal, which is present
inside the out chamber made up of an iron case.
b. Lid/ Door
• The next important component of an autoclave is the lid or door of the autoclave.
• The lid is made airtight via the screw clamps and asbestos washer.
• The lid consists of various other components like:
Pressure gauge
• A pressure gauge is present on the lid of the autoclave to indicate the pressure
created in the autoclave during sterilization.
Pressure releasing unit/ Whistle
• The whistle controls the pressure inside the chamber by releasing a certain
amount of vapor by lifting itself.
Safety valve
• The valve has a thin layer of rubber that bursts itself to release the pressure and
to avoid the danger of explosion.
c. Steam generator/ Electrical heater
• An electrical steam generator or boiler is present underneath the chamber that
uses an electric heating system to heat the water and generate steam in the
inner and the outer chamber.
d. Vacuum generator (if applicable)
• In some types of autoclaves, a separate vacuum generator is present which pulls
out the air from the inside of the chamber to create a vacuum inside the
chamber.

DIAGRAM

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APPLICATIONS
Sterilization can be used for
• Solutions sealed in containers ampules, vials
• Bulk Solutions

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 • Glassware
• Surgical Dressing
• Instruments
• Culture media
ADVANTAGES:
• Rapid, Inexpensive, Effective, Large volumes,
• Steam has Better penetrating power and applicable to wide variety of materials
• Shorter exposure of sterilization
DISADVANTAGES:
• Cannot use for oily preparation (oil base ointment)
• Cannot use for moisture sensitive preparations

COLONY COUNTER:
It is necessary to count the number of viable cell or colonies in different pharmaceutical
formulations. Each microbial cell or spore generally produce single colony on solid culture
media. Colony produced on petri plate is counted by colony counter.

Principle:

Colony counter works on the principle of illuminating the petri dishes by keeping them on an
illuminating surface and thus highlighting the colonies which are further marked off with a felt-
tipped pen by applying a little pressure producing a beep sound and increasing number in the
display count.

Construction:
A colony counter consists of the following parts:
1. Illuminating surface - where the petri plate is kept to be illuminated.
2. Magnifying glass - a magnifying glass is attached to the colony counter to magnify the
petri dish so that easily counting can be done.
3. Counter-pen - a pressure sensor pen which produces a beep sound when lightly
pressed against the surface to count the colonies. Moreover it's connected to the display
which shows the no. of times the pen has been pressed.
4. Display unit - this part simply helps in keeping the number of colonies counted by
pressing the pen.

Some other parts include a switch for switching on and turning off the COLONY COUNTER and
a support to hold the magnifying glass intact just above the surface.

Applications of colony counter:-

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Colony counter find its major applications in industrial, research and education based fields
where bacterial and fungal culture are prepared and studied.

Diagram -

INCUBATOR
Incubator, in microbiology, is an insulated and enclosed device that provides an optimal
condition of temperature, humidity, and other environmental conditions required for the growth
of organisms.

An incubator is a piece of vital laboratory equipment necessary for the cultivation of

microorganisms under artificial conditions.

PRINCIPLE / WORKING –

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 ● All incubators are based on the concept that when organisms are provided with the
optimal condition of temperature, humidity, oxygen, and carbon dioxide levels, they grow
and divide to form more organisms.
● In an incubator, the thermostat maintains a constant temperature that can be read from
the outside via the thermometer.
● The temperature is maintained by utilizing the heating and no-heating cycles.
● During the heating cycle, the thermostat heats the incubator, and during the no-heating
period, the heating is stopped, and the incubator is cooled by radiating heat to the
surrounding.
● Insulation from the outside creates an isolated condition inside the cabinet, which allows
the microbes to grow effectively.
● Similarly, other parameters like humidity and airflow are also maintained through
different mechanisms that create an environment similar to the natural environment of
the organisms.
● Similarly, they are provided with adjustments for maintaining the concentration of CO2 to
balance the pH and humidity required for the growth of the organisms.
● Usual temperature to be maintained is 35– 37°C for bacteria.
CONSTRUCTION –
A microbial incubator is made up of various units, some of which are:

a) Cabinet
● The cabinet is the main body of the incubator consisting of the double-walled cuboidal
enclosure with a capacity ranging from 20 to 800L.
● The outer wall is made up of stainless-steel sheets while the inner wall is made up of
aluminium.
● The space between the two walls is filled with glass wool to provide insulation to the
incubator.
● The insulation prevents heat loss and in turn, reduces the electric consumption, thereby
ensuring the smooth working of the device.
● The inner wall of the incubator is provided with inward projections that support the
shelves present inside the incubator.
b) Door
● A door is present in all incubators to close the insulated cabinet.
● The door also has insulation of its own. It is also provided with a glass that enables the
visualization of the interior of the incubator during incubation without disturbing the
interior environment.
● A handle is present on the outside of the door to help with the manoeuvring of the door.
c) Control Panel
● On the outer wall of the incubator is a control panel with all the switches and indicators
that allows the parameters of the incubator to be controlled.
● The control panel also has a witch to control the thermostat of the device.
d) Thermostat
● A thermostat is used to set the desired temperature of the incubator.
● After the desired temperature is reached, the thermostat automatically maintains the
incubator at that temperature until the temperature is changed again.
e) Perforated shelves
● Bound to the inner wall are some perforated shelves onto which the plates with the
culture media are placed.
● The perforations on the shelves allow the movement of hot air throughout the inside of
the incubator.

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 ● In some incubators, the shelves are removable, which allows the shelves to be cleaned
properly.
f) Asbestos door gasket
● The asbestos door gasket provides an almost airtight seal between the door and the
cabinet.
● This seal prevents the outside air from entering the cabinet and thus, creating an
isolated hot environment inside the cabinet without being interrupted by the external
environment.
g) L-shaped thermometer
● A thermometer is placed on the top part of the outer wall of the incubator.
● One end of the thermometer provided with gradations remains outside of the incubator
so that temperature can be read easily.
● The next end with the mercury bulb is protruded slightly into the chamber of the
incubator.
h) HEPA filters
● Some advanced incubators are also provided with HEPA filters to lower the possible
contamination created due to airflow.
● AN air-pump with filters creates a closed-loop system so that the air flowing inside the
incubator generates less contamination.
i) Humidity and gas control
● The CO2 incubators are provided with a reservoir underneath the chamber that contains
water.

APPLICATIONS
Incubators are used to grow microbial culture or cell cultures.
1. Incubators can also be used to maintain the culture of organisms to be used later.
2. Some incubators are used to increase the growth rate of organisms, having a prolonged
growth rate in the natural environment.
3. Specific incubators are used for the reproduction of microbial colonies and subsequent
determination of biochemical oxygen demand.
4. These are also used for breeding of insects and hatching of eggs in zoology.
5. Incubators also provide a controlled condition for sample storage before they can be
processed in the laboratories.

DIAGRAM -

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HOT AIR OVEN:
This is the most widely used method of sterilization by dry heat. Hot air oven is equipment used
for dry heat sterilization by hot air under atmospheric pressure. Hot air ovens are commonly
available in pharmaceutical laboratories for drying and sterilization.

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CONSTRUCTION AND WORKING:
The modern hot air ovens consist of a double walled chamber of aluminum or stainless steel
separated from the outer case by a thick layer of insulation made of fiberglass.
Insulation is also filled in the hollow flanged door, which carries an asbestos jacket that provides
a tight seal.
Heating is affected by electrical heating elements and thermostats automatically control
temperature.
PRECAUTIONS:
The material should be arranged in a manner which allows free circulation of air between the
objects and it should not be overloaded.
Glassware should be perfectly dry and wrapped in Kraft paper before being placed in the oven.
The oven must be allowed to cool slowly for about two hours before the door is opened, since
the glassware may get cracked by sudden cooling.
PRINCIPLE:
An oven is based on the principle where sterilization is accomplished by dry heat or hot air.
Dry heat is less effective as compared to moist heat because in presence of moisture, proteins
are easily coagulated and moist heat has greater penetrating power than dry heat.
By the process of protein denaturation and oxidation, microbes are destroyed by dry heat. For
normal sterilization work, the oven should be operated at 160 °C for 2 hours.
A time temperature relationship of hot air oven is given -

Temperature ( Celsius) Time (hrs.)

120 8
140 3
150 2.5
160 2
170 1
180 0.5

Substances that are not heat-labile and can tolerate temperature up to 250°C may be sterilized
by hot air oven. Normally the spores as well as the vegetative forms of a microorganisms are
killed in two hours at a temperature of 160°C.

The relation between the temperature and relative time required for sterilization in hot air oven is
given –

Temperature ( Celsius) Time (mins.)

170 60

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 160 120
150 150
140 180

DIAGRAM-

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USES:
1. Hot air oven is used to sterilize glassware forceps, scalpels, scissors, spatula, swabs etc.

2. Some pharmaceutical substances such as glycerin, fixed oil liquid paraffin, propylene glycol,
sulphonamides can also be sterilized using this.

3. Further, dusting powders such as kaolin, talc, zinc oxide, starch etc. are also sterilized using
the hot air oven.
ADVANTAGES OVER AUTOCLAVES-
1. They do not require water and there is not much pressure build up within the oven, unlike an
autoclave, making them safer to work with.
2. This also makes them more suitable to be used in a laboratory environment.
3. They are much smaller than autoclaves but can still be as effective.
4. They can be more rapid than an autoclave and higher temperatures can be reached
compared to other means.

DISADVANTAGES OVER AUTOCLAVES-

1- As they use dry heat instead of moist heat, some organisms like prions, may not be killed by
them every time, based on the principle of thermal inactivation by oxidation.
2- It is not suitable for surgical dressings, rubbers, plastics, volatile and heat labile substances.
Hot air is a bad conductor of heat and its penetration power is low as compared to moist
heat.

LAMINAR AIR FLOW UNIT

It is a carefully enclosed bench designed to prevent contamination of semiconductor wafes,
biological samples or any particle sensitive material.

PRINCIPLE

• A laminar flow unit creates dust free abacterial air environment.

• Air from the room passes through the HEPA (High Efficiency Particulate Absorbing) filters and
is fed into the working chamber by a unidirectional vertical descending flow. From the working
area the air is moved back to the environment

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• Laminar flow hood involve a unidirectional exhaustion of air to the workplace and personnel
whereby filtered air is discharged with a regular and fixed velocity

CONSTRUCTION

•It consists of a filter pad,a fan and a HEPA filter.

• The fan sucks the air through the filter pad where dust is trapped.
• After that the prefiltered air has to pas the HEPA filter where contaminating fungi, bacteria,
dust,etc are removed.
• Sterile air flows into the working area where you can do all work without risk of contamination.

ADVANTAGES
1.It have enough space in sterile area
2.Plant material can stay for longer time in the sterile area because it does not becomes hot.
3.It is easier to use bigger flasks with wider lids in Laminar Air Flow Unit.

DISADVANTAGES
1.It is very expensive.
2.The Laminar Air Flow Unit needs more space.

DIAGRAM-

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APPLICATIONS

There are three general areas in the field of microbiology where laminar flow can be applied:

(i) Product protection:-This area includes activities such as critical sterility tests and assays,
aseptic filling, tissue culture preparation, and other procedures which require that the material
must be kept sterile, but where personnel protection is not a problem. Standard horizontal
laminar flow clean benches can be used for these procedure.

(ii) Personnel protection:-This includes the processing of infectious material and the inoculation
of pure cultures of pathogenic microorganisms where technical personnel must be protected.
For this, a vertical laminar flow cabinet can be used. A supply fan passes air down through an
ultrahigh-efficiency filter into the work area. A second fan exhausts the air through a grated work
surface. Adjustment of the fans to exhaust more air than is supplied results in maintenance of a
slight negative pressure which causes ambient air to move from the operator toward the exterior
periphery of the work area, thereby creating a protective curtain of air. The air can be exhausted
completely or can be partially recirculated if absolute filtration is employed.

(iii) Personnel and product protection:-When exclusion of microbial contaminants and protection
of personnel are required, a vertical laminar flow cabinet can be used.
Laminar flow cabinets are made by several manufacturers and are available as standard items.
A vertical flow unit is slightly higher. When the cost of most laboratory furniture and biological
safety hoods and their capability are considered, laminar flow systems represent a sound
investment. Maintenance requirements are minimal and the life of the filters is from 10 to 15
years.

Precautions:
• Always minimize clutter.
• Always wash your hands and arms before entering.
• Arrange objects in such a way that full benefits of the laminar flow of air can be achieved.
• Avoid spraying or squirting solutions onto the HEPA/ULPA filter.
• Always disinfect your materials before entering the laminar flow cabinet.
• Do not put any waste and other items in the hood.
• Do not wear any jewelry around your hands and wrists.
• Do not bring unnecessary items into the main work area.
• Use materials that were disinfected.

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Date: 03/05/2021

EXPERIMENT NO: 3A
AIM: TO PREPARE AND STERILIZE NUTRIENT BROTH, NUTRIENT AGAR
SLANTS, STABS AND PLATES

REQUIREMENTS:
Apparatus: Conical flask, glass rod, Petri plate, test tube, measuring cylinder,
cotton, pH meter etc.
Chemicals: Beef extract, peptone, glucose, sodium chloride, agar etc.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE

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Composition of Nutrient Broth
SR.
CONTENT QUANTITY ROLE
NO.
1 Beef extract 10 gm
2 Peptone 10 gm
3 Sodium chloride 5.0 gm
4 Distilled water (to make) 1000 ml

Composition of Nutrient Agar

SR.
CONTENT QUANTITY ROLE
NO.
1 Beef extract 10 gm
2 Peptone 10 gm
3 Sodium chloride 5.0 gm
4 Distilled water (to make) 1000 ml
5 Agar 20 gm

OBSERVATIONS
TYPE OF MEDIA SIGNIFICANCE
Nutrient Broth

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Nutrient Agar STAB

Nutrient Agar SLANT

Nutrient Agar PLATE

PROCEDURE
1. Clean, dry and wrap glass wares in a paper and keep it in hot air oven for
sterilization at 160 °C for 30 minutes.
2. Weigh the required quantity of media in a sterile conical flask and make up the
volume with distilled water. Slightly warm the solution.
3. Close the conical flask with sterile cotton plug. Subject it to autoclaving at 121°C for
15 minutes at 15 psi (Pounds per square inch) pressure.

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4. Make your working platform sterile with alcohol. Afterwards, start the burners for
aseptic processing.
5. Remove the glass wares and culture media from oven and autoclave, respectively.
Put them between the burners on your aseptic work platform.
6. Transfer the media in the respective glass wares in different ways viz. :
A. Add 5 ml of sterile NUTRIENT BROTH in sterile test tube and cap it with sterile
cotton plug.
B. Add 5 ml of sterile NUTRIENT AGAR in sterile test tube and cap it with sterile
cotton plug. Keep the test tube in a straight position undisturbed until the agar gets
solidified to for STAB.
C. Add 5 ml of sterile NUTRIENT AGAR in sterile test tube and cap it with sterile
cotton plug. Keep the test tube in a slanting position undisturbed until the agar gets
solidified to for SLANT.
D. Add 10-15 ml of sterile NUTRIENT AGAR in sterile PETRI PLATE in 45 ° position,
spread it uniformly and cap it. Keep the Petri undisturbed until the agar gets
solidified.
7. Observe after solidification.

RESULTS
Nutrient Broth, Nutrient agar slant, stab and plates are prepared and sterilized
successfully.

Date: 10/05/2021

EXPERIMENT NO: 3B
AIM: TO STUDY DIFFERENT INOCULATION TECHNIQUES

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REQUIREMENTS:
Apparatus :Test tubes, inoculating needle, Petri plate, conical flask
Chemicals: Nutrient broth, agar.
Others: caps or cotton plugs etc.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE
• Microorganisms are transferred from one medium to another by sub culturing. This
technique is the routinely used in preparation and maintenance of stock cultures as well
as microbiological test procedures.
• Artificial induction of microbes into the culture medium is called 'inoculation'. Inoculation
is the most important technique for facilitating proper growth of microorganism under
aseptic conditions. Microorganism are always present in the air, laboratory surfaces,
benches and equipments.
• They can serve as source of external contamination and interfere with experimental
results. Hence, to avoid this contamination, use proper aseptic techniques foe sub
culturing .
• The use of laminar air flow device is essential for aseptic handling. It is required for
proper performance of sterility testing, microbial assays, aseptic filling and aseptic
transfer. Any technique or procedure which is carried out in aseptic are so as to avoid
the entry of microbes by any mean and thus maintain the sterility of the sample is called
as aseptic technique. Aseptic transfer of microorganisms can experiment design and for
study of microorganism.
• Solid media can be placed in test tubes, which are then allowed to cool and harden in a
slanted position, producing agar slants.
• The slant provides a growth surface and is an ideal way of maintaining cultures for
study. Similar tubes are allowed to harden in the upright position designed as agar slabs.
Agar slabs are used primarily for the study of the gaseous requirements of

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 microorganisms. A deep or pour is liquefied stab usually contains 18 to 20ml agar
medium. The medium is liquefied and held in water bath at about 50 to 55 degree C.
Microbial cultures are inoculated in broth, slant and stab for growth and incubated at
specific temperature and time. Cultures can be added to it for the isolation and study of
microorganisms.

PROCEDURE
1. An inoculating needle or loop must always be sterilized by holding it in the hottest
portion of the Bunsen burner flame, the inner blue cone, until the entire wire
becomes red hot. Then the upper portion of the handle is rapidly passed through the
flame. Once flamed, the loop is never put down but is held in the hand and allowed
to cool for 10 to 20 seconds. The stock culture tube and the tube to be inoculated
are held in the palm of the other hand and secured with the thumb. The two tubes
are then separated to form a V in the hand.
2. The tubes are uncapped by grasping the first cap with the little finger and the second
cap with the next finger and lifting the closures upward. They must never be placed
on the laboratory bench because doing so would compromise the sterile procedure.
Following removal of the closures, the necks of the tubes are briefly passed through
the flame and the sterile transfer instrument is further cooled by touching it to the
sterile inside wall of the culture tube before removing a small sample of inoculum.
(Note: Once removed, these caps must be kept in the hand that holds the sterile
inoculating loop or needle, thus the inner aspects of the caps point away from the
palm of the hand).
3. Depending on the culture medium, a loop or needle is used for removal of the
inoculum. Loops are commonly used to obtain a sample from a broth culture. Either
instrument can be used to obtain the inoculum from an agar slant culture by carefully
touching the surface of the solid medium in an area exhibiting growth so as not to
gouge into the agar / dipping in the suspension. A straight needle is always used
when transferring microorganisms to an agar deep tube from both solid and liquid
cultures.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 30

4. The cell-laden loop or needle is inserted into the subculture tube. In the case of a
broth medium, the loop or needle is shaken slightly to dislodge the organisms; with
an agar slant medium, it is drawn lightly over the hardened surface in a straight or
zigzag line. For inoculation of an agar deep tube, a straight needle is inserted to the
bottom of the tube in a straight line and rapidly withdrawn along the line of insertion.
This is a stab inoculation.
5. Following inoculation, the instrument is removed, the necks of the tubes are
reflamed, and the caps are replaced on the same tube from which they were
removed.
6. The needle or loop is again flamed to destroy remaining organisms.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 31

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 32
OBSERVATIONS

Before Incubation After Incubation

Nutrient Broth

Nutrient Agar STAB

Nutrient Agar SLANT

Nutrient Agar PLATE

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 33

RESULTS:
Using different inoculation techniques nutrient broth, slant, stab and plates
inoculated successfully.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 34

Date: 10/05/2021

EXPERIMENT NO: 4A
AIM: TO ISOLATE MICROORGANISMS FROM GIVEN BACTERIAL CULTURE BY
POUR PLATE METHOD.

REQUIREMENTS:
Apparatus: sterile test tubes, inoculating loop, Petri plate, spreader.
Chemicals: Nutrient agar,
Others: Incubator, hot air oven, cotton plug or cap etc.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE
Pour plate method is usually the method of choice for counting the number of colony-
forming bacteria present in a liquid specimen. Because the sample is mixed with the
molten agar medium, a larger volume can be used than with the spread plate.

PROCEDURE
1. Prepare sterile nutrient agar and transfer 10 ml in each of the 3 sterile test tubes.
2. From initial, parent bacterial suspension, take one loop of suspension and transfer it
to 1st test tube. Mix well the contents.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 35

3. From above 1st test tube, take one loop of contents and transfer it to 2nd test tube.
Mix well the contents.
4. From above 2nd test tube, take one loop of contents and transfer it to the 3rd test
tube. Mix well the contents.
5. During above serial dilutions, the media should not undergo solidification.
6. Transfer the contents of above three test tubes in respective, sterile Petri plates
aseptically. Make sure that the media is uniformly spread across the Petri base.
7. Allow complete solidification, incubate the Petri plates at 37°C for 24 hrs and count
number of colonies.
8. Calculate to find the number of bacteria in the initial, parent bacterial suspension.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 36

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 37

RESULTS :
Using pour plate method microorganisms are isolated from give bacterial culture.
Parent cell contains

Date: 17/05/2021

EXPERIMENT NO: 4B
AIM: To isolate of pure culture of micro-organisms by multiple streak plate
technique / other techniques.

REQUIREMENTS:
Apparatus: Petri plate, conical flask, beaker, test tube, nichrome wire loop, glass
marking pencil etc.
Chemicals: Sodium chloride, alcohol
Others: Peptone, beef/yeast extract

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 38

PRINCIPLE:
Different technique used for purification of cultures are streak plate method, pour plate
method, spread plate method and micromanipulator. Inoculation of microbial culture to
the surface of the sterile agar plate and spreding it by an inoculating wire loop is called
streak plate method. Streak plate technique is used for the cultivation, isolation and
separation of microorganisms from mixed populations. Streak plate technique is used to
produce well separated colonies of bacteria from mixed suspension and the size,
shape, colour and other physical characteristics of isolated colonies are studied.
One streak plate, two streak plate, four streak plate and multiple streak plate are
commonly used techniques for isolation of cultures.

PROCEDURE
1. Prepare the nutrient agar, autoclave it and transfer it to sterile Petri plate; allow it to
get solidified.
2. Then take a loopful of bacterial culture (only once) and streak it in various labeled
quadrants successively and aseptically.
3. After streaking every quadrant, incinerate the loop, cool it and re-use.
4. Streaking can be done in zig zag or straight streaking pattern.
5. Incubate plates at 37°C for 24 hour and observe the growth.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 39

OBSERVATIONS

Plate Observation (Post Incubation)

RESULTS
Pure culture of micro-organisms by multiple streak plate technique / other techniques was
isolated successfully.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 40

Date: 24/05/2021

EXPERIMENT NO: 5A

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 41

AIM: TO IDENTIFY GIVEN BACTERIAL CULTURE BY MONOCHROME STAINING
(SIMPLE STAINING) METHOD

REQUIREMENTS:
Chemicals: Methyl blue or crystal violet or carbol fuchsin
Apparatus: Staining tray, glass slides, Bunsen burner, inoculating loop, lens
paper, glass marker etc.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE

PROCEDURE
1. Prepare bacterial smear from the given culture.
2. Transfer the stain drop wise on the smear to wet it completely and allow it to react
for 30 - 40 seconds.
3. Tilt the slide so as to remove extra stain in staining pan or washbasin.
4. Using water, wash all excess stain.
5. Air-dry the smear. Use blotting paper for this purpose.
6. Observe the slide under oil immersion objective.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 42

OBSERVATIONS

RESULTS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 43

Date: 24/05/2021

EXPERIMENT NO: 5B
AIM: TO IDENTIFY GIVEN BACTERIAL CULTURE BY NEGATIVE STAINING
METHOD

REQUIREMENTS:
Chemicals: Nigrosin (10%) or congo red (2%)
Apparatus: Staining tray, glass slide, lens paper, Bunsen burner, inoculating
loop.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication. Page no. 43-44.

PRINCIPLE

PROCEDURE

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 44

1. Take a small drop of Nigrosine stain at one end of glass slide.
2. Aseptically take loopful of given bacterial culture and mix it properly with nigrosine
drop.
3. With the edge of another slide, spread the suspension across the whole length of
slide so as to make a thin film.
4. Air dry the film, but don't heat-fix the film.
5. After proper drying of film, add small drop of cedar wood oil at the center of slide and
observe it under oil immersion lens.

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 45

RESULTS
Bacterial cells were seen colourless against the dark background. They were
spherical.

Date: 31/05/2021

EXPERIMENT NO: 5C
AIM: TO IDENTIFY GIVEN BACTERIAL CULTURE BY GRAM STAINING METHOD

REQUIREMENTS:
Chemicals: Crystal violet, safranin, Gram's iodine, ethyl alcohol (95%)
Apparatus: staining tray, Bunsen burner, inoculating loop, glass slide etc.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication. Page no. 48-49.

PRINCIPLE :
Gram staining is one of the most fundamental and widely used technique for the
differential and identification of bacteria. This differential staining technique was

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 46

discovered by Dr. Christian Gram in 1884. It not only reveals the size and shape
of bacteria but is also used to differentiate bacteria into Gram-positive and Gram-
negative cells. Hence, it is known as differential staining.
In this staining the cells are stained with a basic stain ( crystal violet)
and treated with an iodine - potassium iodide mixture to fix the stain. Then the
stain is washed with alcohol or acetone (decolorizer) and counterstained with a
polar dye of a different colour (eg. Safranin). All bacteria take up the initial violet
strain but only Gram-positive cells retain it during the subsequent steps ( Bacillus
anthracis, Clostridium tetani, Staphylococcus aureus etc.) Gram-negative
bacteria are easily decolourised and take up the counterstain and appear as red
colour (Escherichia coli, Salmonella typhi, Vibrio cholerae, Klebsiella pneumoniae
etc.)

PROCEDURE
1. Prepare smear of given bacterial culture
2. Transfer the PRIMARY STAIN (Crystal violet) drop wise on the smear sufficiently to
wet it completely and allow it to remain in contact with smear for about 40 to 60
seconds.
3. Tilt this slide over the washbasin to drain out the excess stain from the slide; rinse it
immediately under slow running tap water.
4. Add few drops of GRAM’ S IODINE solution sufficiently to wet the smear and wait
for one minute.
5. Drain off the excess of iodine solution by tilting the slide; rinse the slide once again
by water.
6. In tilted position, add the decolorizing agent (95 % alcohol) quickly to the smear.
7. Rinse immediately by water to prevent further decolorization.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 47

8. Add COUNTER STAIN (Safranin) on the slide and wait for 1 minute; drain the slide
to remove excess of safranin and rinse it with water.
9. Observe the slide under oil immersion objective.

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 48

RESULTS
Smear prepared from suspension shows red coloured, red shaped bacteria i.e
Gram negative bacilli.

Date: 31/05/2021

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 49

EXPERIMENT NO: 5D
AIM: TO IDENTIFY GIVEN BACTERIAL CULTURE BY ENDOSPORE STAINING
METHOD

REQUIREMENTS:
Apparatus:
Chemicals: Malachite green stain, safranin
Apparatus: slide, water bath, microscope, Bunsen burner, blotting paper.
REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE
Endospore staining is a differential stain that aims at detecting, identifying and
differentiating an endospore from the vegetative cell.

In order to stain a spore the primary stain i.e. malachite green is forced into the
spore by steaming. (here steaming acts as a mordant)

Malachite green is water soluble and has a low affinity for cellular material, so
vegetative cells may be decolorized with water.

Then safranin is added as a counter stain which is only taken by vegetative cells.
Thus, endospores are stained as green while vegetative cells are stained red.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 50

PROCEDURE
1. Prepare the smear; air dry and heat fix it.
2. Keep the slide on the boiling water bath.
3. Cover the smear with a small blotting paper and add malachite green stain to
completely wet it. Continue adding this stain for 2-3 minutes without allowing the
smear to dry.
4. Wash immediately with water. Add counter stain safranin and keep it for 30 seconds.
Again rinse it with water.
5. Observe under microscope using oil immersion objective.

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 51

RESULTS
From the above observation given bacteria contains endospore.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 52

Date: 07/06/2021

EXPERIMENT NO: 6
AIM: TO STUDY MOTILITY of BACTERIA BY HANGING DROP TECHNIQUE

REQUIREMENTS:
Apparatus: Bunsen burner, inoculating loop, depression (cavity) slide, coverslip
etc.
Chemicals: Vaseline or petroleum jelly,

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication. Page no. 61-63.

PRINCIPLE
Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 53
Living microorganisms can be studied to determine the natural size and shape of
cells, cellular arrangement, motility, reactions to various chemicals and response
to environmental factors. Wet mount and hanging drop technique are commonly
used for direct examination of living microorganisms (eg.bacteria, protozoa,
fungi) . Hanging drop technique is routinely used for study of bacterial motility.
Flagella are responsible for the motility of bacteria and this is called as
'organ of locomotion'. The number and arrangement of flagella are characteristic
of each bacteria. Flagella may be arranged on bacterial body as monotrichous,
lophotrichous, amphitrichous or peritrichous. The flagella has three basic parts
as filament, hook & basal body.

PROCEDURE
1. Take a clean cavity slide and cover slip.
2. Put small amount of wax in four corners of cover slip.
3. Put a small drop of given bacterial suspension at the middle portion of cover slip.
4. Place this cover slip above the cavity in such a way that the drop should be in
inverted position, hanging from the roof of cover slip inside the cavity of slide.
5. Care should be taken that the drop should not spread or touch to the base of cavity.
6. Observe the slide under 10 X initially and adjust; then focus under 45 X objective
and observe the movement of bacteria towards the edge.
7. Observation should be carried out as soon as possible before the bacteria get
settled at the bottom of cavity

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 54

OBSERVATIONS

RESULTS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 55

From the given observations, it was found that the given bacterial culture is
motile .
It has peritrichous flagella as observed from its taxis.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 56

Date: 07/06/2021

EXPERIMENT NO: 7
AIM: To perform bacteriological analysis of water by most probable number
(MPN) method

REQUIREMENTS:
Apparatus: Pipettes, glass marking pencil, conical flask, nichrome wire loop,
Durham tubes, slides, Gram stain reagents.
Chemicals: Water sample from tap water, double & single strength lactose
fermentation broth, nutrient agar, eoeosin-methylene blue medium, crystal violet,
Gram's iodine, ethyl alcohol (95per cent), safranin.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 57

PROCEDURE
Presumptive Test
• Take tap water for detection of microbial pathogens in 3 sets, each containing 3
test tubes.
• First Set tubes contain 3 ml of double strength lactose broth
• Remaining 2 sets contains 3 ml of single strength lactose broth
• Aseptically inoculate 0.1 ml in test tube number 1, 4 and 7
• Aseptically inoculate 0.5 ml in test tube number 2, 5 and 8
• Aseptically inoculate 1 ml in test tube number 3, 6 and 9
• Incubate all test tubes at 32°C to 35°C for 24 hours.
• Observe for gas formation and acid production.

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 58

RESULTS
The MPN for given water sample was ___7___per 100 ml as per presumptive test.
Quality of water needs to be further assessed by confirmatory and completed tests.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 59

Date: 10/06/2021

EXPERIMENT NO: 8
AIM: To identify given microbial culture using biochemical tests (INDOLE)

REQUIREMENTS:
Apparatus: Test tubes, glass marking pencil, inoculating wire loop, dropper.
Chemicals: Nutrient broth, tryptophan, Kovac's reagent.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE : Tryptophan an essential amino acid, is oxidised by some bacteria by

the enzyme tryptophanase resulting in the formation of indole, pyruvic acid &
ammonia. The indole test is performed by inoculating a culture into nutrient broth
which contains tryptophan. Production of indole during the reaction is detected
by adding Kovac's reagent (p-dimethylamino-benzaldehyde) which produces a
cherry red reagent layer. The colour is produced by the reagent, which is
composed of p-dimethylamino-benzaldehyde butanol & hydrochloric acid. Indole
is extracted from the medium into the reagent layer by the acidified butanol
component & forms a complex with the p-dimethylamino-benzaldehyde (cherry
red colour).

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 60

PROCEDURE
Prepare and sterilize the nutrient broth.
Pour it in test tube aseptically.
Inoculate it with the given microbial cultures and tryptohan for 5 minutes.
After 24 hours of incubation, add Kovac’ s reagent on the surface of these test tubes
gradually.
Observe for color change.

OBSERVATIONS

Culture Observation

A Deep red colour in the top layer of the test tube.

B Absence of deep red colour.

RESULTS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 61

Development of deep red colour in the top layer of the test tube is indole positive
test.
Absence of deep red colour is indole negative.

Date: 10/06/2021

EXPERIMENT NO: 9
AIM: To check sterility of given marketed samples as per IP
A) Sterile water for injection (S)
B) Ophthalmic drops (O)
C) Tap water (T)

REQUIREMENTS:
Apparatus: Test tube (large size) , conical flask, glass marking pencil, laminar air
flow etc.
Chemicals: Fluid thioglycollate medium[FTM], Alternative thioglycollate medium
[ATM], Soyabean casein digest medium [SCDM] etc.

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 62

PRINCIPLE
Sterility test must be carried out under aseptic conditions designed to avoid
accidental
contaminations of the product during testing by using laminar air flow cabinet.
Fluid thiogly-collate medium is used for clear fluid products for aerobic as well as
anaerobic bacteria. Alternative thioglycollate medium is used for turbid and
viscid products and for devices having tubes with small lumina for checking the
growth of bacteria. Soyabean-casein diges medium is suitable for the culture of
both fungi and aerobic bacteria. All media used for test should comply the test for
sterility and growth promotion test.
Sterility test can be performed by method A (membrane filtration) or method B
(direc inoculation). The given sample or water for injection not having any
antimicrobial activity. hence, nor required for inactivation of given sample. The
given sample quantity is also less than 100 ml. Hence method B: direct
inoculation should be used.

PROCEDURE
DIRECT INOCULATION
1. The test and control tubes are directly inoculated into two types of media to allow
for the detection of both aerobic and anaerobic microorganisms.
2. Prepare the specific quantity of media (FTM or ATM and SCDM) for test as per
guidelines given in IP 2014 and based on sample size.
3. The external surface of ampoules and closures of vials should be cleaned with a
suitable antimicrobial agent.
4. Remove the sample from the test container with a sterile pipette or with a sterile
syringe or a needle.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 63

 5. Transfer the quantity of preparation under examination prescribed in IP 2014
directly into the culture medium under aseptic conditions.
6. Incubate sample containing FTM and SCDM at 30 to 35˚C for not less than 24
hours. Observe the test tubes periodically during the incubation.
7. Intermittent observations as well as a final observation at the end of the testing
period are conducted to detect evidence of microbial growth in terms of turbidity.

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 64

RESULTS :

Date: 12/06/2021

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 65

EXPERIMENT NO: 10A
AIM: To perform the microbiological assay of antibiotic (Penicillin)

REQUIREMENTS:
Apparatus: Petri plate, test tube, glass spreader, pipette, cork borer, conical flask,
laminar air flow, antibiotic zone reader etc.
Chemicals: Marketed preparation: Benzothine Penicillin Injection I.P i.e.
Pencom®[900 mg]

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication. First edition page no. 178.

PRINCIPLE :
This method is based as diffusion of antibiotic from a cavity through a solified
agar layer of petri-plate used for the safety of growth of inoculatoed
microorganism is inhibited in a circular zone around a cavity containing solution
of antibiotics.

PROCEDURE
• 250 mg of formulation powder in 100 ml of tap water for Stock solution [6 units /
ml]
• 1 ml = 6 units
• 1 unit = 1/ 6 = 0.17 ml x 10 ml dilution = 1.7 ml of stock solution
• Make up the volume to 10 ml - in all test tubes

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 66

 • Each Batch = 150 ml x 2 conical flasks = 300 ml Sterile Nutrient Agar
• Each group will need 1 sterile Petri plate
• Autoclaved, sterile nutrient agar is poured in petri plate aseptically.
• After complete solidification, add test microorganism (Staphylococcus aureus) by
using a sterile inoculating loop.
• Now, with the help of sterile plate spreader, spread the inoculated test
microorganism on the culture media.
• With the help of single sterile cork borer, make 5 cavities at equal distance from
each other.
• Now, add the standard antibiotic dilutions in the respective labeled cavities of
plate.
• Spare 1 centre cavity for the unknown test solution. Test solution will be given by
your teacher.
• Allow 5 minutes diffusion time.
• The, incubate all the plates in the incubator at 32°C to 35°C for 24 hours.
• Post incubation, measure the zone of inhibition as the difference between the total
inhibition diameter (mm) and the uniform well diameter (mm).
• Plot the standard assay graph and unknown concentration of given test solution
would be calculated by graphical method.

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 67

PLOT the graph and unknown concentration of given antibiotic.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 68

RESULTS

The concentration of unknown sample is ________ units/ml

Precautions:
1. Do not overflow the solutions while filling up the cavities because it will not give accurate
results.
2. Do not make mistake while calculating the area and diameter of cavities.
3. Continuously close the Petri dish while spreading the bacterial culture on it to minimize
its exposure to external contaminants.

Date: 12/06/2021

EXPERIMENT NO: 10B

AIM: To perform the microbiological assay of antibiotic (Streptomycin)

REQUIREMENTS:
Apparatus:Petri plate, test tube, glass spreader, pipette, cork borer, conical flask,
laminar air flow, antibiotic zone reader etc.
Chemicals: Ambistryn– S [0.75 g] with Streptomycin sulphate IP Injection 750 mg

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 69

REFERENCES:
Pharmaceutical microbiology experiment and technique by "Prof. Chandrakant
Kokare" , carrier publication.

PRINCIPLE :
The microbiological assay of antibiotics may be carried out by the following two
methods:
(1 Method A: Cup-plate or cylinder-plate method.
(ii) Method B: Turbidimetric or tube assay method.
The microbiological assay of streptomycin is based upon a comparison of the
inhibition of microorganisms by measured concentrations of the antibiotics to be
examined with that produced by known concentrations of standard preparation of
the antibiotic having a known activity. Cylinder plate or cup plate method is
generally used for assay of streptomycin. For this assay Bacillus subtilis (NCTC
8236/ATCC 6633) is used as test microorganism and medium E (IP 2014) is used
as medium for the growth of test cultures.

PROCEDURE
• 300 mg of formulation powder in 100 ml of tap water for Stock solution [20 units /
ml]
• 1 ml = 20 units
• 1 unit = 1/ 20 = 0.05 ml x 10 ml dilution = 0.5 ml of stock solution
• 10 units = 5 ml of stock solution
• Each Batch = 150 ml x 2 conical flasks = 300 ml Sterile Nutrient Agar
• Each group will need 1 sterile Petri plate
• Autoclaved, sterile nutrient agar is poured in petri plate aseptically.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 70

 • After complete solidification, add test microorganism (Bacillus subtilis) by using a
sterile inoculating loop.
• Now, with the help of sterile plate spreader, spread the inoculated test
microorganism on the culture media.
• With the help of single sterile cork borer, make 5 cavities at equal distance from
each other.
• Now, add the standard antibiotic dilutions in the respective labeled cavities of
plate.
• Spare 1 centre cavity for the unknown test solution. Test solution will be given by
your teacher.
• Allow 5 minutes diffusion time.
• The, incubate all the plates in the incubator at 32°C to 35°C for 24 hours.
• Post incubation, measure the zone of inhibition as the difference between the total
inhibition diameter (mm) and the uniform well diameter (mm).
• Plot the standard assay graph and unknown concentration of given test solution
would be calculated by graphical method.

OBSERVATIONS

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 71

PLOT the graph and unknown concentration of given antibiotic.

RESULTS
The concentration of unknown sample is ________ units/ml

Precautions:
• Do not overflow the solutions while filling up the cavities because it will not give accurate
results.
• Do not make mistake while calculating the area and diameter of cavities.
• Continuously close the Petri dish while spreading the bacterial culture on it to minimize
its exposure to external contaminants.

Pharmaceutical Microbiology (SYBPharm Sem III) PCI Page 72