School of Pharmaceutical Sciences

PHARMACEUTICAL
MICROBIOLOGY
17BP303T
Pharmacy II Year

Laboratory Manual

Department of Pharmaceutics
STUDENT NAME :

REGISTER NO :

BATCH :

SECTION :

SUBJECT INCHARGE :
INDEX
S. DATE TOPIC REMA T.SI
NO RKS GN
1 01.07 INTRODUCTIO
.24 N
2 01.07 BOD
.24 INCUBATOR
3 08.07 ASEPTIC
.24 HOOD
4 08.07 LAMINAR AIR
.24 FLOW
5 15.07 AUTOCLAVE
.24
6 15.07 HOT AIR
.24 OVEN
7 22.07 DEEP
.24 FREEZERS
FOR MEDICAL
LABORATORI
ES
8 22.07 MICROSCOPE
.24
9 29.07 STERILIZATIO
.24 N OF
GLASSWARE,
PREPARATIO
 N AND
STERILIZATIO
N OF MEDIA
10 29.07 SUB-
.24 CULTURING
OF BACTERIA
AND FUNGUS
11 05.08 PREPARATIO
.24 N OF
NUTRIENT
BROTH
PREPARATIO
N OF
NUTRIENT
BROTH
12 05.08 PREPARATIO
.24 N OF
NUTRIENT
SLAB AND
AGAR SLANT
13 12.08 PREPARATIO
.24 N OF
NUTRIENT
AGAR
14 12.08 INOCULATION
 .24 OF MICRO –
ORGANISMS
IN SOLID AND
LIQUID
MEDIUM
15 19.08 INOCULATION
.24 OF MICRO-
ORGANISM
BY STABBING
AND SLANT
METHOD
16 19.08 ISOLATION
.24 OF PURE
CULTURE BY
STREAK
PLATE
METHOD
17 26.08 ISOLATION
.24 OF PURE
CULTURE BY
SPREAD
PLATE
METHOD
18 26.08 ISOLATION
.24 OF PURE
 CULTURE BY
POUR PLATE
METHOD
19 02.09 PREPARATIO
.24 N OF SMEAR
20 02.09 SIMPLE
.24 STAINING
21 09.09 GRAM
.24 STAINING
22 09.09 ACID FAST
.24 STAINING
23 16.09 SPORE
.24 STAINING
24 16.09 DETERMINAT
.24 ON OF
MOTILITY BY
HANGING
DROP
METHOD
25 23.09 ISOLATION
.24 OF PURE
CULTURE
26 23.09 MICROBIAL
.24 ASSAY OF
 ANTIBIOTICS
BY CUP
PLATE
METHOD
27 30.09 STERILITY
.24 TESTING
28 30.09 MICROBIAL
.24 ASSAY OF
ANTIBIOTIC
DISC PLATE
METHOD
29 07.10 BACTERIOLO
.24 GICAL
ANALYSIS OF
WATER
30 07.10 ISOLATION
.24 OF
ACTINOMYCE
TES FROM
SOIL AND ITS
BIOCHEMICAL
CHARACTERI
ZATION
31 14.10 IMVIC TEST
.24
32 14.10 MR-VP TEST
.24
33 21.1 CITRATE
O.24 TEST
34 21.10 OXIDASE
.24 TEST
35 28.10 CATALASE
.24 TEST
36 28.10 UREASE TEST
.24
37 04.11 TRIPLE
.24 SUGAR IRON
AGAR TEST-
H2S
PRODUCTION
38 04.11 CASEIN
.24 HYDROLYSIS
39 11.11 STARCH
.24 HYDROLYSIS
40 11.11 IDENTIFICATI
.24 ON OF
BACTERIA
USING 16s-R-
RNA
 SEQUENCING

DATE OF INTERNAL
EXTERNAL HOD SIGNATURE
SUBMISSION SIGNATURE
SIGNATURE AND SEAL
EX NO: 01
DATE: 01.07.24
INTRODUCTION
MICROBIOLOGY: -
Microbiology is the study of
microorganisms which are
unicellular and self-dusters.
Microbiology includes virology,
mycology, parasitology, bacteriology
and other branches. Microbes were
directly observed over 300 years
ago. the field of micro biology can be
said to be related to older biological
disciplines such as zoology and
botany.
ANCIENT: -
The existence of unseen micro
biological life was postulated by
Jainism which is based on
Mahaveer’s teaching as early as 6th
century. Paul Dundas notes that
Mahaveer arrested existence of
unsensed microorganisms living in
earth, water, fire.
MODERN: -
Anton van Leeuwenhoek considered
to be first to observe
microorganisms using a microscope.
In 1676, he observed bacteria and
other microorganisms using a single
lens microscope. Robert Hooke made
the first recorded microscope
observation of fruiting bodies of
mould 1665.
MICROBIAL PHYSIOLOGY: -
The study of how microbial cell
functions bio chemically, microbial
growth, metabolism and cell
structure.
CELLULAR MICROBIOLOGY: -
A discipline of cell biology.
MICROBIAL GENETICS: -
The study of how genes are
organized or regulated in microbes
in relation to their cellular functions.
MEDICAL MICROBIOLOGY: -
The study of pathogenic microbes
and the role of microbes in human
illness.
VETERNARY MICROBIOLOGY: -
The study of the role of microbes in
veterinary medicine and animal
taxonomy.
AERO-MICROBIOLOGY: -
Study of air borne microorganisms.
FOOD MICROBIOLOGY: -
Study of microorganisms causing
food spoilage and borne illness
causing microorganisms to produce
food products.
PHARMACEUTICAL MICROBIOLOGY: -
Study of microorganisms causing
pharmaceutical contamination or
spoil.
SOIL MICROBIOLOGY: -
Study of microbes in soil.
WATER MICROBIOLOGY: -
Study of microorganisms in water.
GENERATION MICROBIOLOGY: -
Study of microorganisms those have
some characters as their parents.
NANO MICROBIOLOGY: -
Study of microorganisms of nano
level.
ENVIRONMENTAL MICROBIOLOGY: -
Study of function and diversity of
microorganisms in the natural
environment.
MICROBIOLOGY SAFETY RULES
 All materials and materials
needed for laboratory are to be
kept away from the work area.
 A lab coat should be worn during
lab.
 Cleaning the lab table before and
after working with disinfectant
solution provided.
 Wash the hands before leaving
lab.
 Reusable items are to be placed in
the designated area.
 No eating, drinking out pipetting
is allowed in lab.
 Long hair should be tied back in
lab.
 All accidents, cuts and many
damaged glasses ware or
equipment’s should be reported
to lab inspector immediately.
 Visitors are not allowed into lab.
 Doors and windows are to be kept
closed at all times.
ASEPTIC TECHNIQUE
 This is a technique which prevent
introduction of unwanted
organisms into environment.
 When dealing with microbial
cultures it is necessary to handle
them in such a way that
environmental organisms do not
get introduced into culture.
 Aseptic technique prevents
environmental organism from
entering the culture.
 Doors and windows are kept
closed in the lab to prevent air
current which may cause
microorganism from surface to
become airborne.
 Transfer hooks and needles are
sterilized before and after using
flames to prevent introduction of
unwanted organisms.
 Agar plates are held in manner
that minimize the exposure of
 surface to environment.
 On removing libs from tubes, lids
are held in hand those techniques
are basis of laboratory aseptic
techniques.

REPORT:

EX NO: 02
DATE: 01.07.24
BOD INCUBATOR
PRINCIPLE:
The BOD incubator is a highly used
testing instrument in microbiology
labs. In this testing instrument, the
microbe samples that are used for
testing will consume oxygen. When
more oxygen is consumed by this
testing instrument then, there will
be depletion of oxygen. BOD is
based on the principle that if
sufficient oxygen is available,
aerobic biological decomposition by
microorganisms will continue until
all waste is consumed.
DISCRIPTION:
A biochemical oxygen demand (BOD)
incubator is a laboratory testing
chamber that's used to grow cell
cultures and test
microorganisms. It's a specialized
piece of equipment that's considered
important in hospitals,
pharmaceutical laboratories, and
research laboratories. BOD
incubators simulate conditions that
allow microorganisms to survive,
maintaining temperature, humidity,
oxygen, and carbon dioxide
levels. The BOD test is a chemical
process that measures how quickly
biological organisms use up oxygen
in a body of water. The process
involves:
1. Placing a sample in an airtight
bottle.
2. Incubating the bottle under
specific conditions for a set
 amount of time
3.Measuring the dissolved oxygen
(DO) at the beginning and end of
the incubation period
4. Calculating the BOD from the
difference between the initial and
final DO.
5.A thermostat to protect samples
from overheating.
6.An equipment security system to
protect the cooling system from
overheating.
7.A temperature controller, main
switch, and LEDs on the upper
front panel to indicate heating
and cooling.
8. A soft-touch keyboard.
9. An environmentally friendly
cooling system.
10. Thermal insulation to reduce
temperature dissipation.
11. A magnetically sealed door.
12. Adjustable height stainless
steel racks and trays.
13. A 65 mm port hole with a
 rubber seal to insert sensors for
validation.
14. U-shaped stainless steel
nichrome wire air heaters.
15. An eco-friendly compressor
with CFC-free R 134 A or R 404
refrigerant.
WORKING:
1.Connect to power: Connect the
incubator to a power supply.
2.Turn on: Turn on the main switch
on the mainboard and the switch
on the cabinet.
3.Set temperature: Use the digital
PID temperature controller to set
the temperature, usually at 20°C.
4. Start refrigeration: After the
temperature is set, the
refrigeration system will start.
5. Circulate air: An axial fan will
circulate air inside the chamber.
6. Monitor temperature: A
temperature sensor will sense the
current temperature and send
data to the PID controller, which
 will maintain the set temperature
until the desired time.

REPORT:

EX NO: 03
DATE: 08.07.24
ASEPTIC HOOD
PRINCIPLE:
The principle of an aseptic hood, also
known as a laminar flow hood, is to
create a continuous, unidirectional flow of
HEPA-filtered air over a working station to
prevent contaminants from entering the
sterile workspace.
DISCRIPTION:
An aseptic hood, also known as a laminar
flow hood (LFH) or clean bench, is a
laboratory ventilation device that creates
a sterile work environment to protect
both the lab worker and the materials
being used. LFHs should only be used for
non-infectious materials, like media
preparation, and should not be used with
toxins, volatile chemicals, or materials
that could cause hypersensitivity in the
worker.
WORKING:
22
 LAMINAR AIR FLOW

PRINCIPLE:

The laminar air flow is used for reducing the danger of infection, while working on
pathogenic microbes. In this system air of close cabinet room is made to pass through
High Efficient Particulate Air (HEPA), the HEPA filter does not allow any suspended
particle about 0.3mm dimension
The laminar air flow sucks the air in the room continuously and blows out the air through
a part of filter. The air is blown out in the uniform velocity and is pasted flow line

DESCRIPTION:
The laminar air flow apparatus consists of a platform(or) work table covered from all
sides except front position. The work table of platform is usually fixed to stand which is
to facilitate easy movement of apparatus.

The back of laminar air flow apparatus consists of HEPA filters through which air is
blown. The top of the apparatus consists of an “on” and “off” UV light and turn “on” and
“off” the air flow

WORKING:
 The platform of apparatus must be cleaned with a disinfectant
 Switch on the UV light for about 15min in order to eliminate
microorganisms before performing the experiment
 Switch an air flow and start experiment. This ensures elimination
of microorganisms present on the work bench
 Switch on the air flow and start the experiment and let it continue till
the experiment is complete
 Switch off all the control switches after the completion of the experiment

TYPES:
 Vertical laminar air flow
 Horizontal air flow

23
24
The relation between pressure and temperature

PRESSURE TEMPERATURE

0 100

5 109

10 115

15 121

20 126

25 130

30 135

40 141

25
26
 AUTOCLAVE

PRINCIPLE:

The principle used here is to increase temperature of stream. The water molecules
become more aggregated that increases the penetration considerably. The water boils at 100℃
depending upon vapor pressure of atmosphere. The temperature will be increased. The relation
between pressure and temperature is given on left hand side.
The autoclave is usually operated at 15 to 16sq inches steam pressure for 30min which has been
from above table corresponding at 121degrees centigrade. This temperature for period of 20min is
sufficient to kill all spores and vegetative cells of microorganisms.
DESCRIPTION:
 The autoclave is usually operated or basically a double walled metallic steel vessel made
of thick stainless steel and which has an opening with a tightly fitted closed door
 The opening is meant for keeping materials to be sterilized inside. A pressure
gauge is present used to measure the steam pressure
 In some autoclaves, gauges are present which prevents escape of steam from
chamber & also slow pressure in inner chambers.
 At the top, safety valves are present which prevents escape of steam from chamber and
also preventing explosion of chamber. These are valves to hold and then send it to
sterilizing chamber. There is also an Exit valve below, to let the steam escape from
bottom of the inner chamber.
 A little way there, a thermometer is attached just beyond the exhaust valve to show
temperature of steam as it comes from chamber.
WORKING:
 Load autoclave with materials to be sterilized such as culture media in tubes, flash etc...
 Close and lock the door securely
 Slowly open the air outlet valve to allow the air in chamber to be disposed by the
incoming steam
 Gently open the stream inlet valve to allow the air in to the chamber.
 Observe the increasing temperature in thermometer
 Close the air out let valve as the temperature reaches 100 degrees centigrade. At this,
all the air inside the sterilization chamber would have been dispensed
 Watch the thermometer to see the temperature should not exceed the required
temperature range of modern autoclaves. However, there is temperature regulation
mechanism which does not allow the temperature to go beyond a limit
 For routine sterilization of culture media, pounds of pressure is sufficient and this
should be maintained for 15 min at a temperature ranges at 121degree centigrade. After
you have assured that there is no pressure inside the sterilization chamber, open
the door and remove the material

27
28
 HOT AIR OVEN
PRINCIPLE:

The hot air oven is operated at a temperature of 160-180degree centigrade for a period
of 90min.Its temperature goes above 180degree centigrade then the hot air oven is used
for sterilizing all types of lab glass wares such as test tubes, Petri dish, pipette, flask
bottle etc... Other materials which will not be blend at high temperature may also be
sterilized in hot air sterilization. Petri dish may be put in a metal can on air sterilizer or
wrapped with a paper and placed inside or sterilization.

DESCRIPTION:

Hot air oven consists of double walled, insulated cabinet heated with electricity and is
constructed to with stand at high temperature. The walls of the oven is made up of
stainless steel or aluminum and are designed to prevent heat conduction from inside the
chamber. There is a motor and fan to circulate hot air inside the chamber. There is a
motor and fan filled either at chamber.

The hot air increases the temperature inside the chamber thus sterilizing it. A thermostat
regulates the temperature of the desired level and a thermometer is fitted for recording the
temperature. Hot air oven is used for sterilizing test tubes, conical flasks, Petri dishes,
pipettes etc... The glass wares should be empty. It cannot be used for sterilization of
culture media, alcohol or volatile substances.

WORKING:

 Before sterilization, all the glass wares should be thoroughly washed and
wiped and then mostly wrapped in craft paper
 Only then they are kept inside the chamber.
 After the glassware and other articles to be sterilized are located in hot air
oven temperature should be maintained at this level
 An exposure of at least 1hr is necessary for effective and proper sterilization of
glass wares. A higher temperature may result in breaking of glassware
 After the period of heating, the door of the oven must be opened until the
temperature has fallen at 100℃ because sudden rush of cool air into the oven
may crack the glass ware

29
 arse
focus

Stage will Stage

Clips

30
 MICROSCOPE
PROCEDURE FOR FOCUSING:

Microscope consists of a biconvex lens enclosed in two metal plates with a

magnification of 300x to present day electron microscope capable of magnification
greater than 25000x.

ELECTRON MICROSCOPE:

This is instrument of revolutionary method of microscopy with magnification up to one

million x in this specimen is illuminated by beam of electrons rather than light and
focusing is carried out by electromagnets instead of a set of optics. As electron passes
through the specimen images are formed by directly electron onto photographic film thus
making internal cellular structure visible.

COMPONENTS OF MICROSCOPE

STAGE: - A field platform with an opening in the culture allows the passage of light
from an illuminating source, belong below to the ray lens system above the stage. The
platform provides a surface for the placements of slides.

MECHANICAL STAGE: - It can be moved vertically or horizontally by means of

adjustment contents.

ILLUMINATION: - The light source is positioned by the instrument. some microscopes

are expected with a build in source to provide direct illuminations. Others are provided
with a reversible mirror that has one side flat and other side concave.

CONDENSOR: - This component is found directly under the stage and contains two
sets of lenses that concentrate light. This is equipped with a diaphragm.

BODY TUBE: - It is attached to arm of microscope. The upper surface a eye piece.
Lower end contains objective. tubes may be adjusted using fine adjustment and force
adjustment knobs.

MAGNIFICATION: -
Enlargement or magnification of a specimen is done by: -
 Ocular
 Objective
The objective lens is nearer the specimen and magnifies it producing real image that
projected up to focal plane and then magnified by ocular lens to produce clear image

31
RESOLVING POWER OF RESOLUTION: - It is ability of lens to show two
adjustment objects as discreate entities. It depends upon wavelength of light and numerical
aperture.

NUMERICAL APERTURE: -

It is function of diameter of object lens in related to its focal length.

Resolving power = wavelength of light /2 x N.A

It also depends upon refractive index.

ILLUMINATION: -

Effective illumination is required for efficient magnification and resolving power.

FOCUSSING: -

Adjusting the lens focusing a rule of thumb is that the magnification of the lens increased
the distance between the objective lens and slide called working distance .it decrease
whereas N.A of the objective lens increases.

32
COMPOSITION OF NUTRIENT BROTH:

Ingredient for 100ml

Peptone – 0.5ml
Meat extract – 0.3%
Nacl – 0.5%
Distilled water –Q.s -100ml
pH – 7.4

33
 PREPARATION OF NUTRIENT BROTH PREPARATION OF
NUTRIENT BROTH

AIM: To prepare and submit 10ml of nutrient broth.

REQUIREMENTS: Peptone, meat extract, Nacl, distilled water q.s 100ml, pH 7.4
and mix it in distilled water in a clean conical flask. Plug it with cotton swab.

FORMULA: Nutrient broth, 13g in 1000ml.

COMPOSITION OF NUTRIENT BROTH:

Ingredient for 100ml peptone 0.5%. Heat extracts 0.3% distilled water q.s 100ml. pH 7.4.

PROCEDURE:
 Weigh the required quantity of nutrient broth and mix it in distilled water in a
conical flask plug it with an cotton and sterilize the media in autoclave for 100˚c,
30mins at 1516 pressure.
 wash the test tube on dry it without any water droplet then it should be wrapped
with a help of brown sheet.
 The wrapped glass ware is sterilized by using hot air oven at 160˚c for 1hour.
After the sterilization period, remove the glassware and the culture media from
instrument and it should be taken into aseptic area.
 Clean the laminar air flow bench with the help of alcohol surface and then open
the wrapping glass ware placed in test tube with test tube stands.
 Nutrients is the conical flask is transferred into test tube aseptically in front of
flame, cool the media and then plug it with a help of non-absorbent cotton then
nutrient broth tube is submitted.

REPORT:

34
 COMPOSITION OF NUTRIENT AGAR:

Ingredient for 100ml

Peptone – 0.5%
Agar – 3%
Meat extract – 0.3%
Nacl – 0.5%
Distilled water – q.s to 100ml

35
 PREPARATION OF NUTRIENT AGAR

AIM: To prepare and submit 10ml of nutrient agar.

REQUIREMENTS: Peptones, heat extract, distilled water, pH paper, conical flask,

glass rod, nonabsorbent.

FORMULA: Nutrient agar- 28gm for 1000ml

PROCEDURE:
 Wash the petri dish and sterilize by using hot air oven at 160˚c for 1 hour.
 Weigh the accurate quantity required for medium is mixed with the distilled
water in conical flask. Plug conical flask with non-absorbent cotton wool and
sterilized the media in autoclave at 121˚c.
 For 15min at 15LB pressure molten agar is poured into the sterile petri dish.
 Laminar air flow benches the volume should 3/4th plate.
 Allow medium to reach room temperature which done solidified cover plate
with lid. Nutrient agar plate is ready for further experiment.

REPORT:

36
 INOCULATION OF MICRO – ORGANISMS IN SOLID AND
LIQUID MEDIUM

AIM: To inoculate the given micro-organism in solid and liquid media using aseptic
technique.

REQUIREMENT: Petri dish, test tube, conical flask, glass rod, nutrient agar and
nutrient broth.

PROCEDURE:

PREPARATION OF NUTRIENT BROTH:

 Weigh the required quantities of nutrient broth and mix it with distilled water in a
broth and mix it with cotton and sterile the media in a autoclave at 121˚c for
30minutes at 1516 pressure mean while wash the test tube at culture tube, dry it
without any water droplets.

 Then it is wrapped with the help of a brown sheet, the packet glassware is
sterilized by using hot air oven 160 ˚c for 1 hour.
 After sterilization period, remove the glassware or the culture media from the
instrument and it was taken into the aseptic area, clean the laminar air flow
bench with the alcohol swaps.
 Then opens the wrapping of glassware, put the test tube in a test tube holder,
nutrient broth in the conical flask is transferred.

NOTE:
 The aseptic transfer is done as such the same procedure which is followed in the
nutrient broth. The inoculated plates are kept in incubator at inverted position.
 After the incubation period, remove the plate from incubator and observe the
colonies on the surface at the agar slab.

REPORT:

37
 INOCULATION OF MICRO-ORGANISM BY STABBING AND
SLANT METHOD

AIM: To study the nature of growth of micro-organism by stab and slant tube method.

PRINCIPLE:
In this stabbing method, the organism is stabbed, deep into the agar which shows the
growth depending upon the requirement of oxygen after the incubational period, the slant
method is utilized for pressure the culture in the refrigerator for further use. The
organism is grown on the surface of the slank.

MATERIALS REQUIRED: Test tube inoculating 100x, inoculating medium culture for
inoculation nutrient agar medium.

PROCEDURE:

1. STABBING METHOD:

Nutrient agar is prepared and sterilized by using autoclave. The molten agar is taken in a
sterile test tube and allow it to cool by keeping the test tube in a straight position, after
solidification of agar inoculate the given organism with the help of agar inoculate is
stabbed deep into agar medium. Incubate the inoculated tube at 37˚c for 24hours after
the incubated period, observe the growth of micro-organism in the media.

2. SCANT TUBE METHOD:

Prepare nutrient agar and sterilize it by autoclave. The molten agar is taken in a sterile
test tube and allowed to cool by keeping the tube in a slanting position with the help of
glass rod or folded paper. After the solidification inoculating the given micro-organism
with the help of inoculating loop of by sticking over the sleet surface of agar. Keep it
for inoculation for 37˚c for 24 hrs. After inoculation period observe the growth on the
sleet surface of agar medium.

REPORT:

38
 ISOLATION OF PURE CULTURE BY STREAK PLATE
METHOD

AIM: To isolate the pure culture from the given mixed culture by streak plate method.

MATERIAL REQUIRED: Mixed culture, inoculating loop, sterile nutrient agar and
petri dish.

PROCEDURE:

 Prepare sterile agar plates and mark point with glass marker at the bottom of plate,
using appropriate aseptic techniques

 Pour the culture media in given glassware.

 Remove a cotton swab in culture broth from the mixed culture, with the help of
sterile inoculating loop lift the lid of the plate and streak the organism which in
the inoculating loop by keeping at one point on the surface of the agar.

 The streaking should be plate for 37˚c for 24 hours.

REPORT:

39
 SOLATION OF PURE CULTURE BY SPREAD PLATE
METHOD

AIM: To isolate pure culture from the given culture suspension and to study the nature
of growth of micro- organism by using spread plate method.

PRINCIPLE: The organism is spreads over surface of the agar plate and kept for
incubation which was then observes the colonics on surface of agar plate.

MATERIAL REQUIRED: Petri dish, L-shaped streaker, inoculating loop, nutrient

agar.

PROCEDURE:

 Prepare the required quantity of nutrient agar, media and sterilize it. The molten
medium is poured in the sterile petri dish which it is kept for solidification in the
aseptic room.

 Aseptically transfer a loop full of culture suspension on the surface of solidified agar
by using poor aseptic techniques with the help of L-shaped spreader, spread the
placed culture suspension over the surface of agar plate.

NOTE:

The L-Shaped spreader is made up of glass or plastic which is sterilized with the help
of alcohol. After spreading the organism in the plate is kept for incubation in the
incubator for 24hours at 37˚c. After the incubation growth on the surface of the agar
plates and removes the isolates, colonies strapping of the agar which are taken in the
loop is introduced in the fresh sterile nutrient broth medium and kept for incubation.
The incubation allows the growth of pure culture in the medium which is further used.

REPORT:

40
 ISOLATION OF PURE CULTURE BY POUR PLATE METHOD
AIM: To study the isolation of pure culture and the nature of growth of micro-organism
from the given method.

PRINCIPLE: In this technique the inoculation is introduced into the nutrient broth for
medium before processing it into the plate after the sterilization. The mixed organism will
show thin pointed colonies on surface of agar plates.

MATERIALS REQUIRED: Petri dish, test tube, inoculating loop.

PROCEDURE:

 Prepare the required quantity of nutrient agar and sterilize it. The molten agar is
taken in a different sterile test tube, labeled as test tube A and B cool the media
needs to the room temperature.

 Then aseptically transfer a loopful of culture media suspension in test tube

aseptically mix the culture media by rolling the test tube. The sterile Petri dish
and make up the required with the help of test tube.

 Check the temperature is equal in both the test tube. Allow the media to solidity
the solidified agar position.

 After the incubational period, observe the pin point colony on the surface of the
agar which is transformed into fresh sterile nutrient agar and broth, with the pure
culture is kept for incubation for the growth of pure culture which is used for
further study.

REPORT:

41
 PREPARATION OF SMEAR
INTRODUCTION:

The microscope examination of microorganism is a valuable identification technique in

order to view microbes. It is necessary to prepare the slides of microorganisms.
Microscopic evaluation may be either wet mounts or smears. Wet mount involves
placing cell in a drop of water, adding a cover slip and viewing the material under
microscope.
Wet mounts are temporary preparations and the ability to stain is limited. A smear is a
thin preparation of cell allowed to dry on a slide. This material is then fixed to slide using
heat (or) chemical. A smear is more permanent preparation and may be stained using a
variety of techniques. Smears are made using a variety of techniques. Smears are made
using plain tap water. Bacteria are mixed in water and allowed to dry on the slide to
make a bacterial smear. That is then fixed to slide to make a bacterial smear. This is
fixed to the cell to the slide so that they are not washed off during staining process, as it
kills the cell. So, the slide is hazardous and alter the cell wall for staining.

MATERIALS REQUIRED:

Cleaned glass-slide, Prepared cultures of staphylococcus (E. coli), Inoculating loop,

Laboratory marker.

LABORATORY PROCEDURE:

 Sterilize the inoculating loop or needle into the flame. It must glow red for 3
seconds. A hot loop of needle should be cooled slightly before touching a
bacterial colony to prevent the killing of the cells. Remove the lid from a
bottle/tube, grasp the little finger of the dominant hand to loosen and remove the
lid from the bottle/tube.
 Return the lid to the tube and tighten the lid.
 Glass-slide should be relatively clean and grease free. Work with one sterilized
inoculating loop aseptically. Remove the lid from the bottle and remove a loopful
of water from the bottle. Return the lid to the bottle. Tap the loopful of water on
the center of the curve of labelled slides. Sterilize the loop. Obtain the standard
culture of one of the organisms. Aseptically remove the lid.
 Insert the sterilized loop carefully not much to touch the lid of tube. Touch the
loop of surface of the agar. Do not scrap or dip into agar. Remove the loop in a
drop of water on a appropriately labelled slide.
 Spread the drop on the surface of slide making a uniform preparation of bacteria
and water. Then heat the smear, quicken it will dry. Allow the smear to air dry.
Heat the slide by passing it 10 times on the loop of the flame.

REPORT:
42
OBSERVATION:

Sample Reagent Observation

Sample-1 Methylene blue Spherical shaped,

Blue colour

Sample-2 Saffranine Bean shaped,

Red colour

43
 SIMPLE STAINING
AIM: To determine the morphology of the given organism.

PRINCIPLE:
Bacteria have almost same refractive index as water. This means when you try to
view them using a microscope. They appear to be a stained cell which easier to see
differential stain that gives two or more stains. Categorize cell into groups. Both stain
technique allows the detection of cell morphology or shape. But the differential stain
used in microbiology have 3 basic steps and morphological types: round cells are known
as cocci; rod shaped bacillus and spiral cells are called spirilla.

MATERIALS REQUIRED:
Heat fixed bacterial smears, Methylene blue, Crystal violet, Saffranine, Paper,
Towel, Microscope

PROCEDURE:
 Place the slide on the staining rack and flood the slide. Stain for one
minute. Rinse the slide in tap water.
 The slide is slightly tilted to rinse all the stain from the slide. Grasp the slide to
remove excess water.
 Place a piece of tissue paper on the lab table and place the slide on it. Fold the paper
over the slide and gently go to the slide to remove water. Examine the stained smear
with microscope.

REPORT:

44
 GRAM STAINING
AIM: To determine the morphology of given organism.
PRINCIPLE:
Gram staining is the differential stain and the results are based on the cell wall which is
primarily made up of carbohydrates know as peptidoglycans. Other bacterial cell wall is
thicker and composed of peptidoglycans and diphenyl polysaccharides. Peptidoglycans
are soluble in organic or non-polar solvents such as alcohol or acetone but
lipopolysaccharides are non-polar such as alcohol. The stain can also be used as simple
stain as it covers the cell wall of the bacteria. Gram iodine act as primary moderate stain
also be used as simple stain to make black crystals that is not is easily washed off from
the cell. At this point in this staining process or the cell wall structure displaced by the
discoloration. Alcohol and acetone is used on all organisms. The discoloration does not
affect those with lipid components of the peptidoglycan. The cell of the lipid is dissolved
by acetone or alcohol. This iodine crystal violet complex is washed out of the cell wall.
At counter stain safranin is applied to the cell, which will rise the colorless cells. The cell
that stains the primary stain will appear purple or blue known as composite cells. The
LPS of gram-positive cells not only act for staining but also act as bacterial endotoxin
STANDARD ABBREVIATIONS:
GPC- Gram Positive Cocci
GNC- Gram Negative Cocci
GPB- Gram Negative Bacili
GNB- Gram Negative Bacili
MATERIAL REQUIRED:
Smear (heat fixed)
REAGENTS:
Gram stain, crystal violet, gram iodine, acetone, alcohol and safranin
PROCEDURE:
Cover the label of the tape. Place the slide on the staining surfaces and flood with crystal
violet for one minute. Rinse the slide with tap water, tilting, pour a few drops of iodine
on the slide. Rinse of with the use of water, and flood of with gram iodine for one minute.
Rinse the slide with water with slide tilted, add a drop of acetone or alcohol which
decolorizes. The blue color completely runs from the smear. Immediately rinse with
water add safranin to the slide, wait for one minute. Rinse the safranin from the slide with
tap water, gently tap the slide to excess water. Place a piece of paper or towel on the lab
table and put the slide on it. Hold the paper over the slide tilt the slide containing water.
Examine the stained smear with microscope and record the results.
OBSERVATION:
The cells of S. aureus appear blue and are recognized as non- acid fast.
REPORT:

45
 ACID FAST STAINING
AIM: To determine the given organism is acid fast stain or non-acid fast staining.

PRINCIPLE:
The acid-fast stain is differential stain that measures the resistance of the microbial cell to
decolorize acidic agent develop first by Paul Christian in 1988 & later modified by Linch,
Nelson. This method allows to measure the property of acid fastness by bacteria vitally
used for the identification of microorganism tuberculosis mycobacterium leprae. In this
turning procedure bacteria are classified into acid-fast if they retain their primary stain
after washing with acid, on- acid fasting if they get de-colorized after washing with acid.
On acid fast bacteria retain the counter stain methylene blue. The property of acid
fastness is correlated with a high lipid content in the cell wall.in the acid-fast stain, serve
also as a good identification tool for a number of harmless saprophytic bacteria.

MATERIALS REQUIRED:
Culture of staphylococcus aureus, Acid fast reagent {(a)carbolfuchsin (b) acid alcohol
(c) methylene blue} Staining rack, Hot plate or water bath, Distilled water, Bunsen
burner, Wash bottles, Glass slides, blotting paper, Inoculating loop, Microscope

PROCEDURE:
 Prepare the smear of S. aureus on two
slides. Allow the smear to dry in air
or heat fix
 Heat the slides with a set of steam coming from water bath for about 5-
10 mins Stain the slide with a drop of carbolfuchsin
 Take care not to dry smear.
 Cool the slide and wash with distilled water.
 Decolorize the smear in acid alcohol for about 20-30 mins until they are faint pink
colour. Rinse with distilled water.
 Counter stain with methylene blue for about 80-
90 secs. No heating is required.
 Wash with distilled water.
 Bloat dry with bloating paper carefully.do not wipe the slide.

OBSERVATION:

The cells of S. aureus appears blue and are recognized as non-acid fast.

REPORT:

46
 DETERMINATON OF MOTILITY BY HANGING DROP
METHOD

AIM: To determine the motility of the given sample by using hanging drop method.

PRINCIPLE:
Living bacteria have no color and are small. They are really difficult to see with oil
immersion. All bacteria have some vibrational movements even non- motile. The
Brownian movement is caused by water molecule in solution. Knocking against each
other is the microorganism kinetic energy which is inherent to all molecular causing this
kind of movements, on the other hand, those bacteria with flagella will be apparently
moving about the field of vision, although not all bacteria will be moving about the field of
vision. Some cell will move straight across moving about the field of vision of some will
run tumble across the field in slow motion.

MATERIALS REQUIRED:
Fresh culture, depression slide, cover slip, petroleum jelly

PROCEDURE:
 Place a drop of bacteria in the middle of the cover slip.
 Place a thin line of petroleum jelly around the edge of the
cover slip. Turn the depression slide upside down, gently
touch the cover slip.
 The jelly holds the cover slip to the slide to keep the suspension from
drying out. The slide is placed under microscope to the motility.

REPORT:

47
 STERILITY TESTING
AIM: To perform sterility test for the given sample

PRINCIPLE:
Sterility needs freedom from living organism aseptically prepared parentally product be
issued with confidence. Unless contamination have been carried out to show the risk of
contamination from a limit processing is very low. Sterility test should be effective
against bacteria, mould and yeast. Reducing agent sodium thioglycolate dentatae
substance to increase the viscosity of agar on oxidation reduction indicator.
Nutrient pancreatic digestion of casein, yeast factor extract. Amino acid induced
because it encourages the growth of certain clostridium. This formula is suitable for the
detection of certain anaerobes. thioglycolate formula is 20% usually rendered by
heading on a water bath until the pink color disappear. The treatment must not be
repeated because frequent penetration give rise to toxic degenerating product The
following of some of the for which compains with the test is required readymade injection,
inducing suspension both aqueous and oil soluble for injection, including a number of
materials from biological source.
E.g.: heparin hydroxide antibiotics: -
1) the fluid thioglycolate medium was prepared and sterilized at 1800c for 15 hrs.
2) the given sample was inoculated in medium aseptically.
3) it was incubated at 370c for 7 days.

REPORT:

48
 ISOLATION OF ACTINOMYCETES FROM SOIL AND ITS
BIOCHEMICAL CHARACTERIZATION
AIM: To isolate and perform biochemical test on actinomycetes

MATERIAL REQUIRED:
Soil sample, Tween 80, Actinomycetes isolation agar, Nutrient agar with and without
antibiotics, SD agar with and without antibiotics, Streptomycin, Amphotericin,
Nitrus reduction medium, Gelatin medium, H2S Reduction medium, Waksman
medium

PRINCIPLE:
Soil is the major source of various microorganisms as it’s providing nutrients. Principle of
the experiment involves the isolation of actinomycetes from soil by exploiting its ability
to produce antibiotics. When a soil sample is spread over a suitable solid media
actinomycetes grow as direst colonies with a clear zone around them which is due to the
inhibition of surrounding microorganism by them. This technique is called crowded plate
technique. Soil sample were collected from different places. The sample were collected
from fertile land where there are no aromatic plants. 1 gm of soil is collected at least half
feet beneath the surface.

PROCEDURE:

Soil is collected at a place which is fertile land and at the depth of 1feet. The 1 gm of soil
is dispensed in 100 ml distilled water containing 5 drops of tween 80 and shake in a
mechanical shaker for 15 min. Then it is serially diluted in a 10-fold dilution manner up
to 10-7 in sterile water. All the dilution is spreaded over the nutrient agar with and
without the antibiotics (Streptomycin). Also spread in SD agar with and without the
antibiotics (Amphotericin). The dilution is also spreaded on actinomycetes isolation
agar. All the plates are incubated at 260C for 7 days.

BIOCHEMICAL TESTS

1. MELANIN PRODUCTION TEST:

Most of the soil pigments produce melanin pigment in Waksman medium.

Yeast extract 1.0g

L-Tyrosome 1.0g
Sodium Chloride 18.5g
Agar 16.0g
Water upto1000ml
Ph 6.8

49
PROCEDURE:
Medium was prepared, sterilized and slants were made. The isolates were streaked over
the slants and incubated for 4 days at 370c.During the period of incubation the formation
of pigments was observed at every 12-hour intervals.

OBSERVATION:
After the specified incubation period, the slant showed production of pigments.

2. NITRATE REDUCTION TEST:

Nitrate reduction property of the isolate was evaluated with organic nitrate Broth.
Peptone 0.5g
KNO3 0.1g
Meat extract 0.3g
Distilled water 100 ml
PH 7.0

PROCEDURE:
10 ml of sterilized broth and a loop full of spores were added and incubated at 37oc for
one week from the 5th day. The tubes were observed for nitrate reduction properly.
The reagents used
are 2-napthalene
solution Sulphonic
acid.
To 1ml of broth under examination 2 drops of reagents a) and b) were added.

OBSERVATION:
A Positive show pink red color.

3. ACID PRODUCTON TEST

PROCEDURE:

To 10ml of sterilized broth the test organism was inoculated and incubated at 280c were
inoculated for 15 days and at a every 12 hours interval the colour was observed. (Blue-
yellow)
GLUCOSE NUTRIENT BROTH
Meat extract 3.0g
Peptone 5.0g
Glucose 10.0g
Distilled water 1000ml
Ph 7.6

50
Colour Brome thymol blue.

51
OBSERVATION:
At the end of 10th day, the change in colour (blue-yellow) was observed.

4. GELATIN LIQUIFICATION TEST:

Protein gelatine is hydrolyzed by exo-enzyme secreted by most of the soil isolates.
The nutrient gelatine medium supports the growth of micro-organism. The solid state
of the medium depends on the gelatine remaining in the medium.

NUTRIENT GELATINE MEDIUM

Peptone 5.0g
Beef extract 3.0g
Gelatin 120g
Distilled water 1000ml
Ph 6.8

The soil isolates were inoculated in sterile medium by stab culture technique. Then it is
incubated at room temperature for 10 days.

OBSERVATION:
After 10 days of incubated the gelatin medium get solidifies.

5. H2S PRODUCTION TEST:

Numerous actinomycetes are able to ferment the proteins accompanied by the production
of H2S.
Ferric ammonium citrate 0.5g
Dibasic calcium citrate 1.0g
Sodium thiosulphate 0.02g
Meat extract 1.0g
Agar 2.0g
Distilled water 1000ml
Ph 7.0
PROCEDURE:
Media was prepared, sterilized and inoculated with the spores, incubated at 37 0c for 5
days. Presence of greenish brown, bluish brown, black colour formation checked every
12 hrs.

OBSERVATION:
Black colour is produced.

REPORT:

52
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