LIST OF EXPERIMENTS
S.
No.
CONTENTS
Page No
MICROBIOLOGY
1.
Laboratory rules and regulation
14
2.
Preparation and cleaning of glass wares
15
3.
Sterilization Techniques
17
4.
Preparation of culture media- Nutrient agar
20
5.
Preparation of culture media – Nutrient broth
21
6.
Pour Plate technique
22
7.
Spread Plate technique
23
8.
Streak Plate technique
24
9.
Smear Preparation
25
10.
Simple Staining
26
11.
Gram’s Staining
27
12.
13.
Motility of bacterial cell
Antibiotic sensitivity test
1. LABORATORY RULES AND REGULATIONS
There are some rules and regulation which must be observed for the successful
completion of the laboratory exercise for the safely and convenience of the others
working in the laboratory.
1. Always wear a lab coat before entering a laboratory for protecting cloths
from accidental discoloration by staining solutions.
2. Before and after each laboratory period clean the work bench with
disinfectant like Lysol, phenol or 90% ethanol.
3. Keep laboratory bench clean off everything (like pencil, book and paper)
except laboratory period.
4. Never eat or drink inside the laboratory.
5. If a live culture spilled, cover the area with a disinfectant such as mercuric
chloride for 15 min and then clean it.
6. Never place pencil, label or any material in your mouth.
7. In the event of personal injury such as cuts or burns inform your instructor
immediately, since bacteria enter through open injury.
8. Long hair should be tied back to minimize contamination by culture and fire
hazards.
9. Be careful of laboratory burner and turn off when not in use.
10. All the microbial culture should be handled as potential pathogens.
11. The waste paper and contaminated glass wares should be kept in places
provided.
12. Wash your hands with detol soap and water before leaving the lab.
13. Broth culture should never be mouth pipette.
14. Aseptic technique must be seriously observed at all the times.
15. Always keep culture tubes in an upright position in each the rack or basket.
16. Label all the plates, tubes culture properly before starting the exercise in
sequence.
17. Keep the lab window and door closed when the experiment is in progress.
18. As you perform the exercise, record your data in ink and mark sketches and
tables in pencil.
19. Keep your lab notebook up to date.
2. PREPARATION AND CLEANING OF GLASSWARES
Glass wares used in laboratories have to be properly cleaned, washed and
sterilized before and after use.
Improper cleaning and sterilization may interfere with results and may cause
contamination. Newly purchased glass wares also have to be cleaned and washed
properly as they may be the source of chemical or microbial contamination
Before sterilization and after use, glass wares like petri plates, conical
flasks and test tubes have to be washed using the following procedures
NEW GLASS WARES:
New glass wares require special attention because of the resistant spores
present in straw and other packaging materials. These materials may give out some
alkali which is sufficient to interfere with the growth of certain organisms.
Therefore it should be placed in 1% HCl for overnight then washed with tap water
followed by distilled water and autoclaved.
Screw capped tubes are subjected to a special cleaning process till the
surface alkali is removed. The tubes may be used without further treatment as
received from the manufactures.
CLEANING OF GLASSWARE FOR LABORATORY USE:
All contaminated glassware should be autoclaved before cleaning. If this is
not possible, glass containers with discarded culture can be replaced in 3% Lysol or
simple disinfectant after use. Glass containers contaminated with spore bearing
organisms must be autoclaved. The discarded spores must be placed in a hot
detergent solution. The containers placed in hot detergent solution / treated as
recommended by manufacture / finally rinsed in de-ionised water. The glass ware is
then dried in hot air oven at 100 ºC for 3 hrs.
CLEANING OF GLASS WARE FOR BIOCHEMICAL WORKS:
Remove any grease with petroleum then wash with water. Then place the
glassware in chromic acid solution for 24 hrs after that wash with distilled water
and then dry in hot air oven at 100 ºC for 3 hrs.
CHROMIC ACID CLEANING SOLUTION:
Dissolve 63gms of potassium dichromate by heating with 35ml of water,
cooled and add Conc. Sulphuric acid and make upto one liter. This fluid should be
handled
with
care.
Rubber
gloves
and
apron
should
be
worn.
CLEANING OF PIPETTES:
Place the contaminated pipette in a 3% Lysol solution. The Lysol solution
should be placed in a plastic container about 40cm in height and 10cm in diameter,
the tips of the pipette should be broken and then it is rinsed with tap water, if
necessary they can be placed in a detergent solution of chromic acid solution. Then
wash with water followed by de-ionized water. To neutralize pipettes, place them in
an aluminum pipette cane and keep it in a hot air oven at 160 ºC for 3 hrs.
NOTE:
Accurately, calibrated volumetric glass wares should never be heated in an
oven, since the expansion and contraction makes the graduation inaccurate.
3. STERILIZATION TECHNIQUES
Sterilization is the process which elimination of living organisms including spores
from treated substances or any objects. This can be accomplished by moist heat,
dry heat and open flame sterilization.
MOIST HEAT STERILIZATION
Autoclave is a device used in the laboratory for moist heat sterilization. It
is normally operated at 15 lbs/ square inch steam pressure for 15 min. and thus
produced a temperature inside the autoclave of 121ºC (250 ºF). Sterilization in an
autoclave is done with saturated steam under pressure. Saturated steam is the
water vapour at the temperature at which it is produced.
Autoclave is a double walled cylindrical metallic vessel, made of thick
stainless steel or copper, one end of which is open to receive the material to be
sterilized. Autoclave lid is provided with pressure gauge for noting the pressure,
steam cock (exhaust valve) for air exhaustion of the chamber. Autoclave is
provided with controls for adjusting the pressure and temperature and a safety
valve to avoid explosions. The articles to be sterilization are kept loosely in a
basket, provided with roles all around for the free circulation of steam. To
increase the area of materials to be sterilized, metallic separators having holes can
be used.
USES
This is the most efficient and common instrument used for sterilizing solid
and liquid media for microbial cultures, heat stable liquids- usually the common
media ingredients, heat resistant instruments and equipments, glass wares and
rubber products. Scalpels and other sharp metal instruments are damaged by
constant flaming and are usually sterilized by autoclaving.
DRY HEAT STERILIZATION
Hot air oven is based in the principle where sterilization is accomplished by
dry heat or hot air. Sterilization is accomplished by exposure of item to 150-180
ºC for 2-4 hrs. This consists of an insulated cabinet which is held at a constant
temperature by means of an electric heating mechanism and thermostat. It is
fitted with a fan to keep the hot air circulating at a constant temperature and
thermometer for recording the temperature. For proper circulation of the hot air,
the shelves are perforated. For normal sterilization work, the oven should be
operated at 160 ºC and most glass wares will require a period of two hrs for
complete sterilization.
The schedule of time and temperature for sterilization with dry air
Temp (ºC)
120
140
150
160
170
180
Time(min)
480
180
150
120
60
20
USES
Commonly used for sterilizing glass wares like petridishes, test tubes, pipettes,
metal instruments that can tolerate prolonged heat exposure, oils, powder, waxes
and other articles that are either spoiled or not effectively sterilized by the moist
heat of the autoclave.
OPEN FLAME STERILIZATION:
Contaminated materials that are combustible may be disposed off by burning. The
inoculation needle or loops and flaming the tips of the tubes and conical flasks are
directly heated in an open flame or Bunsen burner or an alcoholic lamp.
LAMINAR-FLOW HOOD:
This equipment used for reducing the danger of infection while manipulating
infectious microorganisms and for preventing contamination of sterile materials.
In a closed room where handling of microorganisms, like transfer and
inoculation are done constantly and in operation involving lyophilization and opening
of lyophilized culture, large amount of floating microbial cells and aerosol may be
produced.
Radiation and other treatments may not be effective always in keeping such
room free of particles and droplets containing microorganisms. To keep such room
free of all microbes carrying particles, a new kind of technology has been
developed known as laminar air-flow technique. This technique involves passing the
air of a closed room of cabinet through high efficiency particulate air (HEPA)
filters pack. When air thus passes through, becomes free of all particles above
0.3µm dimension. This also involves sucking of air from the room and blowing out
the air through a back of filters with uniform velocity and in parallel flow line.
Both horizontal and vertical laminar air –flow systems are now used in
microbiological and pharmaceutical laboratories. Hospital rooms are now designed
with laminar air-flow system. The advantage of the system is that in operation
involving inoculation, transfer of culture and opening of lyophilized culture no
closed chamber is necessary. Instead the operation can be done on platform
provided with the laminar air flow unit making it easier for handling.
USES:
THE LAMINAR AIR FLOW
(a) Separates contaminants from the interior space by creating a curtain of
filtered air across the front opening. The worker has complete access to the
materials inside the hood. Air is circulated through filters.
(b) Before it for exhausted, laminar air-flow hoods are standard equipments in
tissue culture laboratories and are required for contaminant of genetically
engineered microorganisms.
Room air is filtered before entering the working chamber and moves
in a single direction. Contaminated air from the chamber is filtered again
before being either re-circulated in the system or exhausted to the outside.
4. PREPARATION OF CULTURE MEDIA – NUTRIENT AGAR
AIM:
To prepare the nutrient agar medium for bacterial growth.
REQUIREMENTS:
i.
250ml conical flask
ii.
Distilled water
iii.
pH meter
iv.
Electronic balance
v.
Non- absorbent cotton.
MEDIUM COMPOSITION (G/100ML):
a. Yeast extract (1g)
b. Peptone (1g)
c. NaCl (0.5g)
d. Agar (2g)
e. Distilled Water(100ml)
PROCEDURE:
1. A clean glass conical flask was taken with 100ml distilled water.
2. The medium components were weighed accurately and dissolved one by
one in distilled water. (Care was taken to see that the chemical was
added to distilled water only if the pervious chemical was dissolved
completely).
3. The pH was adjusted to 7.2 using 1N NaOH solution.
4. The mouth of the flask was then plugged with non-absorbent cotton, and
then it was covered with brown paper then tied with a thread tightly.
5. The flask was then autoclaved at 121ºC for 15 min.
RESULT:
Nutrient agar plate was prepared for the cultivation of bacteria.
5. PREPARATION OF CULTURE MEDIA – NUTRIENT BROTH
AIM:
To prepare the nutrient broth medium for bacterial growth.
REQUIREMENTS:
i.
250ml conical flask
ii.
Distilled water
iii.
pH meter
iv.
Electronic balance
v.
Non- absorbent cotton.
MEDIUM COMPOSITION (G/100ML):
a. Yeast extract (1g)
b. Peptone (1g)
c. NaCl (0.5g)
d. Distilled Water(100ml)
PROCEDURE:
1. A clean glass conical flask was taken with 100ml distilled water.
2. The medium components were weighed accurately and dissolved one by
one in distilled water. (Care must be taken to see that to see that the
next chemical was added to distilled water only if the previous chemical
added was dissolved completely).
3. The pH was adjusted to 7.2 using 1N NaOH solution.
4. The mouth of the flask was then plugged with non-absorbent cotton, and
then it was covered with brown paper then tied with a thread tightly.
5. The flask was then autoclaved at 121ºC for 15 min.
RESULT:
Nutrient broth culture media was prepared for the cultivation of
bacteria.
6. POUR PLATE TECNIQUE
By pour plate technique, individual colonies can be observed throughout the
nutrient agar medium. The colonies will be of different size, shape and color. From
the individual colony, pure colonies can be isolated.
MATERIAL REQUIRED:
Test tubes, pipettes, sterile distilled water, Bunsen burner, test sample etc.
MEDIA USED:
Nutrient agar medium
PROCEDURE:
1. 1ml of the liquid sample was transferred into the test tube which
contains 9ml of distilled water blank. Here the dilution is 10-1 and this
has to be mixed properly.
2. Using a sterile pipette, 1ml from 10-1 dilution was transferred to the
next test tube which contains 9 ml distilled water (10-2)
3. Subsequent dilutions were made up to 10-5
4. Then 1ml of each dilution was transferred to each sterile Petri plates.
5. Then the molten nutrient agar was poured into the plates and was
rotated gently to ensure a uniform distribution of cells in the medium.
6. The step 5 was repeated for each dilution.
7. Then the media was allowed to solidify.
8. After solidification, the plates were incubated for 18 - 24 hrs at
optimum temperature in an inverted position.
CALCULATION:
CFU =
RESULT:
Total Number of organism present is a given sample = _________________
cfu/ml
7. SPREAD PLATE TECHNIQUE
This is another method, which is also used for the isolation of individual
colonies. In this technique, microorganisms are spreaded over the solidified agar
medium with a sterile L-shaped glass rod while the pertidrsh is spun on a turn
table. The principle is, as the petridish spuns, at some stage single cells will be
deposited with the bent glass rod on the agar surface. Some of these cells will be
separated from each other by a distance sufficient to allow the colonies that
develop to be free from each other.
MATERIAL REQUIRED:
Nutrient agar medium L rod, tubes, petriplates, distilled water etc.
PROCEDURE:
1. 9ml of distilled water was taken in different test tubes and was
sterilized.
2. After sterilization, 1ml of the sample was added to the first tube(101
)
3. From the above, 1ml of diluent was taken and added to the second
test tube (10-2). In this method all the test tubes were serially
diluted.
4. About 0.1ml of sample was taken from each dilution and applied to the
surface of nutrient agar plate and spreaded uniformly by using sterile
‘L’ rod.
5. After inoculation, the petriplates were incubated at optimum
temperature for 24hrs in an inverted position.
CALCULATION:
CFU =
RESULT:
Total Number of organism present is a given sample = _________________
cfu/ml
8. STREAK PLATE TECHNIQUE
The streak plate method is a qualitative isolation method. If a microbial
culture is applied to the surface of agar in a petriplate and spreaded with a loop or
a bent needle it is called streaking and the plate thus prepared is called as streak
plate. Ther streaking ‘thins out’ the bacteria on the agar surface so that the
individual bacteria are separated from each other. The streak plate technique can
be done by the following methods.
i.
ii.
Phase Streaking
Continuous streak
MATERIALS REQUIRED:
Plates with sterile agar medium, inoculation needle, etc.
PREPARATION OF AGAR PLATES FOR STREAKING:
About 15-20ml of melted sterile nutrient agar medium was allowed to
solidify.
A. PHASE STREAKING:
1. A loop full of organisms was taken in the inoculation needle and
spreaded evenly on a small area of the agar plate.
2. Then three or four parallel streaks were made.
3. The inoculation needle was then sterilized and 3 or 4 streaks were
again made at right angles from that of the first streak.
4. The loop was flamed again and the step 3 was again repeated.
5. Then the streaked plates were incubated in an inverted position at
optimum temperature for 24 hrs.
B. CONTINUOUS STREAKING:
A part of the inoculums was taken in the inoculation loop and spreaded well on
the agar plate and the streak was done in one continuous motion.
The plates were then incubated at optimum temperature for 18-24 hrs in an
inverted position.
RESULT:
Isolated
observed on final
Isolated colonies
maintain as a
colonies were
streak path area.
were selected and
pure culture.
9. SMEAR PREPARATION
The preparation of a smear is required for many laboratory procedures,
including the Gram-stain. The purpose of making a smear is to fix the bacteria onto
the slide and to prevent the sample from being lost during a staining procedure. A
smear can be prepared from a solid or broth medium. Below are some guidelines for
preparing a smear for a Gram-stain.
1. Place one needle of solid bacterial growth or two loops of bacterial
growth in the center of a clean slide.
2. If working from a solid medium, add one drop (and only one drop) of
water to your specimen with a water bottle. If using a broth medium, do
not add the water.
3. Now, with your inoculating loop, mix the specimen with the water
completely and spread the mixture out to cover about half of the total
slide area.
4. Place the slide on a slide warmer and wait for it to dry. The smear is now
ready for the staining procedure.
RESULT:
Thin bacterial smear was prepared for staining.
SIMPLE STAINING
AIM:
To elucidate morphological shapes and arrangements of bacterial cells by
using simple stain technique.
PRINCIPLE:
In simple staining, the bacterial smear is stained with a single reagent. Basic
stains with a positively charged are used because the bacterial nucleic acids and
certain cell wall components carry negative charge that strongly attracts and bind
to the cationic chromogen. The commonly used basic stains are methylene blue,
crystal violet and carbol fuchsin.
MATERIALS REQUIRED:
Bacterial sample, Methylene blue stain, Microscope slides, inoculation loop,
Bunsen burner etc.
PROCEDURE:
1. By means of a sterile inoculation loop, a drop of the bacterial suspension
was transferred to the center of the slide.
2. The drop over the slide was spreaded to about 1sq.inch as a thin film and
allowed to air dry.
3. Then the smear was fixed by passing the slides rapidly over the flame.
4. The smear was then flooded with several drops of methylene blue and
allowed to remain for 1-2 min.
5. The slide was washed after 2 min in a gentle stream of water to remove
the excess of stain and the slide was air dried.
6. Then the slide was examined under oil immersion microscope.
Simple Staining
RESULT:
The bacterial cells stains blue and the cells were in ______________
Shape and are referred as ____________________
10.
GRAM’S STAINING
AIM:
To differentiate the two principal groups of bacteria ie. Gram positive and
gram negative on the basis of the gram stain.
PRINCIPLE:
Most bacteria can be differentiated by the gram reactions due to difference in
their cell wall structure.
GRAM POSITIVE BACTERIA
Cell wall has a large amount of peptidoglycan.
GRAM NEGATIVE BACTERIA
They have less amount of peptidoglycan in their cell wall. They have
lipopolysaccharide containing a compound known as lipid A or endotoxin.The
organisms that retain purple color with crystal violet and are not decolorized by
acetone iodine are called gram positive bacteria.The organisms that loose their
color of crystal violet after being treated with acetone iodine are called gram
negative bacteria.
Differential staining requires the use of atleast three chemical agents that
are applied sequentially to the heat fixed smear. The first reagent used is called
the primary stain. Its function is to impart its color to all the cells. When iodine
solution is applied, it replaces the chloride in the crystal violet molecule and a
complex is formed which becomes insoluble in water. This complex is formed in
both the gram negative cells. This is due to the difference in the composition of
the cell wall. The gram positive cell wall having a lower lipid content becomes
dehydrate, making the pore size small and thus not allowing the get dehydrated
and thus the pore size remaining the same allows the complex to leak out on
applying the counter stain, the cells that have last the primary stain appearing pink.
The gram positive cells that retain the primary stain colour appears violet.
MATERIALS REQUIRED:
Bacterial Sample, Microscopic slide, Microscope, inoculation loop, blotting
paper, spirit lamp etc.
REAGENTS:
a. Crystal violet
b. Grams iodine
c. 95% ethyl alcohol
d. Safranin
PROCEDURE:
1. The smear of the organisms was made in a clean glass slide, an dried and
then it was heat fixed.
2. The fixed smear was covered with crystal violet stain for 30-60 seconds.
3. Then the stain was rapidly washed off with distilled water
4. Again the smear was covered with gram’s iodine solution for 30-60
seconds.
5. Using the distilled water, the iodine was washed off.
6. The smear was then decolorized rapidly with 95% alcohol and washed
immediately with distilled water.
7. The smear was again covered with safranin for 1 min.
8. The above stain was washed off with distilled water.
9. The slide was then allowed to air dry.
OBSERVATION:
The smear was examined microscopically with 40x and 100x objective for
morphology and arrangement of cells and microphotograph was made.
RESULT:
The bacteria appeared _________________ color, ________________
shape and are referred as________________________.
12.MOTILITY TEST- HANGING DROP SLIDE
AIM:
To gain expertise in determining the motility of living bacteria.
PRINCIPLE:
Most motile bacteria move by use of flagella, thread like locomotor
appendages extending outward from the plasma membrane and cell wall. Motile
cells may have one flagellum, but most have several. Each flagellum has a very rigid,
helical structure and actual motility results from the rotation of the flagellum in a
manner similar to that of a boat propeller. Motility by means of flagella is
particularly important for the identification of microorganisms, for instance Bacilli
are motile except for the anthrax, which is instead capsular, therefore flagella
could be produced to enable the organisms to run away from danger and to move
from less favorable to more favorable environment.
Materials Required :





Clean depression slides and coverslips
Petroleum jelly / Vaseline
Inoculating loop
Toothpicks
Cultures:- 24- to 48-hour Nutrient broth cultures of Pseudomonas
aeruginosa
Procedure:
1. With a toothpick, spread Vaseline or Petroleum jelly on the four corner of a
clean coverslip.
2. After thoroughly mixing one of the cultures, use the inoculating loop to
aseptically place a small drop of the bacterial suspension in the center of a
coverslip
3. Lower the depression slide, with the concavity facing down, onto the
coverslip so that the drop protrudes into the center of the concavity of the
slide .Press gently to form a seal.
4. Turn the hanging drop slide over and place on the stage of the microscope so
that the drop is over the light hole. Examine the drop by first locating its
edge under low power and focusing on the drop. Switch to the high-dry
objective (40 x). To increase the contrast and to see the bacteria clearly,
close the diaphragm as much as possible.
5. Distinguish between actual motility and Brownian movement
6. Discard the coverslips and any contaminated slides in a container with
disinfectant solution.
Hints:
1. Always examine a hanging drop preparation immediately, because the
organisms become less motile with time.
2. Particular care must be taken to avoid breaking the coverslip since it is more
vulnerable when supported only around its edges
3. Always make sure the specimen is on the top side of the slide.
Expected Results:
Actively motile organisms (Pseudomonas aeruginosa) were observed
OBSERVATION:
The smear was examined microscopically with 40x and 100x objective for
morphology and arrangement of cells and microphotograph was made.
RESULT:
Actively motile organisms organism name were observed