INDEX

PART 1: Basics
Exp 1.1: Introduction to microbiology laboratory and practices

PART 2: Bacterial isolation, identification and maintenance

Exp 2.1: Isolation of pure cultures of bacteria from various samples and cell count
Exp 2.2: Grams staining of bacteria
Exp 2.3: Preparation of glycerol stock for long term preservation
Exp 2.4: IMViC test for biochemical characterization of bacteria
Exp 2.5: Test of hydrolytic enzymes (pectinase, cellulase, amylase, protease) in bacteria
Exp 2.6: Fluorogenic detection of E. coli

PART 3: Microbial population

Exp 3.1: Coliform counts in contaminated water sample
Exp 3.2: Dehydrogenase activity assay for qualitative determination of microbial population
Exp 3.3: Enumeration of microbial cells in air microflora

PART 4: Abiotic and biotic Factors

Exp 4.1: Bacterial growth curve
Exp 4.2: Effect of pH, temperature, salt and radiation on growth of microorganisms
Exp 4.3: Effect of various antibiotics on microbial growth
Exp 4.4: Effect of various metals on microbial growth

PART 5: Fungal microbiology

Exp 5.1: Isolation of pure cultures of fungus
Exp 5.2: Staining of fungus- Lactophenol cotton blue staining
Exp 5.3: Study of hydrolytic enzymes (pectinase, cellulase, amylase, protease) in fungi
Exp 5.4: Determination of size of fungal spore using micrometry
Exp 5.5: Isolation of arbuscular mycorrhizal fungi from rhizospheric soil samples
Exp 5.6: Production of citric acid from fungus using laboratory fermentor

Exp 1.1: Introduction to microbiology laboratory and practices

Preface
As well as causing a familiar range of diseases in animals and plants and problems in food
spoilage and deterioration of other materials, microbes are also our invisible allies. Indeed,
life on Earth would not be sustainable without the benefits that many of them provide. The
teaching of such an important subject as microbiology cannot be achieved effectively without
enhancing the theory with hands on experience in the laboratory. The purpose of this
manual is to provide teachers and technicians with good techniques in practical microbiology
to ensure that investigations proceed safely and achieve the required educational aims
successfully.
Safety guidelines
The small size of microbes and the consequent need to deal with cultures that contain many
millions of microbial cells require special procedures for their safe use. Activities involving
micro-organisms are must be carefully managed and controlled following strict guidelines
drafted by an authorized regulatory body. Teachers, students and technicians have a duty to
comply with any safety instructions given by their employers under the Health and Safety at
Work guidelines. The guidelines are straightforward and largely commonsense and, as such,
are not an obstacle to conducting interesting microbiological investigations in a institutional
laboratory.
Risk Assessment
Factor
Level of practical work

Relevance
Degree of risk of microbial culture; expertise of teacher; age
range of students
Choice of micro-organisms
Present minimum risk; refer to list of suitable cultures
Source of cultures
Reputable specialist supplier or approved environmental sample
Type
of Adequate containment of cultures; class practical work vs.
investigations/activities
teacher demonstration
Composition
of
culture Possibility of selecting for growth of pathogens
media
Volume of cultures Increased risk with increase in volume of
liquid culture
Laboratory facilities
Suitability for level of practical microbiological work
Equipment
Adequate for purpose
Incubation conditions
Possibility of selecting for growth of pathogens
Disposal procedures
Ensures elimination of risk to others
Expertise of technicians and
Competence and level of training in techniques and procedures
teachers
appropriate to level of practical work
Student age and discipline
Appropriate to level of practical work; confidence in class
discipline

Good microbiological laboratory practice (GMLP)

Training in GMLP is aimed at developing proficiency in containing any uncontrolled spread
of microbes in order to protect:
1. Practical investigations from becoming contaminated with microbes from external sources
2.The operators (students, teachers and technicians) from the very small possibility of
infection. It is important to arrange the workplace carefully to ensure safe and effective
operations.

Spills: Spillages of cultures must be reported immediately to the teacher or technician to be

dealt with quickly. The keeping of a record of all such incidents is recommended. Spilled
cultures and surrounding debris (e.g. glass, cotton wool plugs), if any, must not be touched
with unprotected hands. Wearing disposable gloves, disinfect the area by covering the spill
with several layers of paper towel/cloth soaked in a suitable disinfectant and leave for 15-30
minutes. Spill debris should then be swept into a dustpan using paper towels. All disposable
material should then be transferred to a suitable container, e.g. an autoclave/roasting bag, for
autoclaving and disposal. The dustpan must be decontaminated either by autoclaving or by
soaking (at least 24 hours) in Hypochlorite (sodium chlorate I).
Broken glass: Observe an appropriate disposal procedure for broken glass if present. It should
be swept carefully into a suitable container, autoclaved and disposed of in a puncture proof
container.
Splashes on clothing and the skin: Contaminated clothing should be soaked in disinfectant.
Splashes on the skin should be treated as soon as possible; washing with soap and hot water
should be sufficient, but if necessary the skin can be disinfected.
Aerosols: Spillages also carry a risk of generating aerosols (an invisible mist of small
droplets of moisture) which may contain microbes and might be inhaled. The risk of spillages
occurring is lessened by using cultures grown on agar instead of in liquid media whenever
possible. Care should also be taken to avoid generating aerosols during practical work.
Resources
Equipment:
Equipment
Loop (wire/plastic)
Straight wire

Use
Routine inoculation of agar slopes/deeps and small volumes of
liquid media (up to ca 10cm3); making streak plates
Inoculation from very small colonies; transfer of small inocula
from liquid media for nutritional work
3

Spreader (glass/plastic/metal) Making lawn/spread plates

Forceps (metal/plastic)
Transfer of sterile paper/antibiotic discs; also plant material,
e.g. short lengths of root with nodules
Pipette (calibrated)
Transfer of measured volumes/drops of culture/sterile solutions
Teat (For manual pipette)
Filling and emptying pipettes safely (never pipette by mouth)
Test tube
Small volumes (ca 5-10 cm3) of liquid media/agar slopes/sterile
solutions for inoculation (held in test tube rack; dry nonabsorbent cotton wool plug or plastic cap prevents
contamination)
Universal bottle (wide neck); Volumes of liquid and agar media/sterile solutions up to ca 20
McCartney bottle (narrow
cm3 for inoculation or for storing sterile media or stock cultures
neck)
on agar slopes (stay upright on bench; plastic screw cap
prevents contamination and reduces evaporation during long
storage)
Conical flask
Large volumes of liquid media for inoculation and liquid/media
for short-term storage(non-absorbent cotton wool plug prevents
contamination but does not reduce evaporation during long
storage)
Petri dish (plastic/ glass)
Plastic: pre-sterilized for streak/spread/lawn/ pour plates;
Glass: only to be used after sterilization by autoclaving
Marker pen
Labelling Petri dishes, test tubes, flasks, bottles and microscope
slides
Personal protective
Clean laboratory coat/apron: protection of clothing,
equipment
containment of dust on clothing; Safety spectacles: not
considered essential when dealing with suitable cultures and
observing GMLP but may be required by local regulations and
for dealing with chemicals
Apparatus:
Apparatus
Bunsen burner and Laminar
air flow chamber

Impervious sheet or tray

Autoclave/pressure cooker

Gas ring
Autoclavable bag
Hot air oven
Discard pots containing
disinfectant
Microwave oven
Incubator
Water bath

Use
Bunsen burner is used for sterilization of wire loops and (with
alcohol) metal forceps and spreaders; Laminar air flow chamber
is used for maintaining aseptic condition by filtering air through
HEPA filters.
Provides individual student working area if the bench surface is
not appropriately sealed
Sterilization of media, solutions and equipment before use and
contaminated items afterwards; melting solidified agar media
for use.
Steam generation in autoclave
Holds contaminated items in autoclave to contain spillages
Drying of glass and plastic wares after autoclaving
Disposal of used pipettes and slides of non-stained
microscopical preparations
Melting solidified agar media for use (but not in vessels with
metal caps and not for sterilization)
Incubation of cultures for optimum growth
Suitable temperature for keeping melted agar media molten for
use (ca 50C); accurate temperature control
4

Thermometer
pH meter
Cupboard
Refrigerator/Cold room
Microscope
Materials:
Material
Culture media ingredients

Disinfectants

Ethanol (70%)
Autoclave indicator tape

Sterilizer control tube/strip

Non absorbent cotton
Spillage kit

Checking incubator/water bath temperatures

Checking and adjusting pH values of media
Storage of culture media and stock cultures
Storage of heat-labile materials and preservation of cultures
Microscopical observations

Use
Stock of a range of culture media in dehydrated form
(tablets/powder); available as complete media and as separate
ingredients
Treatment of work surface before and after use and spillages;
disposal of used pipettes and microscope slides; in soap form
for hand washing
Sterilization of metal forceps and glass spreaders by ignition
and common disinfectant
Changes colour in response to heat to distinguish those items
that have received heat treatment (but is not an indicator of
effective sterilization)
Changes colour when correct temperature has been applied and
held for the required length of time to effect sterilization
Wool Plugs for test tubes, flasks and pipettes
Dealing with spilled cultures

Preparation of culture media

Rehydrate tablets or powder according to manufacturers instructions. Before sterilization,
ensure ingredients are completely dissolved, using heat if necessary. Avoid wastage by
preparing only sufficient for either immediate use (allowing extra for mistakes) or use in the
near future. Normally allow 20-25 cm3 medium/ Petri dish. Dispense in volumes appropriate
for sterilization in the autoclave/pressure cooker. Agar slopes are prepared in test tubes by
allowing sterile molten cooled medium to solidify in a sloped position. Bottles of complete,
sterile media are available from suppliers but are expensive.
Pouring a plate
1. Collect one flask of sterile molten agar from the autoclave/ hot air oven/water bath.
2. Hold the flask in the left hand; remove the cotton plug with the little finger of the right
hand.
3. Flame the neck of the flask. Transfer the flask from left hand to your right hand
4. Hold the Petri dish in your left hand and lift the lid of the Petri dish slightly with left hand
fingers and pour the sterile molten agar into the Petri dish and replace the lid.
5. Flame the neck of the flask and replace the lid.
6. Gently rotate the dish to ensure that the medium covers the plate evenly.
7. Allow the plate to solidify.
8. Seal and always incubate the plate in an inverted position.
Note: The base of the plate must be covered, agar must not touch the lid of the plate and the
surface must be smooth with no bubbles.
Storage of media
Store stocks of prepared media at room temperature away from direct sunlight; a cupboard is
satisfactory but refrigerator or cold room is ideal. Media in vessels closed by cotton wool
5

plugs that are stored for future use will be subject to evaporation at room temperature; avoid
wastage by using screw cap bottles. Re-melt stored agar media in boiling water bath, pressure
cooker or microwave oven. Sterile agar plates can be pre-poured and stored in well-sealed
plastic bags (media-containing base uppermost to avoid heavy condensation on lid).
Sterilization vs. Disinfection
Sterilization means the complete destruction of all the micro-organisms including spores,
from an object or environment. It is usually achieved by heat or filtration but chemicals or
radiation can be used.
Disinfection is the destruction, inhibition or removal of microbes that may cause disease or
other problems e.g. spoilage. It is usually achieved by the use of chemicals.
Sterilization
Autoclave/pressure cooker: The principle of sterilization in an autoclave or pressure cooker is
that steam under pressure is used to produce a temperature of 121C which if held for 20
minutes will kill all micro-organisms including bacterial endospores.
Sterilization of equipment and materials
1. Wire loop: Heat to redness in Bunsen burner flame.
2. Empty glassware and glass (not plastic!) pipettes and Petri dishes: Either, hot air oven,
wrapped in either greaseproof paper or aluminium and held at 160C for 2 hours, allowing
additional time for items to come to temperature (and cool down!). Or, autoclave/pressure
cooker.
Note: plastic Petri dishes are supplied in already sterilized packs; packs of sterile plastic
pipettes are also available but cost may be a consideration.
3. Culture media and solutions: Autoclave/pressure cooker.
4. Glass/metal spreaders and metal forceps: Flaming in alcohol (70%)
Disinfectants
Choice, preparation and use of disinfectants: Specific disinfectants at specified working
strengths are used for specific purposes. The choice is now much more straightforward as the
range available from suppliers has decreased.
Inoculation and other aseptic procedures
There are several essential precautions that must be taken during inoculation procedures to
control the opportunities for the contamination of cultures, people or the environment.
1. Operations must not be started until all requirements are within immediate reach and must
be completed as quickly as possible.
2. Vessels must be open for the minimum amount of time possible and while they are open
all work must be done close to the Bunsen burner flame where air currents are drawn
unidirectional.
3. On being opened, the neck of a test tube or flask must be immediately warmed by flaming
so that any air movement is unidirectional and the vessel held as near as possible to the
horizontal.
4. During manipulations involving a Petri dish, exposure of the sterile inner surfaces to
contamination from the air must be limited to the absolute minimum.
5. The parts of sterile pipettes that will be put into cultures or sterile vessels must not be
touched or allowed to come in contact with other non-sterile surfaces, e.g. clothing, the
surface of the working area, outside of test tubes/bottles.

6. Using a wire loop: Wire loops are sterilized using red heat in a Bunsen flame before and
after use. They must be heated to red hot to make sure that any contaminating bacterial
spores are destroyed. The handle of the wire loop is held close to the top, as you would a
pen, at an angle that is almost vertical. This leaves the little finger free to take hold of the
cotton wool plug/screw cap of a test tube/flask.
Flaming procedure for wire loop:
The flaming procedure is designed to heat the end of the loop gradually because after use it
will contain culture, which may splutter on rapid heating with the possibility of releasing
small particles of culture and aerosol formation.
a. Position the handle end of the wire in the light blue cone of the flame.
b. This is the cool area of the flame. Draw the rest of the wire upwards slowly up into the
hottest region of the flame, (immediately above the light blue cone).
c. Hold there until it is red hot.
d. Ensure the full length of the wire receives adequate heating.
e. Allow to cool then use immediately.
f. Do not put the loop down or wave it around.
g. Re-sterilize the loop immediately after use.
Flaming the neck of flasks and test tubes:
a. Loosen the cotton plug of the flask/test tube so that it can be removed easily.
b. Lift the flask/test tube with the left hand.
c. Remove the plug of the flask/test tube with the little finger of the right hand. (Turn the
flask/test tube, not the plug)
d. Do not put down the flask/test tube cotton plug.
e. Flame the neck of the flask/test tube by passing the neck forwards and back through a
hot Bunsen flame.
f. Replace the cotton plug using the little finger. (Turn the bottle, not the plug)
Streak plate
The loop is used for preparing a streak plate. This involves the progressive dilution of an
inoculum of bacteria or yeast over the surface of solidified agar medium in a Petri dish in
such a way that colonies grow well separated from each other. The aim of the procedure is to
obtain single isolated pure colonies.
1. Loosen the top of the flask containing the inoculum.
2. Hold the loop in the right hand.
3. Flame the loop and allow to cool.
4. Lift the flask/test tube containing the inoculum with the left hand.
5. Remove the cotton plug of the flask/test tube with the little finger of the right hand.
6. Flame the neck of the flask/test tube.
7. Insert the loop into the culture broth and withdraw. Hold the loop as still as possible.
8. Flame neck of the flask/test tube.
9. Replace the cotton plug on the flask/test tube using the little finger.
10. Hold the petri dish in your left hand and partially lift the lid of the Petri dish using left
hand fingers containing the solid medium.
11. Hold the charged loop parallel with the surface of the agar; smear the inoculum
backwards and forwards across a small area of the medium (streaked area A).
12. Remove the loop and close the Petri dish.
13. Flame the loop and allow it to cool. Turn the dish through 90 anticlockwise.

14. With the cooled loop streak the plate from area A across the surface of the agar in three
parallel lines. Make sure that a small amount of culture is carried over.
15. Remove the loop and close the Petri dish.
16. Flame the loop and allow to cool. Turn the dish through 90 anticlockwise again and
streak from B across the surface of the agar in three parallel lines (to C).
17. Remove the loop and close the Petri dish.
18. Flame the loop and allow to cool. Turn the dish through 90 anticlockwise and streak loop
across the surface of the agar from C into the centre of the plate (to D).
19. Remove the loop and close the Petri dish. Flame the loop.
20. Seal and incubate the plate in an inverted position.
Labeling of Petri dish:
Label the half of the dish that contains medium; use abbreviations and keep them to the edge
of the plate so as not to interfere with the later observation of colonies. The same applies to
the pour and spread plates described below. Either marker pens or self-adhesive labels are
suitable. There are two approaches to making a streak plate: (1) with the base (containing
medium) placed on the working surface, lift the lid vertically (i.e. still covering the base) the
least amount that will allow access of the loop; (2) with the lid placed on the working surface,
lift out the base, invert it and inoculate the upwards - facing agar surface. The second method
is best reserved for older students working in a relatively dust and draught-free laboratory; it
is the one used by professional microbiologists.
Pour plate
A pour plate is one in which a small amount of inoculum from broth culture is added by
pipette to a molten, cooled agar medium in a test tube or bottle, distributed evenly throughout
the medium, thoroughly mixed and then poured into a Petri dish to solidify. Pour plates allow
micro-organisms to grow both on the surface and within the medium. Most of the colonies
grow within the medium and are small in size; the few that grow on the surface are of the
same size and appearance as those on a streak plate. If the dilution and volume of the
inoculum, usually 1 cm, are known, the viable count of the sample i.e. the number of
bacteria or clumps of bacteria, per cm can be determined.
Using a spreader
Sterile spreaders are used to distribute inoculum over the surface of already prepared agar
plates. It is advisable to use agar plates that have a well-dried surface so that the inoculum
dries quickly. Dry the surface of agar plates by incubating the plates for several hours, e.g.
overnight at 37C.
Sterilization of spreader using alcohol
a. Dip the lower end of the spreader into a small volume of 70% alcohol contained in a
vessel.
b. Pass quickly through a Bunsen burner flame to ignite the alcohol; the alcohol will burn
and sterilize the glass.
c. Remove the spreader from the flame and allow the alcohol to burn off.
d. Do not put the spreader down on the bench.
Spread plate
Spread plates, also known as lawn plates, should result in a culture spread evenly over the
surface of the growth medium. This means that they can be used to test the sensitivity of
bacteria to many antimicrobial substances.

The spread plate can be used for quantitative work (colony counts) if the inoculum is a
measured volume, usually 0.1 cm3, of each of a dilution series, delivered by micropipette.
1. Loosen the lid of the bottle containing the broth culture.
2. Hold a sterile pipette in the right hand and the flask/test tube containing the broth culture in
the left.
3. Remove the plug of the flask/test tube with the little finger of the right hand and flame the
neck.
4. With the pipette, remove a small amount of broth.
5. Flame the neck of the flask/test tube and replace the plug.
6. Hold the Petri dish in the left hand and partially lift the lid of the Petri dish containing the
solid nutrient medium.
7. Place a few drops of culture onto the surface about 0.1 cm3.
8. Replace the lid of the Petri dish.
9. Place the tip of the micropipette in a discard jar.
10. Dip a glass spreader into alcohol, flame and allow the alcohol to burn off.
11. Lift the lid of the Petri dish to allow entry of spreader.
12. Place the spreader on the surface of the inoculated agar and, rotating the dish with the left
hand move the spreader in a top-to-bottom or a side-to-side motion to spread the inoculum
over the surface of the agar. Make sure the entire agar surface is covered. This operation
must be carried out quickly to minimize the risk of contamination.
13. Replace the lid of the Petri dish.
14. Flame spreader using alcohol.
15. Let the inoculum dry.
16. Seal and incubate the plate in the inverted position.
Incubation
For guidance on optimum incubation temperatures refer to appropriate literature. If required,
the lid and base of an agar plate should be taped together with 2-4 short strips of paraffin as a
protection from accidental (or unauthorized!) opening during incubation. Agar plates must be
incubated with the medium-containing half (base) of the Petri dish uppermost otherwise
condensation will occur on the lid and drip onto the culture. This might cause colonies to
spread into each other and risk the spillage of the contaminated liquid. The advantages of
incubators are that they may be set at a range of temperatures and reduce the possibility of
cultures being interfered with or accidentally discarded.
At several instances, the temperature of an incubator varies from the set temperature,
oscillating by several degrees in the course of use. Water baths are used when accurately
controlled temperatures are required, e.g. for enzyme reactions and growth temperature
relationships, when temperature control of incubators is not sufficiently precise. They should
be used with distilled or deionised water to prevent corrosion and emptied and dried for
storage.
Note: Overlong incubation of cultures will result in massive formation of spores which
readily escape, particularly from Petri dishes, and may cause contamination problems in the
laboratory and be a health hazard. This can occur in an incubator, at room temperature and
even in a refrigerator but at slower rate.
Clearing up
Working surfaces must be cleared after use. If cultures have been used the benches must be
swabbed with disinfectant. Discarded cultures, empty media tubes and all contaminated
material must be placed in the appropriate labelled receptacles. Discard containers must be
9

carefully and securely packed and never overloaded. Plastic Petri dishes must never be
stacked above the lip of the discard container. Cultures and contaminated paper towels,
gloves etc. must be autoclaved at 121C for 15 minutes before disposal. Slides, pipettes and
Pasteur pipettes must be discarded in the appropriate containers of Hypochlorite (sodium
chlorate 1). They must be soaked for at least 24 hours before disposal. Never discard sharp or
broken items in a way which would endanger anyone. After sterilization, all materials can be
disposed of with normal waste. Care must be taken that glass is adequately packaged to
prevent injury. Before leaving the laboratory, laboratory coats must be removed and hands
washed with water and soap followed by application of commercial sanitizers.
Maintenance, preparation and sub-culturing
Obtaining suitable cultures
Micro-organisms which are non-pathogenic and approved for use in schools and colleges
present minimum risk given observance of GMLP. The list is not definitive; other organisms
may be used if competent advice is taken. Ensure that all the guidelines are followed with
proper consultation with experienced microbiologist because recommendations are altered
from time to time with changes in experience and assessment of the risks. Cultures must be
obtained from a reputable specialist supplier. Isolation of cultures from the environment may
be conducted if appropriate to the level of work.
Pure cultures
The ability to keep pure cultures from becoming contaminated during inoculation and use is a
key feature of GMLP. This skill is crucial for reasons of safety and for maintaining the
scientific integrity of an investigation. Clearly, it is also vital skill to recognize when a culture
has become contaminated.
Maintaining stock cultures
It may be convenient to maintain a stock of a pure culture instead of re-purchasing it when
needed. Most of those considered suitable for use are also relatively easy to maintain by subculturing on the medium appropriate for growth but maintenance of stock cultures needs to be
well organized with attention to detail. Cultures on streak plates are not suitable as stock
cultures. Slope cultures in test tubes are preferred because the tube length reduces
evaporation and drying out and cannot be accidentally knocked off (cf. a streak plate culture).
Slope cultures are preferred to broth (i.e. liquid medium) cultures because the first sign of
contamination is much more readily noticed on an agar surface. As soon as there is adequate
growth, store the cultures at lower temperature in either a refrigerator or cold room. Keep on
the lookout for contamination.
Checking cultures for contamination
Evidence for a culture being pure or otherwise is given by the appearance of colonies on a
streak plates and of cells in a stained microscopic preparation. There should be uniformity of
colony form and cell form (and consistency with the appearance of the original culture!). It is
sensible to check purity on suspicion of contamination of the working stock culture from time
to time. If a culture becomes contaminated, it is not advisable to try to remedy the situation
by taking an inoculum from a single colony from a streak plate of the mixed culture because
of the possibility of (1) not being able to distinguish between the colony forms of the
contaminant and the original culture, and (2) culturing a variant of the original culture that
does not behave as the original culture did. Instead, go back to the permanent cryopreserved
stock cultures.

10

Exp 2.1: Isolation of pure cultures of bacteria/Fungus from various samples and cell count
Exp 5.1: Isolation of pure cultures of fungus

INTRODUCTION
Nutrient agar: It is used for growth of nonfastidious bacterium. It is useful as it remains
solid even at high temperature. Bacterium grows on the surface and is clearly visible as
small colonies.
MGYP agar: Used for cultivation of fungi (M=Malt extract, G=glucose, Y=yeast,
P=peptone).
Glucose is the source of carbon, yeast extract provides vitamins, amino acids and peptone
is the source of nitrogen. Overall this provides an acidic medium, favourable for the
growth and metabolism of fungi.
Microorganism present in a sample may be high thus to avoid the overcrowded growth on
plate, samples can be serially diluted.
Serial dilution:
It is a stepwise dilution as shown in the fig of a substance in a solution. Usually the dilution
factor at each step is constant, resulting in a geometric separation of the concentrations in a
logarithmic fashion.
Serial Dilution

Isolation of microorganisms

Methods for isolation of a microorganisms from any source are as follows:

Spread plating technique: In this technique, the sample is appropriately diluted (serial
dilution) and a small amount (0.1 mL) is transferred to the centre of an agar plate and is
spread evenly over the surface with the help of a sterile glass/metal rod.
Pour plate technique: In this technique the required dilution is first introduced into a petri
dish and the nutrient medium which is kept liquid by keeping it in water bath at 500C is
poured over it, which is then mixed by gentle agitation of the plate. When the nutrient
medium solidifies, the plate is incubated. In this method, the colonies can be seen on the
surface as well as beneath the surface of the medium.

11

Streaking: The purpose of streaking is to obtain pure individual bacterial/fungal cells

(colony-forming units) on a nutrient medium. Mainly three type of streaking is methods are
used.
1. Simple streaking: In this, culture is taken with inoculation loop and is spread on the petri
plate in a horizontal zig-zag pattern starting from the top till the bottom of the plate.
2. T- Streaking: In this, streak one half of your agar plate with the bacteria, then flame your
loop. After the loop has cooled, in one of the quadrants left, run your loop into the first
region three times, then streak the quadrant. Flame your loop and let it cool again. Run
your loop through the last quadrant into the last region (quadrant) you just streaked one
time and streak the last quadrant with your loop.
3. Quadrant streaking: Mark your agar plate into four quadrants either with your indelible
marker or with your imagination. Flame your loop, let it cool, then remove one colony
from the mixed culture and streak the first quadrant. Flame your loop and let it cool. Drag
your loop from the second quadrant back into the first quadrant three times and streak the
second quadrant. Flame your loop and let it cool. Drag your loop from the third quadrant
back into the second quadrant three times and streak the third quadrant. Flame your loop
and let it cool. Drag your loop from the fourth quadrant into the third quadrant one time
and then streak the fourth quadrant with one wavy line.
Simple streaking

T- Streaking

Quadrant streaking

Inoculation loop: It is a simple tool used to retrieve an inoculum from a culture of microorganisms and transfer the inoculum onto a petri plate after which it is used for streaking. The
loop of wire at the tip may be made of platinum or nichrome.
Identification of bacteria by morphological characteristics

12

MATERIALS REQUIRED
Measuring cylinder, Erlenmeyer flask, Test tubes (10), Petri plates, Cotton plugs, Nutrient
agar, MGYP Agar, Chloramphenicol, Distilled water, Brown paper, Water/soil sample,
Inoculation loop, Glass/Metal spreader, Autoclave, 70% alcohol solution, Laminar air flow.
PROCEDURE
Preparation of culture media and test tubes for serial dilution
Take a 500ml flask and rinse it with distilled water thrice and prepare 250ml of nutrient
agar (Nutrient broth; 3.25gm and Agar powder; 5gm) and MGYP agar supplemented with
chloramphenicol.
Add 250ml distilled water and place the cotton plug properly and cover it with help of
brown paper and thread.
Take 10 test tubes and rinse with distilled water and pipette 9ml of distilled water in each.
Place the cotton plugs and put all in standing position in beaker.
Wipe the petri dishes using 70% alcohol and stack them together and cover with brown
paper.
Put all the above items for autoclaving. After autoclaving, pour the medium in the petri
dishes and wait for 15 min for the medium to solidify.
Serial dilution
Take 1g (soil sample) or 1ml (water) of sample and add to a test tube containing 9ml of
water.
Using micropipette, aliquot 1ml of sample from the test tube and add it to another 9ml
water in the next test tube. Repeat this procedure for the remaining tubes also.
Label the test tubes 10-1, 10-2.. 10-6.
Spreading of culture and counting
Take 0.1ml of inoculum using micropipette and spread it over the petri plate using sterile
glass rod.
For fungi, inoculum is taken from test tubes labelled 10-1, 10-2, 10-3 and for bacteria
inoculum is taken form test tubes labelled 10-4, 10-5, 10-6.
After spreading, the bacterial plates are kept for incubation at 37C for 12hrs and the
fungal plates are kept for incubation at 28C for 72hrs.
After the colonies have grown, the number of colonies of bacteria and fungi are counted
using a plate counter.
Streaking
In order to isolate a pure strain from the culture, using an inoculating loop pick up an
isolated colony and by simple streaking, streak onto the petri plate and keep the plates for
incubation at 37C for bacteria for 12hrs and 28C for fungi for 72hrs.

OBSERVATIONS
The following figures below show the results obtained after spread plate and streaking.

13

Bacterial colonies after spreading

Pure culture of bacteria after streaking

Fungal colonies after spreading

Pure culture of fungi after streaking

CALCULATIONS
Calculate the number of bacteria (CFU) per millilitre or gram of sample by dividing the
number of colonies by the dilution factor. The number of colonies per ml reported should
reflect the precision of the method and should not include more than two significant figures.
For example, suppose the plate of the 10-6 dilution yielded a count of 130 colonies. Then, the
number of bacteria in 1 ml of the original sample can be calculated as follows:
Bacteria (per ml) = (130) x (10-6) = 1.3 x 108 i.e. 130,000,000

Bacterial colonies can be counted by using colony counter.

Each colony that can be counted is called a colony forming unit (cfu) and the number of
cfu's is related to the viable number of bacteria present in the sample.

RESULT
No of cells of bacteria in the sample are _______ / g or ml
No of cells of fungi in the sample are _______ / g or ml
PRECAUTIONS
The inoculation loop should be sterilized properly using flame and alcohol.
While performing serial dilution, the test tubes should be opened near the flame
The spreader should be properly sterilized with alcohol and flame and cooled properly.
While working in the laminar air flow room, hands should be properly wiped with 70%
alcohol.
Switch on the UV light at least 20 minutes before for radiation sterilization of laminar air
flow chamber and take care that fluorescent light should not be switched on immediately
after UV light.

14

Exp 2.2: Grams staining of bacteria

INTRODUCTION
Staining depends on the fact that bacteria differ chemically from their surroundings and thus
can be stained to contrast with their environment. The first step in the procedure involves
staining with the basic dye crystal violet. This is the primary stain. It is followed by treatment
with an iodine solution, which functions as a mordant; that is, it increases the interaction
between the bacterial cell and the dye so that the dye is more tightly bound or the cell is more
strongly stained. The smear is then decolourized by washing with an agent such as 95%
ethanol or isopropanol-acetone. Gram-positive bacteria retain the crystal violet-iodine
complex when washed with the decolourizer, whereas gram-negative bacteria lose their
crystal violet-iodine complex and become colour- less. Finally, the smear is counterstained
with a basic dye, different in colour than crystal violet. This counter stain is usually safranin.
The safranin will stain the colourless, gram-negative bacteria pink but does not alter the dark
purple colour of the gram-positive bacteria. The end result is that gram-positive bacteria are
deep purple in colour and gram-negative bacteria are pinkish to red in colour.
Mechanism of action:
The mechanism is based on the differences in the structure of cell walls of gram positive or
gram negative bacteria and how each reacts with the various reagents. Crystal Violet, the
primary stain, stains both gram positive and gram negative cells purple because the dye enters
the cytoplasm of both types of cells. When Iodine (mordant) is applied, it forms large crystals
with the dye that are too large. The application of alcohol makes gram positive cells
impermeable to CV I complex. However, in Gram negative cells it dissolves the outer
membrane leaving small holes in peptidoglycan layer through which CV I complex can pass.
Since gram negative are colourless after this, they are made visible by adding Safranin which
makes them pink. The basis of this difference in the stains of the specimen is because of the
lipid content in the cell walls of the bacteria. The bacterial cells that show Gram positive
result have lower lipid content as compared to the Gram negative bacteria. So, when the
decolourizer is added to the slides containing the bacterial smear, the decolourizer dissolves
the lipids in the cell wall leading to lesser restriction on the complexes to pass through the
membrane and hence the CV-I complex contained in the cell now washes away unrestricted.
Due to this, a void is created in the cell paving way for the next stain to fill up the interior.
This is why the Gram negative bacteria appear to be pink in colour (due to safranin stain). In
case of the Gram positive bacteria, the lipid content is low and hence it does not affect the
cell wall in a major way. Therefore there is no way the large CV-I complex can escape the
cell. And this is why the Gram positive cells appear to be purple-violet in colour.
MATERIALS REQUIRED
Safranin, Iodine, Crystal Violet, Decolourizer (95% ethanol), Inoculating loop, Clean Slide,
Distilled water, Microscope.
PROCEDURE
Take a clean slide and using a sterilized loop, transfer a little amount of bacteria to the
corner of the slide on top of a drop of water.
Spread it evenly and heat fixed it.
Add two drops of Crystal Violet and the smear is covered with iodine.

15

 Crush of the iodine after a few minutes and then wash off the smear with alcohol
(decolorizing agent).
After waiting for two minutes, the alcohol is rinsed off the slide is then stained with
safranin.

OBSERVATION
The bacteria are ____________________ colored, ___________ shaped and arranged in
_________________ .
RESULT
The bacteria are found to be GRAM _____________________ .
PRECAUTIONS

The bacteria should be heat fixed properly or else or it will be washed off.
The inoculating loop should be heat fixed properly and it should be properly sterilized.
Large amount should not be taken.
Excess stain should not be added to the smear.
Old cultures should not be used because Gram-positive cultures may often turn gram
negative if they get too old.

16

Exp 5.2: Staining of fungus- Lactophenol cotton blue staining

INTRODUCTION
Fungi can be branched into three main divisions: yeasts, molds, and mushroom.
Yeasts are unicellular fungi that are spherical, ellipsoidal, or oval in shape, and usually (with
the exception of the dimorphic yeasts such as Candida) do not form hyphae (fungal
filaments). Yeasts commonly reproduce asexually by budding, a process in which a new cell
(called a daughter cell) is formed by the parent cell from a protuberance called a bud. When
yeasts reproduce sexually, they produce several types of sexual spores (e.g., ascospores).
Molds are multicellular, filamentous fungi. The techniques of culturing and observing fungi
differ from the methods used to study bacteria. The macroscopic aggregation (colony) of
mold cells is called a thallus. A thallus is composed of a mass of strands called a mycelium;
each strand is a hypha. Vegetative hyphae grow on the surface of culture media. They form
aerial hyphae, called reproductive hyphae, which bear asexual reproductive spores or conidia.
The hyphae that grow below the surface of culture media are called rhizoidal hyphae. The
hyphal strand of some molds may be separated by a crosswall called a septum. Hyphae that
contain septa are called septate hyphae. Molds with hyphae that lack septa are called
coenocytic hyphae.
Fungal cell wall is made up of chitin instead of peptidoglycan like in bacterial cells.
Methylene blue binds to the fungal cell wall and gives them a Prussian blue colour. It can
also be used as an indicator to determine if the cells are alive or not.
Lactophenol Cotton Blue is one of the most common stains used to stain fungi. It is made of
lactophenol, which is the fixing and mounting fluid, and methyl blue (cotton blue). The
organism suspended in lactophenol cotton blue is killed by the presence of phenol. The high
concentration of phenol deactivates lytic cellular enzymes and therefore the cell doesn't lyse.
Cotton blue is an acid stain that stains the chitin in the fungal cell wall.
MATERIALS REQUIRED
Fungi culture MGYP plates, lactophenol cotton blue stain, Toothpicks, Glass slide, Cover
slip, Laminar air flow, microscope.
PROCEDURE
Clean the glass slide with soap and wipe it with 70% alcohol.
In the laminar air flow, place a drop of lactophenol cotton blue in the middle of the glass
slide.
With the help of toothpick transfer the fungus from the petri plates to a corner of the slide
and spread it out evenly in the stain.
Use the toothpick, separate the hyphae so that they don't overlap. This process is called
shearing.
Gently put a cover slip on the top of the slide and observe under the microscope, taking
care that no bubbles form in the process.
Using a filter paper, carefully remove the excess stain around the cover slip.
Place the slide under a microscope and focus under low as well as high magnification.

17

OBSERVATION
Fungal colonies can be distinguished morphologically base on their back colouration, the top
colouration, spore forming structure, colour of spore, presence of caenocytic cells etc.
(Record presence of septae, hyphae, conidiospores etc. )

RESULT
On the basis of microscopic observation the fungus can be classified as _________________.
PRECAUTIONS
Be careful to not allow any air bubbles while putting cover slip.
Spread the fungi properly and stain it adequately. Do not use too much of culture.
Shear enough such that there is no overlapping.
Handle the cover slip with care. It breaks easily.
Do not use excess dye or lactophenol.
Keep the culture inverted as far as possible else the spores will spread. Do not disturb the
plate too much.
Lacto phenol cotton blue is acidic. Skin contact should be avoided.

18

Exp 4.1: Bacterial growth curve

INTRODUCTION
The four phases (lag, logarithmic, stationary, and death or decline) of growth of a bacterial
population can be determined by measuring the turbidity of the population in a broth culture.
Turbidity is not a direct measure of bacterial numbers but an indirect measure of biomass,
which can be correlated with cell density during the log growth phase. Since about 10 7
bacterial cells per millilitre must be present to detect turbidity with the unaided eye, a
spectrophotometer can be used to achieve increased sensitivity and obtain quantitative data.
The construction of a complete bacterial growth curve (increase and decrease in cell numbers
versus time) requires that aliquots of a
shake-tube culture be measured for
population size at intervals over an
extended period. The bacterial population
will be plotted on graph paper by using the
optical density of the broth v/s time. The
resulting growth curve can be used to
delineate stages of the growth cycle. It also
makes possible the determination of the
growth rate of a particular bacterium under
standardized conditions in terms of its
generation time-the time required for a bacterial population to double.
The spectrophotometric measurements of the developing turbidity in a bacterial culture taken
at regular intervals. These samples serve as an
index of increasing cellular mass. The graphical
determination of generation time is made by
extrapolation from the log phase, as illustrated in
figure 45.1. For example, select two points (0.2
and 0.4) on the absorbance (A) scale that represent
a doubling of turbidity. Using a ruler, extrapolate
by drawing a line between each absorbance on the
ordinate, and the plotted log or exponential phase
of the growth curve. From these two points, draw
perpendicular lines to the time intervals on the
abscissa. From these data, the generation time can
be calculated as follows:
Generation time = t (A of 0.4) t (A of 0.2); Generation time = 90 min - 60 min = 30 min.
MATERIALS REQUIRED
Nutrient broth, test tubes, micropipette, cotton plugs, laminar air flow, log phase E. coli
culture, incubator, spectrophotometer.
PROCEDURE
Separate the 20 sterile nutrient broth tubes (10 ml each) into 10 sets of two each. Using the
marker, label each set as to the time of inoculation (t= 0h, t= 2h, t= 4h, t= 6h, t= 8h, t=
10h, t= 12h, t= 14h, t= 16h and t= 24h) and the replicate as A and B respectively.
19

 Using a sterile micropipette, transfer 0.1 ml of the log phase E.coli culture to the all the
labelled tubes containing 10 ml of nutrient broth.
On a chart tabulate the tube number, time and date at which the optical density of the
respective tube needs to be measured.
Remove 0h time tubes from the incubator and measure absorbance (A) of this broth
immediately at 550 to 600 nm.
After every two hours, remove the respective tube from incubator and measure its
absorbance at 550 to 600 nm for the next 24 hours.
Plot a graph taking time (in hours) at x-axis and absorbance at y-axis.

OBSERVATION
Tube

Time/Date

Absorbance
(Rep A)

Absorbance
(Rep B)

Average

0h
2h
4h
6h
8h
10h
12h
14h
16h
24h
RESULT
On the basis of experimental observation and graphical representation of bacterial growth
curve, the generation time of bacteria is _________________.
PRECAUTIONS
Be sure to maintain good aseptic technique when making transfers and report any spills to
your instructor.
Carefully clean and decontaminate your work area at the end of the experiment.
The bacterial growth curve study can be very easily run using 96 well plate and ELISA
reader (spectrophotometer).

20

Exp 4.2: Effect of pH, temperature, salt and radiation on growth of microorganisms
INTRODUCTION
Effect of salt: Since bacteria are separated from their environment by a selectively permeable
plasma membrane, they can be affected by changes in the osmotic pressure or water
availability of their surroundings. Osmotic pressure is the force developed when two
solutions of different solute concentrations are separated by a membrane that is permeable
only to the solvent. If a bacterium is placed in a hypotonic solution (low solute, high-water
content), water will enter the cell and cause it to burst unless something is done to prevent the
influx. Most bacteria have rigid cell walls that
maintain the shape and integrity of the cell; thus,
hypotonic solutions are not harmful to these
bacteria. When bacteria are placed in a hypertonic
solution (high solute, lower water content), water
leaves, and the plasma membrane shrinks away
from the wall, a process known as plasmolysis. This
dehydrates the cell, and it ceases to grow. A few
bacteria, called halophiles, are able to tolerate high
(hypertonic) salt concentrations. Bacteria that can
live in very salty environments are called extreme
halophiles to distinguish them from the moderate
halophiles that live in the sea. In an isotonic
solution, the concentration of solutes is the same
(iso means equal) outside and inside the
bacterium. The bacterium is in osmotic equilibrium
with its environment and does not change volume.
This tolerance (or lack of it) can be observed by the
amount (or lack) of growth of the three bacterial species.
Effect of pH: The pH affects the activity of enzymes, especially those that are involved in
biosynthesis and growth. Each microbial species possesses a definite pH growth range and a
distinct pH growth optimum. Acidophiles have a growth optimum between pH 0.0 and 5.5;
neutrophiles between 5.5 and 8.0; and alkalophiles 8.5 to 11.5. In general, different microbial
groups have characteristic pH optima. The majority of bacteria and protozoa are neutrophiles.
Most molds and yeasts occupy slightly acidic environments in the pH range of 4 to 6; algae
also seem to favour acidity. Many bacteria produce metabolic acids that may lower the pH
and inhibit their growth.
Effect of temperature: Each microbial species
requires a temperature growth range that is
determined by the heat sensitivity of its
particular enzymes, membranes, ribosomes,
and other components. As a consequence,
microbial growth has fairly characteristic
temperature dependence with distinct cardinal
temperaturesminimum,
maximum,
and
optimum. Minimum growth temperature is the
lowest temperature at which growth will occur;
maximum growth temperature is the highest
21

temperature at which growth will occur; and optimum growth temperature is the temperature
at which the rate of cellular reproduction is most rapid. The optimum temperature for the
growth of a given microorganism is correlated with the temperature of the normal habitat of
the microorganism. For example, the optimum temperature for the growth of bacteria
pathogenic to humans is near that of the temperature of human blood (35 to 37C). Most
bacteria can be classified into one of three major groups based on their temperature
requirements. Psychrophiles can grow at 0C and have an optimum growth temperature of
15C or lower; the maximum is around 20C. Mesophiles have growth optima between 20
and 45C. The majority of bacteria fall into this category. Thermophiles can grow at
temperatures of 55C or higher.
Effect of UV radiation
The excitation of electrons in DNA molecules often results in the formation of extra bonds
between adjacent pyrimidines (specifically thymine) in DNA. When two pyrimidines are
bound together in this way, it is called a pyrimidine dimer. These dimers often change the
shape of the DNA in the cell and can cause problems during replication. The cell often tries
to repair pyrimidine dimers before replication, but the repair mechanism can also lead to
mutations as well.
MATERIALS REQUIRED
Bacterial culture, nutrient agar, petri plates, test tubes, inoculation loop, pH adjusted nutrient
broth (pH 3, 7 and 9), salt concentration adjusted nutrient broth (0.5%, 5% and 10% NaCl),
Laminar Air Flow, Incubator
PROCEDURE
Effect of temperature
Test tubes having nutrient broth solutions are properly labelled as 4C, 37C, 55C
respectively.
All the tubes are inoculated with the bacterial culture and incubated at respective
temperatures as labelled for 12-16 hours.
Effect of salt
Test tubes of nutrient broth having NaCl concentration 0.5%, 5%, 10% including control
are labelled.
The bacterial culture is inoculated in each tube and incubated at 37C for 12-16 hours.
Effect of pH
Test tubes of nutrient broth having pH adjusted at 3, 7 and 9 including control are labelled.
The bacterial culture is inoculated in each tube and incubated at 37C for 12-16 hours.
Effect of UV radiations
Two nutrient agar petri dishes are labelled as UV treated and untreated.
The bacterial culture is spread on both the plates.
The UV treated plate is kept open in laminar air flow bench with UV lamp on for 30 mins.
After 30 mins, both plates are incubated at 37C for 12-16 hours.

22

OBSERVATION
The samples are fed into ELISA wells to estimate growth of bacteria is compared by
observing the turbidity (spectrophotometrically) in the wells.
Sample
Media
Con
Temp 4C
Temp 37C
Temp 55C
pH 3.0
pH 7.0
pH 9.0
Salt 0.5%
Salt 5.0%
Salt 10.0%

Well No.

OD

The plate treated with UV radiations after spreading of bacterial sample showed
________________________________________ compared to the one not treated with UV.

RESULTS
Record conditions that support optimum growth of bacteria.

23

Exp 4.3: Effect of various antibiotics on microbial growth

INTRODUCTION
Antibiotics, also known as antibacterials, are types of medications that destroy or slow down
the growth of bacteria. The Greek
word anti means "against", and the
Greek
word bios means
"life"
(bacteria are life forms). Their mode
of action is to interfere with microbial
metabolism, thereby producing a
bacteriostatic or bactericidal effect on
the
microorganisms,
without
producing a like effect in the host
cells. Antibacterial antibiotics are
commonly classified based on their
mechanism of action, chemical
structure, or spectrum of activity.
Most target bacterial functions or
growth processes. Those that target the bacterial cell wall (penicillins and cephalosporins) or
the cell membrane (polymyxins), or interfere with essential bacterial enzymes
(rifamycins, lipiarmycins, quinolones and sulfonamides) have bactericidal activities. Those
that target protein synthesis (macrolides, lincosamides and tetracyclines) are
usually bacteriostatic (with the exception of bactericidal aminoglycosides). Further
categorization is based on their target specificity. "Narrow-spectrum" antibacterial antibiotics
target specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas
broad-spectrum antibiotics affect a wide range of bacteria.

MATERIALS REQUIRED
Petri plates, 12 hour old culture of bacteria, assorted antibiotic discs, nutrient agar, forceps,
spreader.

24

PROCEDURE
First prepare the petri plates containing nutrient agar media and spread them with
100uL of 12h old culture of respective bacteria. Leave the plates under incubation for
15 min inside the laminar hood.
After incubation, using forceps place the assorted antibiotic disc on the petri plates.
Make sure that all the disc of each antibiotic is firmly placed on the agar surface.
Incubate the plates overnight at 37 C in upright position.
After incubation, using a ruler measure the zone of inhibition (ZOI).
OBSERVATION
Diameter (ZOI)
Antibiotic
Gram +ve

Gram -ve

RESULTS
Record among various antibiotics tested, which one showed highest and lowest activity
against gram positive and negative bacteria respectively.

25

Exp 4.4: Effect of various metals on microbial growth

INTRODUCTION
Certain metals fulfill cellular functions that cannot be met by organic molecules, and these
metals are therefore indispensable for the biochemistry of life in all organisms. Specific metal
ions are crucial for the structure of cell membranes and DNA; approximately half of all
known proteins are predicted to be dependent on metal atoms for their structure and their
participation in key cellular processes, such as electron transfer and catalysis. Nonetheless,
these essential metals are lethal to all cells when present in excess. Furthermore, certain nonessential metals such as silver (Ag),
mercury (Hg) and tellurium (Te) are
extremely poisonous to most bacteria
and have microbicidal activity at
exceptionally low concentrations.
Because of their potent toxicity to
bacteria and yeast, particular metals
have been used as antimicrobial agents
since
ancient
times.
Today,
antimicrobial
metal
compounds
including metallic surfaces and
coatings (which are used, for example,
on indwelling medical devices),
chelates and nanomaterials have a
multitude of applications in industry,
agriculture and healthcare. These innovations were made possible following the discovery
that certain metals disrupt antibiotic-resistant biofilms exert synergistic bactericidal activity
with other biocides, inhibit metabolic pathways in a selective manner and kill multidrugresistant bacteria. The mechanisms of toxicity of metals in microbe are specific to particular
metal species. Mainly, a metal can act in following ways to kill any cell; (a) Metals can lead
to protein dysfunction; (b) They can also lead to the production of reactive oxygen species
(ROS) and depletion of antioxidants; (c) Certain metals have been shown to impair
membrane function; (d) Some can interfere with nutrient assimilation; (e) They can also be
genotoxic.

Addition of solution of metal

at different concentration

MATERIALS REQUIRED
Petri plates, 12 hour old culture of bacteria, metal solutions with defined metal concentration
(FeSO4.7H2O, ZnSO4.7H2O, CuSO4.5H2O; 4 g/L and AgNO3; 2 g/L), nutrient agar,
well borer, spreader.
26

PROCEDURE
First prepare the petri plates containing nutrient agar media and spread them with
100uL of 12h old culture of respective bacteria. Leave the plates under incubation for
15 min inside the laminar hood.
After incubation, using well borer puncture wells in the agar. Make sure that well are
evenly distant from each other. Remove the agar plugs and clear the formed wells.
Using micropipette, aliquot different metal solution in respective wells to achieve a
specific metal concentration in each well.
Incubate the plates overnight at 37 C in upright position. After incubation, using a
ruler measure the zone of inhibition (ZOI).
Calculate minimum inhibitory concentration for each metal against given bacteria,
wherein minimum inhibitory concentration (MIC) is the lowest concentration of an
antimicrobial metal (in g) that will inhibit the visible growth of a microorganism after
overnight incubation.
OBSERVATION
Metal

Ag

Fe

Zn

Cu

Diameter (ZOI)

Concentration (g)

Gram +ve

Gram -ve

10
20
30
40
50
20
40
60
80
100
20
40
60
80
100
20
40
60
80
100

RESULTS
On the basis of experimental findings, the calculated minimum inhibitory concentration of
various metals is as follows.
Ag:......................... g, Fe: ..................... g, Zn: ......................... g, Cu: ......................... g

27

Exp 3.1: Coliform counts in contaminated water sample

INTRODUCTION
There are a wide variety of disease-causing organisms which can live in water and one of the
primary responsibilities of the water treatment plant operator is to ensure that none of these
pathogens reach the consumer in their drinking water. However, testing for the presence of
each of these disease-causing organisms individually would be prohibitively expensive and
time-consuming. Instead, operators usually test for the presence of coliform bacteria.
Coliform bacteria do not cause disease, but they are reliable indicators of the presence of
disease-causing organisms. A special group of coliform bacteria, known as fecal coliform
bacteria, live in the intestinal tracts of warm-blooded animals. Fecal coliform bacteria are
usually present in water only when human or animal wastes have come in contact with the
water. The number of fecal coliform bacteria present in a sample is a good indicator of the
amount of animal pollution present in the water. Since most waterborne disease-causing
organisms originate in human or animal bodies and are discharged as part of body wastes,
water with a large quantity of fecal coliform bacteria is likely to contain disease-causing
organisms and is not safe to drink. A faecal coliform is a facultative anaerobic, rod shaped,
gram negative, non-sporulating bacterium. They are capable of growing in presence of bile
salts and produce acid and gas from lactose within 48 hrs. at 35 C.
Eosin Methylene Blue is a slightly selective stained media for growing Gram-negative
bacteria. It is a blend of two stains, eosin and methylene blue. A common application of this
stain is for differential microbiological medium, which slightly inhibits the growth of Grampositive bacteria and provides a colour indicator distinguishing between organisms that
ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella). Organisms
that ferment lactose display "nucleated colonies" with dark red centres. Lactose fermentation
produces acids, which lower the pH. This encourages dye absorption by the colonies, which
are now coloured reddish brown. Lactose non-fermenters may increase the pH by
deamination of proteins. This ensures that the dye is not absorbed. The colonies will be
colourless. Sometimes, certain rapid lactose fermenters will give a distinctive metallic green
sheen (due to the metachromatic properties of the dyes).
One of the test for measuring coliform numbers (quantity) in water is the membrane filter
technique. This technique involves filtering a known volume of water through a special
sterile filter. These filters are made of nitrocellulose acetate or polycarbonate, are 150 m
thick, and have 0.45 m diameter pores. When the water sample is filtered, bacteria (larger
than 0.45 m) in the sample are trapped on the surface of the filter. The filter is then carefully
removed, placed in a sterile petri plate on a saturated with EMB agar-based medium, and
incubated for 20 to 22 hours at 35C. One assumes that each bacterium trapped on the filter
will then grow into a separate colony. By counting the colonies one can directly determine
the number of bacteria in the water sample that was filtered.

28

MATERIALS REQUIRED
Water Samples, EMB agar, petri plates, spreader, micropipette, filtration assembly,
membrane filters, vacuum pump, colony counter.
PROCEDURE
Prepare the petri plates containing EMB agar media.
Spread 100uL of the respective water sample on EMB agar plate.
Simultaneously, filter the respective water samples through a membrane filter attached
to a vacuum pump. Place the membrane filter on the EMB agar surface. Gently tap on
the filter disc to ensure that it is firmly fixed over the agar surface.
Incubate the plates 20-24 hours at 35 C.
After incubation, count only those colonies that either have a pink to dark red
deposition or have a metallic sheen.

OBSERVATION
Water sample

Number of Coliforms

RESULTS
On the basis of experimental findings, following water samples which have less than 4
coliform colonies (100 ml water) were found to be portable and suitable for human
consumption.

29

Exp 3.2: Dehydrogenase activity assay for qualitative determination of microbial population

INTRODUCTION
Dehydrogenase are enzymes that are found in all living organisms. These enzymes take part
in many reactions involving the transfer of pairs of electrons. In catabolic reactions, i.e.,
reactions involving the modification of complex or high-energy compounds to simpler or low
energy compounds, dehydrogenases catalyze the transfer of electron pairs from some
substrate to NAD+ forming NADH. NADH then transfers the electrons to another compound,
thereby serving as an electron transfer intermediary. In anabolic reactions (the opposite of
catabolic reactions), NADP+ is involved instead. Two hydrogen atoms tag along to keep
the charge balanced; it is the electrons that are being transferred. As dehydrogenases also take
part in the electron transfer system of aerobic organisms, the activity of these enzymes is a
measure of respiration along with general metabolic activity.
By far the most commonly used version of the dehydrogenase assay is that of Casida et al.
(1977). This assay usually involves incubating soil mixed with a solution of the competitive
NAD+ inhibitor, 2,3,5-triphenyltetrazolium chloride (TTC) with soil in air-tight containers to
exclude oxygen. Here the TTC serves as the ultimate electron acceptor during respiration.
Exclusion of oxygen favours the transfer of the electrons to TTC, reducing the pale yellow,
slightly water-soluble compound to the insoluble red dye triphenyl formazan (TPF). After
incubation, the soils are extracted with a solvent (methanol) to remove the TPF. Following
extraction, the concentration of TPF in the soil is determined by absorption
spectrophotometry. The dissolved analyte, TPF, absorbs light in the visible region of the
spectrum, with 485 nm best for analytical purposes. Over a certain range of concentration, the
degree to which the TPF absorbs 485 nm light is a linear function of concentration of TPF in
the methanol solution.
MATERIALS REQUIRED
Fresh soil sample, glass test tube with a tightly fitting lid, analytical weighing balance,
spatula, deionized water, 3% (w/v) TTC, 1% (w/v) glucose, micropipette, methanol.
PROCEDURE
Collect fresh soil samples from various places. Ensure that collected soil is free from
any biological debris, stone and plastic.
Weigh 1g of soil and transfer to assigned test tube. In one tube add 1 mL of water
instead of soil to be used as blank.
To each test tube, add 0.5 mL of 1% (w/v) glucose and 0.2 mL of 3% (w/v) TTC. Close
the mouth of the test tubes.
Incubate the test tubes at 28-30 C for 24 hours.
After incubation, add 10 mL of ice cold methanol in each tube and incubate the tubes at
4 C for 3-4h.
Filter the content in the tubes using a whatman filter paper and collect the filtrate in a
fresh test tube.
Determine the absorbance of the collected filtrate at 485 nm using a spectrophotometer.

30

OBSERVATION
Formula for calculating g of TPF produced per gram of soil = OD485nm x 17.196429
S. No.

Soil Sample Detail

OD at 485 nm

g of TPF produced
per gram of soil

A
B
C
D
E
F

RESULTS
On the basis of experimental findings, the highest and lowest dehydrogenase activity was
observed in ................................................ and .................................................. soil samples
respectively.

31

Exp 3.3: Enumeration of microbial cells in air microflora

INTRODUCTION
Aerobiology has been defined as the study of aerosolization, aerial transmission, and
deposition of biological materials. A collection of airborne biological particles is called a
bioaerosol. Bioaerosols are generated by a wide variety of natural and human-made processes
including coughing, sneezing, wave action, splashes, wind, cooling towers, ventilation
systems, etc. Inhalation, ingestion, and dermal contact are routes of human exposure to
airborne microorganisms, but inhalation is the predominant route that results in adverse
human health effects. Airborne Legionella pneumophila, Mycobacterium tuberculosis, and
some pathogenic viruses are known to be transmitted by aerosols. Asthma, hypersensitivity
pneumonitis and other respiratory illnesses are also associated with exposure to bioaerosols.
Deterioration of building materials, offensive odours, and adverse human health effects are
associated with microbial contamination of indoor environments, such as residences, offices,
schools, health care facilities, enclosed agricultural structures (barns and crop storage areas)
industrial facilities and recycling facilities. Sources and reservoirs of microorganisms are
present within these settings, including building materials and furnishings, pets, plants, and
air-conditioning systems. Fungi, which can colonize drier surfaces than bacteria, tend to grow
in a wide variety of building materials, such as wallboard, ceiling tiles, carpeting and vinyl
flooring.
Temperature and relative humidity are the two most important factors affecting the survival
of microorganisms in the airborne state. In general, bacteria and fungi are more stressed as
the rate of evaporation increases, which occurs as relative humidity deceases and temperature
increases. Thus, increased survival is favoured at higher relative humidity and lower
temperatures. The effect of relative humidity varies more with virus type with some surviving
better at high relative humidity and others better at low relative humidity.
MATERIALS REQUIRED
Petri plate, nutrient agar, Martin Rose Bengal Agar (MRBA), chloramphenicol.

PROCEDURE
Prepare petri plates containing nutrient agar and MRBA supplemented with
chloramphenicol.
The poured petri plates were opened for 2 mins at desired places to harbour the air
microflora.
For allowing the growth of bacteria, petri plates containing nutrient agar incubated at
37 C for 24 hours while for growth of fungi, petri plates containing MRBA were
incubated at 28 C for 3-4 days. Collect fresh soil samples from various places. Ensure
that collected soil is free from any biological debris, stone and plastic.
Count the number of colony forming unit (cfu) in each case.

32

OBSERVATION
Air sample

Colony forming unit

Bacteria

Fungi

RESULTS
On the basis of experimental findings, the highest and lowest air microflora was observed in
................................................ and .................................................. air samples respectively.

33

Exp 2.6: Fluorogenic detection of E. coli

INTRODUCTION
Latest and economical range of diagnostic product HiDtect Rapid Identification Disc
has been developed which helps
in
direct
identification
of
microorganisms from clinical
samples, water samples, food
samples, environment etc.
HiDtect Rapid Identification
Discs is simple to perform. It
eliminates the use of Selective
Medias
and
further
study
elaborate
biochemical
tests.
Unique discs are developed which
finds application in various
sectors of Food & Dairy industry,
Water industry, Pharmaceuticals
Laboratory Testing, Cosmetic
industry,
Environmental
&
Sanitary
Testing,
Clinical
Diagnosis etc. In either fields it
involves the use of Enriched and
Selective media. The isolate is
often presumptively identified
morphologically
by
special
staining methods and plated out
on diagnostic media. Further
elaborate biochemical tests are
done for confirmation of the isolate recovered. Overall the whole procedure is time
consuming, expensive and laborious. Hence there is continuous search for a technology that
could aid in rapid, reliable, simple and economical diagnosis worldwide. HiDtect Rapid
Identification Discs have been developed which enables rapid and reliable detection of
microorganisms.
MATERIALS REQUIRED
DT007 HiDtect E. coli Fluorogenic Identification Disc
PROCEDURE
It involves routine inoculating and isolation technique followed by replication and
direct identification.
Step I Inoculation, Isolation and Incubation
Inoculate the organisms from sample on any of general purpose media like
Nutrient Agar
Adopt any of surface plating methods as; Spread Plate Method, Quadrant (four or
five) streak pattern or T streak method so as to obtain isolated colonies from inoculums.
Incubate at 35-37 C for 18-24 hours. Check for bacterial growth
34

 Step II Replication and Identification

Place the HiDtect Rapid Identification disc of choice (suspected organisms) on
the surface of Agar plate. Perform this step for maximum of 30 seconds to 1 Min.
Mark the corresponding orientation of paper. This is replication technique.
Incubate the replicated identification disc in empty sterile Petri dish at 35-37 C for 1-4
hours.
Observe for development of colour under UV light and interpret.
OBSERVATION

RESULTS
Detection of E.coli was carried out using DT007 discs.

35

Exp 2.4: IMViC test for biochemical characterization of bacteria

INTRODUCTION
The IMViC tests are a group of individual tests used in microbiology lab testing to identify an
organism in the coliform group. A coliform is a gram negative, aerobic or facultative
anaerobic rod which produces gas from lactose within 48 hours. The presence of some
coliforms indicate fecal contamination. Except for the lowercase "i", which is added for ease
of pronunciation, each of the letters in "IMViC" stands for one of these tests. "I" is for indole
test; "M" is for methyl red test; "V" is for Voges-Proskauer test, and "C" is for citrate test.
The lower case "i" is merely for "in", as the Citrate test requires coliform samples to be
placed "in Citrate". These tests are useful in distinguishing members of Enterobacteriaceae.
INDOLE TEST
In this test, the organism under consideration is grown in tryptone water broth. It contains
tryptophan, which under the action of enzyme tryptophanase is converted to an Indole
molecule, pyruvate and carbon dioxide. To test the broth for indole production, Kovac's
reagent is added after incubation. A positive result is indicated by a pink/red layer forming on
top of the liquid.

METHYL RED AND VOGES-PROSKAUER TEST

These tests both use the same broth for bacterial growth. The broth is called MRVP broth.
After growth, the broth is separated into two different tubes, one for the methyl red (MR) test
and one for the Voges-Proskauer (VP) test. The methyl red test detects production of acids
formed during metabolism using mixed acid fermentation pathway using pyruvate as a
substrate. The pH indicator Methyl Red is added to one tube and a red color appears at pH's
lower than 4.2, indicating a positive test (mixed acid fermentation is used). The solution
remaining yellow (pH = 6.2 or above) indicates a negative test, meaning the butanediol
fermentation is used.

36

The Voges-Proskaeur test uses alpha-naphthol and potassium hydroxide to test for the
presence of acetylmethylcarbinol (acetoin), an intermediate of the 2,3-butanediol
fermentation pathway. After adding both reagents, the tube is shaken vigorously then allowed
to sit for 5-10 minutes. A pinkish-red color indicates a positive test, meaning the 2,3butanediol fermentation pathway is used.

CITRATE TEST
This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate
as its sole carbon source. The agar contains citrate and ammonium ions (nitrogen source) and
bromothymol blue as an indicator. The citrate agar is green before inoculation, and turns blue
as a positive test indicator, meaning citrate is utilized.

MATERIALS REQUIRED
Nutrient broth tubes for stock cultures, dropper, bunsen burner, test tubes, inoculation loop,
1% tryptone broth (5mL/tube) for Indole test, Kovac reagent, MRVP broth tubes (peptone 7g;
dextrose 5g; potassium phosphate 5g; water 1000 mL; 5mL/tube), methyl red pH indicator,
V-P reagent I (napthol solution), V-P reagent II (40% potassium hydroxide), Simmon's citrate
agar slants.

37

PROCEDURE
Indole test Procedure:
Each culture of bacteria is inoculated in each test tube containing tryptone broth and then
incubated over a period of 24-48 hours at 37C.
After incubation, few drops of Kovacs reagents are added to it with the help of dropper.
The observations are recorded.
Methyl Red Procedure:
The bacterial cultures are inoculated into test tubes containing MRVP broth using
inoculation loop and kept for 24-48 hours of incubation period at 37C.
After this, the indicator Methyl red is added to each test tube and the change is observed.
Vogues-Proskauer Procedure:
The bacterial cultures are inoculated into test tubes containing MRVP broth using
inoculation loop and kept for 24-48 hours of incubation period at 37C.
After this, the reagent VP reagent I and VP reagent II are added to each test tube and the
changes are observed.
Citrate Utilization Procedure:
The bacterial cultures are streaked into test tubes containing Simmons citrate agar using
inoculation loop and kept for 48 hours of incubation period.
The change in colour of the medium after incubation is observed and noted.
OBSERVATION
S.No.
Culture 1
Culture 2
Culture 3
Culture 4

Indole test

Methyl Red test

Vogues-Proskauer test

Citrate Utilization test

PRECAUTIONS
While adding Kovacs reagent, wear gloves.
Dont leave tryptone tubes for inoculation for more than 24 hours as indole may be
further metabolised.
No more than 5 drops of Methyl red should be added.
After incubation, check if the test tubes are turbid so that bacteria growth is there.
After adding both the reagents, the test tube needs to be shaken vigorously.
After shaking the tubes, allow it to stand for 10-15 minutes.
The simmon citrate agar should be in a slant fashion to allow for maximum surface area
for inoculation of the culture.
The test tubes should be kept for incubation at 37C for atleast 24 hours.

38

Exp 2.3: Preparation of glycerol stock for long term preservation

INTRODUCTION
A bacterial culture in a capped tube is in a closed environment. Though the culture may start
healthy, given time the number of viable cells will decrease to zero. The goal of preserving
the cultures is to slow that death rate so that when the culture is revisited, some of the cells
are still viable and available for culturing. The reasons the cells die can be numerous, but in
every instance are based on the inherent chemistry of the cells and their environment. If the
deleterious chemical reactions can be slowed or halted, then the overall culture will remain
viable for a longer period of time.
There are two basic approaches to slowing the rate of deleterious reactions in a culture of
bacteria. The first is to lower the temperature which decreases the rate of all chemical
reactions. This can be done using refrigerators, mechanical freezers, and liquid nitrogen
freezers. The second option is to remove water from the culture, a process which can be
tricky and involves sublimation of water using a lyophilizer. The common methods are as
follows.
1. Cold Storage on Agar slant cultures: Agar slants that are prepared can be streaked with
individual culture and incubated overnight. The cultures are then stored at 5-10C. It must
be sub-cultured every six months.
2. Agar Slant Culture covered with mineral oil: Agar slants after streaking can have the
incubated cultures covered with mineral up to 1cm. It must be sub-cultured every 1 year.
3. Storage in saline suspension: Bacteria require salts for various metabolic activities, but a
high concentration of salt acts as a bacteriostatic. Cultures preserved in 1% salt
concentration must be sealed tightly to prevent evaporation. The tubes can be stored at
room temperature.
4. Preservation by drying in vacuum: The culture are dried over CaCl2 in a vacuum, then
stored in a refrigerator. The organisms survive longer than air dried cultures.
5. Lyophilization: In this method the microbial suspension is put in small ampules and
process through drying under vacuum. Vacuum drying causes the sublimation of cell
water. The ampules are then sealed off in vacuum. The cultures are then stored at 4C.
These cultures remain viable for up to 10 years.
6. Cryopreservation using glycerol: Bacteria on an agar plate can be stored at 4C for a few
weeks. However, if you want to store bacteria for a longer time, you will need to establish
glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage
to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be
stored stably at -80C for many years. Glycerol is used as it is a cryoprotectant. Cells at 39

70C if kept in some other buffer or media, will freeze causing ice formation inside and
outside the cells causing their death as the ice crystals rupture the membranes present.
Glycerol forms strong hydrogen bonds with water molecules, competing with waterwater hydrogen bonds. This disrupts the crystal lattice formation of ice unless the
temperature is significantly lowered. The minimum freezing point temperature is at about
36 F / 37.8 F corresponding to 70% glycerol in water. Glycerol is therefore used as it
allows for the lowering of temperature as well as increasing the viscosity.
MATERIALS REQUIRED
Nutrient broth for bacterial stock culture, Potato dextrose broth for fungal stock culture,
Sterile eppendorf tube or cryo-vial, 80% (v/v) Glycerol, Micropipette with autoclaved tips,
Bacterial Culture (12 hours old), Fungal culture Inoculating loop
PROCEDURE

Add 200 l of sterile 80% (v/v) glycerol solution to the eppendorf tube using a cut tip.
Put 800 l of stock culture in a sterile eppendorf tube with the help of Micropipette.
Mix the culture ampules by capping and then inverting the tubes.
After mixing allow the tubes to stand for about 30-60 seconds.
Freeze on liquid nitrogen or directly into 70C.
Store in a freezer at 70C.
To recover/revive the bacteria scrape the frozen surface of the culture with a sterile
inoculating loop, and then immediately streak the bacteria that adhere to the loop onto the
surface of an LB agar plate. Incubate the plate overnight at 37C.

OBSERVATION
The number of viable cells remain almost the same after preserving in glycerol stock for
many weeks as in the original culture.
RESULT
Creating bacterial glycerol stocks helps in maintaining the number of viable cells for longer
periods of time.
PRECAUTIONS

It is very important that you shake the glycerol before freezing. Make sure it is uniform.
Be sure to label both lid and tube before freezing.
The freezer should not be self-defrosting.
The ampules must be transparent.
The inoculation loop must be sterilized properly during revival process if the glycerol
stock is to be used again.

40