EXERCISE NO.

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COMMON LABORATORY APPARATUS / EQUIPMENTS, INSTRUMENTS, CHEMICALS
AND THEIR USE IN MICROBIOLOGY LABORATORY

INTRODUCTION:
Any individual working in laboratory must learn the use and care of laboratory
Instruments. Most instruments, for their efficient use, require some measure of training
and skill. Instruments are delicate and should be handled carefully. Hence it is advisable
to read the ‘instruction manual’ carefully before its operation or should contact the
person who is familiar to the instrument. It provides information regarding operation,
maintenance, trouble shooting and essential knowledge for repair jobs.
In microbiology laboratory, following instruments are used:
1. Weighing Balance 7. Water Bath
2. pH Meter 8. Orbital Shaker
3. Autoclave/ Steam Sterilizer 9. Colony Counter
4. Hot Air Oven 10. Centrifuge
5. Laminar Air Flow Cabinet 11. Refrigerator
6. Incubator

1. WEIGHING BALANCE:
Several types of balances such as single pan
balance, top pan balance, analytical balance are available
for weighing different ingredients required during course
of experimentation. Accuracy of weighing is determined
by sensitivity of the balance, which may be as low as
0.0001 gram (1 milligram). Electronic balances are easy
to handle and are more accurate and sensitive.
1. Electronic balances are highly sensitive and even a
little movement of air may affect the accuracy of the
measurements.
2. After every use clean the pan with dry clean tissue
paper and keep in dust proof chamber.

2. pH METER:
As a convenient way of expressing hydrogen ion concentration, S.P.L. Sorensen
introduced the symbol pH in 1909. ‘pH is defined as the negative value of the logarithm
to the base 10 of the hydrogen ion concentration or the logarithm (to the base) of the
reciprocal of the hydrogen-ion concentration’.
pH = -log 10 [H+] = log 10/[8+] , or [H+] =10 –pH

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 The most reliable way to measure the pH of a solution is to use pH meter. A pH
meter measures the differences in potential between two solutions of different pH
value.
PRINCIPLE:
When two solutions containing hydrogen
ion are separated by the bulb of a glass pH
electrode that is hydrogen sensitive, an
electrical potential is developed across the thin
glass separating the two solutions. If the solution
inside the bulb is of fixed hydrogen ion
concentration, the potential across the glass will
change as the hydrogen ion concentration of the
other solution varies. This difference in potential
can be measured by making an electrical
connection between the internal element of the
glass electrode and a reference electrode
(potential of which is known). pH meter should be calibrated using standard solutions of
pH 4.0, 7.0 and 9.0.

3. AUTOCLAVE/ STEAM STERILIZER:

Autoclave was developed by Charles Chamberland in 1879, which tremendously
promoted growth of microbiology discipline.
PRINCIPLE:
Increase in pressure of steam is directly proportional to the increase in
temperature. In other words, it is called as ‘Steam under Pressure’. Moist heat has more
penetration power, kills living cells by degrading nucleic acid or by denaturing enzymes
and other essential proteins. It may also disrupt cell membrane. Autoclave is used for
sterilization of various utilities specially the media under saturated steam pressure at
any selected point between 10-20 Pounds per square inch (psi).
Autoclaves may be either vertical or horizontal. It is a double or triple walled unit
mounted on a sturdy metal (Steel) body. The heating element is fitted at the base of the
cylinder. The space between the outer and steam jacket is insulated to minimize
temperature loss. The lid is fitted with rubber gasket and tightened by wing nut/radial
locking system. All autoclaves are fitted with standard accessories such as water
indicator, pressure gauge, temperature gauge, steam release valve, safety valve etc.
Generally sterilization is carried out at 15 psi for 20 minute and time here is considered
after the pressure is achieved.
Relationship between pressure and temperature is as follows.
Pressure Saturated steam* Pressure (psi) Saturated steam*
[OC] [OC]

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 05 109 20 126
10 115 25 130
15 121 30 135
(*Pressure-temperature relationship holds true only if the chamber is completely filled with saturated
steam only.)

PRECAUTIONS:
 Check water level whether it is up to marked level or not. If not, make it up to the mark.
 Screw down lid, tighten diagrammatically opposite wing in pairs so that the lid is clamped evenly on
the gasket.
 Let steam flow for at least 3-5 minutes to remove all air from the chamber to obtain correct pressure
and temperature.
 Time is calculated after the chamber reached the desired temperature.
 Over sterilization should be avoided because it leads to hydrolysis of the components.
 Let the pressure and temperature normal and then only open the chamber.
ARTICLES STERILIZED IN AUTOCLAVE:
 Routine Microbiological media.
 Solution having water.
 Sugar solutions are sterilized at 10 psi pressure for 30 min.
 Linen cloth, bandages, apron, surgical cotton etc.
 Petri dishes, pipettes and other glass wares can be sterilized.
4. HOT AIR OVEN:
It is a dry heat type of sterilizer. It is a vertical steel cabinet with double or triple
walled body of aluminum or stainless steel. The body is constructed with heating
elements between the walls either at the bottom of the box (for bottom heated model)
or on all three sides of the body (universal heating models). Door is provided with
synthetic rubber gasket to make it air tight. The range of temperature inside varies from
50°C to 300°C or more. Digital temperature controller cum indicator is provided with
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power switch to control temperature inside the chamber. The temperature required for
sterilization may vary with the time of exposure to dry heat as mentioned bellow:
Temperature Time
120°C 8 hours
140°C 3 hours
160°C 1 hour
180°C 20 minutes

PRINCIPLE:
It is based on the principle that the dry-heat or hot
air destroys microorganisms by oxidizing their bio-chemical
constituents. Normally 165-170°C (329- 338°F) temperature
for 2 hours is sufficient to kill the live spores by dry heat.
ARTICLES STERILIZED IN OVEN:
Glassware like Petri-dishes, pipettes, glass syringes,
surgical instruments, mineral oils, Paraffin oil, petroleum
jelly, talcum powder etc.

5. LAMINAR AIR FLOW CABINET:

Laminar flow provides aseptic or micro-organism free environment for
performing various activities such as pouring of sterilized media in sterilized plates,
isolation and transfer of microorganisms. During applications of different
methodologies, require aseptic or sterilized environment.
The cabinet is fabricated out of thick board of sun mica or made up of stainless
steel. Interior surface of working platform/table (popularly known as hood) is of stainless
steel with the sun mica clad at the top. Sides of the panels are of thick transparent
plexi/acrylic glass duly framed. The unit is fitted with both pre filter and High Efficiency
Particular Air (HEPA) filter. Air is drawn through pre filter by blower and passed through
HEPA filters particles of size 0.3 micron or larger.
The working area is illuminated by florescent light fitted with the unit. U.V. light is
also fixed underneath the sun mica clad at the top and it is switched on 10-20 minutes
before working. Cock for gas, vacuum line is also provided at the outer layer of the top
clad.
PRECAUTIONS:
1. Proper care is taken not to expose any part of the body to the U.V. light as the
exposure may be carcinogenic or mutagenic.
2. Before and after every use, clean the platform and disinfect with alcohol.
3. Keep the blower on while working.

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4. Vacuum line should be monitored time to time.

6. INCUBATOR:
Incubators are the cabinets in which desired temperature can be constantly
maintained. There are two type of incubators used in microbiology laboratory as under:
(A) BACTERIOLOGICAL INCUBATOR: It is used for incubation of bacterial cultures. Most of
the modem incubators are heated electrically with the help of
heaters provided usually at the bottom of the cabinet. The
incubator is set to the desired temperature which is normally
the optimum temperature of most microorganisms.
Normally the temperature of incubator is constantly
maintained at 37°C. This is due to the fact that; (i) most of the
routine bacteria studied are mesophilic and (ii) the human
pathogens growing optimally at this temperature (because the
normal human body temperature is also 37°C).

(B) BOD INCUBATOR:

The Biological Oxygen Demand (BOD)
incubator maintains a range of temperature
below and above the ambient temperatures
required for growth and multiplication of
various microorganisms. Temperature inside
the may be maintained from 5°C to 50°C with

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an accuracy of ± 1.0°C. BOD incubator is provided with both heating and cooling system.
It is also equipped with air circulating fans for uniform distribution of temperature
inside. Fluorescence light is also installed inside the chamber for illumination. The light is
attached with timer for regulating illumination period. An inlet nozzle is also installed for
monitoring air mixture concentration inside and humidistat for control of humidity (55-
95%) by natural mist. The front side is installed with automatic/manual control panel
consisting mains, temperature indicator cum controller, heat energy regulator etc.
Generally used for incubation/maintenance of fungal cultures, rearing insect &
nematodes etc.

7. WATER BATH:
Water bath is a constant temperature
device which is electrically operated. The
temperature is maintained constantly at a
desired value with the help of automatic
thermostat in the range of 0°C to 100°C.
Water baths are of two types one is Incubator
or serological water bath and another one is
boiling water bath.
Water maintains better control of the
temperature in comparison to the air. Due to
this reason, most serological procedures
require incubation in water bath rather than in incubator.
8. ORBITAL SHAKERS:
It has the facilities for continuous variable shaking speed with motor shaft and
maintaining the uniform temperature. A control panel is mounted on the top of the
chamber accommodated with digital display cum control for shaking in form of RPM
(Rotation/Revolution Per Minute) and temperature (heating and cooling).
USE:
It is used for mass multiplication of microorganisms. Due to optimum
temperature, aeration and agitation rapid growth is obtained in a very short time. It can
also be used for mixing of certain liquids.

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 9. COLONY COUNTER:
The instrument is used when large numbers of colony counts
are to be done. The Petri dish resets on this glass and is
illuminated by a strong light source placed inside the metal
cabinet. A simple magnifying lens (5 X) is fitted at the top
corner, which can be adjusted vertically as well as radially. To
count colonies, place the open dish with glass side up, over
the illuminated screen, count colonies using the magnifying
lens and a hand held counter. Mark Total number of colonies
or colony forming units per milliliter (cfu/ml) can be
calculated by multiplying the average number of colonies per
countable plate by the reciprocal of the dilution.
10. CENTRIFUGE:
Centrifuge is a device used for separation of
particulate matter (having different densities) from a liquid
medium by spinning it at high speed. As a result the
centrifugal force of spinning pushes the solid particles of
higher density outwards, which settle at the bottom of the
centrifuge tube and form a pellet. The body of the device is
equipped with microprocessor controller for regulating the
speed in revolution per minute (rpm). The centrifuge speed
varies according to type of centrifuge, normal laboratory
table centrifuge has maximum speed up to 5000 rpm, high
speed cooling centrifuge has maximum speed up to 25,000 rpm in which temperature
can be controlled up to 4°C, while ultra centrifuge has maximum speed up to 1,00,000
to 1,50,000 rpm.

11. REFRIGERATOR/DEEP FREEZER:

A refrigerator is an instrument, which maintains the temperature of a material at
a low level then the temperature of the surrounding environment. A normal laboratory
refragirator has temperature range between 0˚C to 10˚C. Deep freeze has all
specifications similar to that of refrigerator except, the temperature is maintained below
0°C therefore, puff insulation and more than one compressor is installed.
PRINCIPLE:
Low temperature slows down the biochemical activities/metabolism of the cell and
exerts a micro biostatic effect. Hence, the growth of the organism is inhibited.
Use:

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 Refrigerator is used in the laboratory for preservation of microbial culture. It is
also used for storage of bacteriological media, reagents, blood, serological aid,
antibiotics, vaccines and many other heat labile substances.

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 Exercise No.
Title: General procedure for isolation of soil microorganisms

Objective: To isolate and count the fungal and bacterial microorganisms from soil by serial
dilution method
Materials required:
 Erlenmeyer flask
 1 g of soil sample
 Sterile 1ml pipettes
 Sterile glass stirring rod
 Petri plates containing about 20 ml of PDA/NA media
 Glass vials
 Measuring cylinder
 Strips of Parafilm
 Paper towel
 Disinfectant
 Marking pens
Methods:
1. Mark the dilutions on the vials (10-1, 10-2, 10-3, 10-4, 10-5, 10-6,10-7,10-8) and Petri plates
(10-4, 10-5, 10-6,10-7).
2. Add 1 g soil sample to the Erlenmeyer flask containing 10 ml of sterilized distilled
water.
3. Shake well using vertex for one minute.
4. Using a sterile pipette, take 1 ml suspension from Erlenmeyer flask and place it in a vial
containing 9 ml of sterilized distilled water. (10-2 dilution).
5. Shake well using vertex for one minute.
6. Repeat steps 4 and 5 for six other dilutions (10-3, 10-4, 10-5, 10-6,10-7, 10-8).
7. Starting with the weakest dilution (10-8), pipette 1ml onto each of 20 Petri plates
containing PDA/NA.
8. Spread over the entire surface using the sterile glass spreader.
9. Follow the same steps for the 10-7 to 10-4 dilutions.
10. Seal the Petri plates with the Parafilm.
11. Incubate the Petri plates at room temperature.
12. Observe the incubated Petri plates after 24, 48 and 72 hours.
Results:
 Record the number of colonies per Petri plate.
 Calculate the number of microorganisms per gram of soil using the following formula.

Total number of colonies

Cfu/g =
Dilution x Volume taken (ml)

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Serial dilution:
A laboratory technique in which a substance is decreased in concentration in a series of
proportional amounts.
A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution
factor at each step is constant, resulting in a geometric progression of the concentration in a
logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M.

How to calculate CFU (colony forming unit):

Colony forming unit is a measure or count of viable cells unlike direct microscopic
methods where all cells are counted either viable or non-viable. The results are read as CFU/mL
(Colony Forming Unit per millilitre) for the liquid cultures, whereas CFU/g (Colony Forming
Unit per grams) for the solid cell cultures.

The method:
First of all if the culture is broth, dilutions are made and suitable dilution is selected for
spreading and colonies are counted on the plate that has been spread with the selected dilution
and then using this formula the colony forming units are counted:

Bacteria per mL= Number of colonies present on the plate x Dilution factor
Volume of culture plate

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Example:
5ml of Bacterial Culture is added to 45ml of sterile diluent. From this suspension, two
serial, 1/100 (10-2) dilutions are made, and 100µL is plated onto agar plate. After overnight
incubation, 150 colonies were counted on the plate. Calculate CFU/mL of the original Sample?
Solution:
First thing that we need to find out is the dilution of the original sample or how much is the
sample is diluted.
Here in this problem the original sample or the bacteria culture was first mixed in a diluent,
hence we need to find out this dilution factor first
That is: Final Volume/Sample volume
Final volume = Volume of bacterial culture added to diluent + Volume of diluent
= (5 + 45) mL
= 50mL
Sample Volume = 5mL
Dilution factor = 50/5
=10/1
= 101
Now, the two serial dilutions of 1/100 i.e. 10-2 were made
Hence,
Total Dilution Factor= 101 x 102 x 102
= 105
Now,
CFU/mL= (Number of bacterial colonies counted on plate x Dilution Factor) / Volume of
culture plate
CFU/mL= (150 x 105) / 0.1
= 1.50 x 108
So, total colony forming unit = 1.5 x 108 per mL

Significance of Colony Forming Unit calculations:

Colony Forming Unit is a unit used in microbial counting to estimate the number of
viable microbes in a sample. Viable simply means the microbes that are able to divide and are
alive.
This method lets us count only the live cells unlike other methods that counts number of cells
irrespective of the viability of the cells.

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Microbiological media used for isolation of different microorganisms

Sr. No. Organism Media

1. Entomopathogenic fungi PDA (Potato Dextrose Agar)
2. Trichoderma PDA (Potato Dextrose Agar) or Trichoderma Specific
Medium-(TSM).
3. Pseudomonas King’s B Medium
4. Bacillus spp. Nutrient Agar/broth
5. Rhizobium Congo Red Yeast Extract Mannitol Agar (CRYEMA)
6. Azotobacter Jenson’s Agar Medium
7. Azospirillum Nitrogen Free Bromo Thymol Blue medium
(NFBTB)
8. PSM Pikovaskaya’s medium or PKVK medium

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 Exercise No.
Title: Methods of application of biofertilizers.
Based on type of microorganism, the bio-fertilizer can also be classified as follows:

 Bacterial Biofertilizers: e.g. Rhizobium, Azospirilium, Azotobacter, Phosphobacteria.

 Fungal Biofertilizers: e.g. Mycorhiza
 Algal Biofertilizers: e.g. Blue Green Algae (BGA) and Azolla.
 Actinimycetes Biofertilizer: e.g. Frankia.

1. Seed inoculation OR seed treatment

This is the most common practice of applying biofertilizers. In this method, the
biofertilizers are mixed with 10% solution of jaggary. The slurry is then poured over the seeds
spread on a cemented floor and mixed properly in a way that a thin layer is formed around the
seeds. The treated seeds should be dried in the shade overnight and then they should be used.
Generally, 750 grams of biofertilizer is required to treat the legume seeds for a one-hectare area.
2. Seedling root dip
The seedling roots of transplanted crops are treated for half an hour in a solution of biofertilizers
before transplantation in the field. In this method, the seedlings required for one acre are
inoculated using 2–2.5 kg biofertilizers. For this, a bucket having adequate quantity of water is
taken and the biofertilizer is mixed properly. The roots of the seedlings are then dipped in this
mixture so as to enable the roots to get inoculum. These seedlings are then transplanted. This
method has been found very much suitable for crops like tomato, rice, onion, cole crops and
flowers.
3. Main field application
This method is mostly used for fruit crops, sugarcane and other crops where localized
application is needed. At the time of planting of fruit trees, 20 g of biofertilizer mixed with
compost is to be added in the ring of one sapling. The same quantity of biofertilizer may be
added in the ring soil of the seedling after it has attained maturity. Sometimes, biofertilizers are
also introduced in the soil but this may require four to ten times more biofertilizers. Before use,
the inoculants should be incubated with the desired amount of well decomposed granulated
farmyard manure (FYM) for 24 hours. The FYM acts as nutrition medium and adjuvant
(carrier) for biofertilizers.
4. Self-inoculation or tuber inoculation
This method is exclusively suitable for application of Azotobacter. In this method, 50 liters of
water is taken in a drum and 4–5 kg of Azotobacter biofertilizer is added and mixed properly.
Planting materials required for one acre of land are dipped in this mixture. Similarly, if we are
treating potato, then the tubers are dipped in the mixture and planting is done after drying the
materials in the shade.

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