Measurement of microbial growth

Microbial cells are usually counted using a Petroff-Hauser bacterial counter (after
placing it under a phase-contrast or dark-eld microscope). This counter consists
of a thick slide containing one block, which is divided into ten sub-blocks with
grooves. The depth of each groove is 0.2 mm. Each sub-block has the capacity
to retain a denite number of micro-organisms. Thus, the number of microbes
in a block can be determined. It is observed that 1 ml of liquid pure culture of
bacterium medium may contain about 5 000 million bacteria.
The growth of any micro-organism (e.g. bacteria) can also be measured by
counting the number of colonies developed on Petri dishes. Colony counting is a
commonly practised method in microbiological laboratories.
There are many techniques for counting viable cells that are able to divide and
form offspring. The usual method is to determine the number of cells in a given
sample capable of forming colonies on a suitable agar media. The method involves:
serial dilution;
spreading of diluted suspension on plates, and counting of colony-forming units on
plates.

Serial dilution
A laminar air-ow chamber is used in order to achieve serial dilution of the broth
culture of the strain or biofertilizer sample suspension. For plate counts, the
countable range is generally 30300 cells/ml. To achieve this concentration, the
procedure is:
1. Set out 8 tubes, each containing 9 ml of sterile water.
2. Dilute 1 ml of broth culture or biofertilizer sample suspension (1 g of sample in 9
ml of water) in steps (10^-1 to 10^-8) with a sterilized 1-ml serological pipette
equipped with a rubber bulb of 1 ml capacity.
3. Suck up broth culture or sample suspension from tube 1 to the 1-ml mark.
4. Immediately expel the broth culture or sample suspension back into the tube
with sufcient vigour to effect a thorough mixing.
5. Repeat sucking up and expelling 5 times, and then transfer 1 ml to tube 2.
6. Take a new sterile pipette, attach the rubber bulb, and remove 1 ml to tube 3.
7. Repeat this procedure using a fresh sterile pipette each time until the dilution
series is completed.
8. After completion of serial dilution, put an aliquot of the diluted sample on a
pour plate, spread plate or drop plate with specied nutrient agar media (as
described below). After incubation at 28 !} !2 C for 35 days, each viable cell
will give rise to one distinct colony, which is counted and calculated for the
number of viable cells per millilitre of suspension. The microbes so grown
are taken for further multiplication and testing for their efciency/quality.
Spreading of diluted suspension on plates and colony counting
Pour plate method
In this method, sterilized molten medium is kept ready in conical asks placed in
a water-bath at a constant temperature of 48 !. The procedure is:
1. Remove dry sterilized Petri dishes from paper packs and stack them in a
laminar air-ow chamber.
2. For each dilution, three Petri dishes will be required. Stack the plates in sets
of three each and label them with the help of a glass marker pen.
3. Using a fresh sterile pipette, pour 1 ml of aliquot from the last dilution (say,
10-8) into each of the three Petri dishes.
4. Using the same pipette, pour similar aliquots from the next two dilutions
(say, from 10^-7 and 10^-6) into three Petri dishes for each dilution.
5. Pour about same volume of molten medium into each of the plates.
6. Immediately after pouring, move the plates gently in a whirling motion to
mix the contents.
7. Allow the medium to solidify, and incubate at 28 !}" 2! for 35 days.
8. Count the colonies after 35 days.
Chapter 7 Biofertilizer assay and production 141
9. Multiply the average number of colonies by the dilution factor. If the average
number of colonies at 10^-8 dilution is 60, then the sample had a concentration
of 60 10^8 = 6 10^9 cells/ml.
Spread plate method
Using the same serially diluted samples prepared for the previously described
pour plate method, the procedure is:
1. Begin with the 10-7 dilution, and deliver 0.1 ml of the sample into each of
4 plates of yeast extract mannitol agar (YEMA) medium previously dried at
37 ! for about 2 hours.
2. Using the same pipette, dispense 0.1-ml samples from the 10^-6 and 10^-5
dilutions, in that order.
3. Prepare a glass spreader by bending a 20-cm glass rod of 4 mm diameter to
the shape of a hockey stick, dip it into alcohol and hold on ame, then cool
the spreader by touching it on the surface of a separate YEMA plate.
4. Lift the cover of each Petri dish just enough to introduce the spreader, and
place it in position on the agar surface.
5. Spread the sample evenly over the agar surface, sterilizing and cooling the
spreader between samples.
6. Incubate as before.
7. Calculate the number of viable cells as outlined for the pour plate method,
adjusting for the smaller volume plated (0.1 ml instead of 1.0 ml). For
example, if 60 colonies were counted on a plate inoculated with 0.1 ml of a
10^-7 dilution, the results should be 60 10 10^7 = 6 10^9 cells/ml.

Drop plate method
For the drop plate method, the procedure is:
1. Select three-day-old agar plates, which have been dried enough to absorb
some moisture, or dry the agar plates in a bacteriological incubator at 37 !
for 2 hours.
2. Take a xed-volume or variable-volume microlitre pipette and set the volume
30 !l.
3. Sterilize appropriate-sized microtips in a microtip box and keep them
ready.
4. Take two Petri dishes containing solidied and dried agar media and divide
the bottom of each plate into eight equal parts.
5. Using a microlitre pipette, deliver one drop (30 !l) of diluted suspension in
one part.
6. From the last dilution (say, 10-8), deliver four aliquots of 30 !l in each of the
four parts of the Petri dish. Use the remaining four parts for the next dilution
(say, 10-7). Repeat the process for two further dilutions.
7. Incubate the plates in incubator. In this case, as the area used is very small,
observations are to be recorded at the earliest. Otherwise, overlapping of
colonies will make counting difcult.