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Measurement of microbial growth

Microbial cells are usually counted using a Petroff-Hauser bacterial counter (after

placing it under a phase-contrast or dark-field 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 definite 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-flow 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 30–300 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 sufficient vigour to effect a thorough mixing.

5. Repeat sucking up and expelling 5 times, and then transfer 1 ml to tube 2.



7. Allow the medium to solidify, and incubate at 28 !}" 2! for 3–5 days.

8. Count the colonies after 3–5 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 flame, 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 fixed-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 solidified 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 difficult.

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