Practical 1 (SBB 2105)

Determination of microbial numbers through cultivation

1. Quantifying soil microbes using the spread-plate technique
Introduction
The counting techniques are based on the assumption that a single colony develops from each
cell and that all colonies are formed from a single mother cell. However, sometimes microbes
clump and a single colony may grow from several microbes clustered together. The viable
count is thus invariably less than the total cell number in a sample.
From most samples (e.g. soils), a suspension must be prepared first and diluted. Usually a 10-
fold dilution series is applied. Different media can be used for a given sample. A known
volume (0.1 ml) of the dilutions is plated onto the surface of a suitable solid growth medium.
After incubation for a specified period of time, the colonies are counted and the microbial
numbers in the original sample can be determined.

Materials and equipment:

Agar plates
Glass spreader
(Alcohol for sterilisation)
Pipettes, sterile pipette tips
9 mL sterile distilled water in test tubes (7)
Vortex mixer
Bunsen burner
Incubator

Procedure:
1. Make a 10-fold dilution series from an environmental sample (soil): measure 1 g soil
sample into a test tube containing 9 ml sterile water (mix thoroughly with vortex
mixer, 10-1 dilution), pipette 1 ml from this suspension into a test tube containing 9 ml
sterile water, mix thoroughly with vortex, pipette 1 ml from this latter suspension into
another test tube containing 9 ml sterile water, mix thoroughly Figure 1. (until the
desired degree of dilution is reached , 10-7).
 Figure 1. Serial dilutions

2. Spread 0.1 ml from the given each dilution onto the surface of agar plates by
pipetting 0.1 ml from the appropriate tube of the dilution series onto the centre of the
agar surface; rinse the glass spreader with alcohol (remove any excess alcohol) and
sterilise the rod by flaming (take the rod away from the flame while the alcohol
burns); cool down the glass spreader by touching the medium surface (without
touching the liquid containing bacteria); spread the liquid evenly over the surface
(while spreading dish should be opened only slit like) (Fig. 2). Do this in duplicates

Fig. 2. Spreading on the surface of an agar plate.

3. Incubate Petri dishes at 28°C for 24-48 hours.
4. Count the number of discrete colonies, average the numbers and calculate the CFU
value of the sample.
5. Results of different dilutions should also be averaged. Give the CFU values of the
original sample in CFU/g units.

2. Quantifying soil microbes using the pour-plate technique

Procedure:

1. Using the same dilution series above, pipette 1.0 ml sample into empty labelled
Petri dishes.
2. Pour 20-25 mL sterile, melted (ca. 50°C) medium into the inoculated Petri dishes
and mix them softly. Let it solidify.
3. Incubate Petri dishes at 28°C for 24-48 hours.
4. Count the number of discrete colonies, average the numbers and calculate the
CFU
value of the sample.
5. Results of different dilutions should also be averaged. Give the CFU values of the
original sample in CFU/g units