EXPERIMENT 45
Isolation and enumeration of microorganisms from soil by the serial
dilution-agar plating method (or viable plate count method)
Though various methods are available to isolate and enumerate microorganisms (bacteria,
fungi, protozoa, algae and viruses) from soil, food stufs, milk and water. The serialdilution
agar plating method or viable plate count method is one of the commonly used
for the isolation and enumeration of fungi and bacteria which are the procedures
most prevalent
microorganisms. This method is bassed upon the principle that when material containing
154
Techniques for Isolation and
Cutivation Enumeration of Microorganisms 155
microorganism is cultured each viable microorganisms will develop into a colony, hence
number of colonies appearing on the plates
the
the sample. represent the number of living organisms
presentin
In serial dilution agar-plate method, a known amount (10 mL or 10 g) of material is
su_pended or agitated in a known volume of sterile water blank (90 mL of so to make
volume to 100 mL) to make a microbial
thetotal made by Suspension. Serial dilutions 102, 10,...
107 are pipetting measured volumes (usually 1 mL or 10 mL) into additional
dilution blanks (having 9 mL, 99 mL or 90 mL sterile water) (see Fig. 5.2 and Fig. 11.1).
each
Finally I mL aliquot of various dilutions are added to sterile Petri dishes (triplicate formedia
dilution) to which are added 15 mL (approximately) of the sterile, cool, molten (45°C)
(Nutrient agar for bacteria, Glycerol yeast agar for actinomycetes and Czapek-Dox agar or
Sabouraud agar medium, supplemented with chlorotetracycline or streptopenillin, 10ug/mL,
106 for
for fungi). The dilutions 102 to 10S are seiected for enumeration of fungi. 10-3 to
actinomycetes and 10-4 to 107 for bacteria relative to their proportion in soil. Upon
solidification, the plates are incubated, in an inverted position for 3-7 days at 25°C. The
number of colonies appearing on dilution plates are counted, averaged and multiplied by the
Ailution actor to find the number of bacteria/cells/spores per gram (or millilitre) of the
sample.
No. of bacteria/cells/mL or g
Number (average of 3 replicates)of colonies x Dilution factor
Dry wt. of soil
Dilution factor = Reciprocal of the dilution (e.g., 107= 10)
as a diluent, in place
Currently, 0.85%% sodium chloride (NaCI) is being preferred Figure 11.1 illustrates
suspension of soil sample.
of sterile water, for the preparation of microorganisms per gram of soil by viable plate
the various steps for estimating numnber of
count method.
Requirements
" Soil sample
cooled (45°C) media: nutrient agar, glycerol yeast agar, Sabouraud agar
" Molten and
(2 flasks each)
Aureomycin (Chlorotetracycline)
sulphate)
" Streptopenicillin (or streptomycin
" 90 mL sterile water blanks (7)
"Sterile Petri dishes (36)
" Sterile syringe with needle
" Sterile 10-mL pipettes (7)
" Magnetic shaker
" Colony counter
" Bunsen burner/Spirit lamp
" Wax marking pencil.
156
Procedure
field, mix thoroughly to mal..
1. Collect soil samples at random, minimum five, from a
a composite sample for microbiological analysis.
and sterile Petri dish.
2. Label 90-mL sterile water blanks as 1. 2. 3, 4, 5, 6, and 7 w
as 10 (3 plates), 10-3 (6), 104 (9), 10-5 (9), 106 (6) and 10 (3) with a
pencil.
10g Soil
+ 1/10 Soil dilution
90mL0.85% NaCI
1mL 1 mL 1mL 1mL
5
10 10 10 10
9mL 0.85% NaCI Spread 1 mL on replicate
Petri plates containing
1.0 mL previous dilution appropriate medium
Incubation of plates at 37°C for 24-28 hours
in an inverted position
Count number of colonies of microorganisms
in each plate using Quebec colony counter
Calculate the number of microorganisms per g of soil
Mean plate countx Dilution Factor
Viable cells/g dry soil =
Dry weight of soil
FIGURE 11.1 Procedure for estimating number of microorganisms per gram of soil by
viable plate count method (serial dilution agar plating technique) (Modified from Tate,
R.L,1995, Soil Microbiology. John Wiley &Sons, New York).
Cultivation Techniques for Isolation and Enumeration of 157
Microorganisms
3. Add 10 g sample of fincly pulverized, air dried soil into numbered Iwater blank
to make 1:10 dilution (10 ).
4. Vigorously shake the dilution on a mapnetic shaker for 20-30 minutes to obtain
uniform suspension of microorganisms.
5. Transfer 10 ml of suspension from flask number intowater blank number 2with
asterile pipette under aseptic conditions to make 1:100 (10 2) dilution and shake it
well for about 5 minutes.
6. Prepare another dilution 1:1000 (10 ) by pipeing 10 ml. of the suspension into
water blank number 3, using afresh sterile pipette and shake it.
7. Make further dilutions 104 to 107 by pipetting 10 mL suspension into additional
water blanks (4, 5, 6, 7) as prepared above.
8. Transfer l mL aliquots cach from 10 dilution blank into 3sterile Petri dishes, from
10 dilution blank to 6sterile Petri dishes, from 10to 9sterile Petri dishes, from
10 to 9 Petri dishes, from 10 to 6Petri dishes and from 107 to 3 Petri dishes.
9. Add 15 mL of the cooled medium (45°C) to each Petri dish and mix the inoculum
by gentle rotation of the Petri dish. The three media are to be added to various
dilutions as follows:
(a) For bacteria-nutrient agar medium to 12 plates with 104 to 107 dilutions.
(b) For actinomycetes glycerol yeast agar medium supplemented with aureomycin
to plates with 103 to 106 dilutions.
(c) For fungi -Sabouraud agar medium supplemented with streptopenicillin to
plates with 102 to 105 dilutions.
10. Upon solidification of the media, incubate allthe plates inan inverted position at
25°C for 2-7 days.
Observations
1. Observe the plates for number and distribution of colonies of bacteria fungi and
actinomycetes from each dilution.
2. Select plates from the appropriate dilution which contain colonies in the range of
30 to 300 and make plate counts using a colony counter.
3. Since the dilution plates are replicates of each other, ditermine the average of the
triplicate microbial counts.
4. Record your results in the chart.
Results
Calculate the number of organisms (bacteria, actinomycetes and fungi) per g of the soil by
applying the formula:
Mean plate count xDilution factor
Viable cells/g dry soil = Dry weight of soil
For example,if 160 bacterial colonies were counted (average of 3replicates) in 1:100000
ailution of a soil sample (8 g dry wt. basis), the number of colonies per g of the soil
would be:
Culture and Microbial
Experiments in Microbiolog, Plant Pathology, Tissue
158
160 × 100000
Biotechnolo
=2000000 = 2 x 10° bacteria/g
Number of colonies of bacteria, actinomycetes and fungi in each dilution plato
Organism Dilution Dilution Number of Average Organisms per
factor colonies/plate number gm of soil
of colonies
per dilution
No. of colonies x
Dilution factor
3 Dry wt. of soil
Bacteria 10-4 104
10-5 105
10-6 106
10-7 107
Actinomycetes 10-3 103
10-4 104
10-5 105
10-6 106
Fungi 10-2 102
10-3 103
104 104
10-5 105
*Formerly actinomycetes were considered a separate group related to fungi, however, they are now
included in the domain Bacteria.
Typicaly bacteria are found in soil at densities of 108 to 109 colony-(forming unis
(CFUS)) associated with 10 to 10 actinomycetes propagules and 10 fungal and protozoan
propagules each per gram of dry soil.
Precautions
1. Isolation of microorganisms should be done from a composite sample collecte
randomly from a field.
2. Soil should be in a powdered form.
3. Each dilution must be thoroughly shaken before removing an aliquot for subseque
dilution.
4. Use separate sterile pipettes for each dilution. mixed
5. Immediately after the addition of the medium to a plate, inoculum is to be
with circular and side to side movements (i.e. swirling) of the Petri dish.
6. Aureomycin and streptopenicillin should not be added in molten media whie
too hot and to be added just before pouring of the media (at 45-50°C) to P
