Determination of Microbial Counts
Colony count
methods
Determination of
total viable Membrane filtration
count
Most probable
number (MPN)
Determination
of the Direct microscopic
microbial count
counts
Direct fluorescent
filter technique
Determination
of total count Flow cytometry
ATP determination
Turbidimetric methods
Colony count methods
Pour plate method Surface colony count
Pour - plate method (Dilution – plate method)
This method determines the number of living
microorganisms in a sample to be counted.
In this method, a definite weight or volume of
the sample is shaken in a sterile dilution fluid. A
measured amount of the suspension or dilution is
mixed with suitable solid medium in Petri dishes.
After solidification of the inoculated medium, the
plates are incubated at the optimum temperature
for 48 – 72 hours. During the incubation,
microorganisms will grow both on the surface and
within the medium.
Pour - plate method (Dilution – plate method)
After the incubation:
q The plates that contain from 30 to 300 bacterial
colonies are used to calculate the number of
total bacteria per the unit of the sample (g, ml or
cm2).
q The plates that contain from 15 to 150 fungal
colonies are used to calculate the number of
total fungi per the unit of the sample (g, ml or
cm2).
It must be considered that, each colony
arises from one cell or group of cells
according to the arrangement of cells.
SO
The count is measured by
CFU / sample unit
cfu / g solid sample
cfu / ml liquid sample
cfu / cm2 surface area
Each colony will arise from group of cells
Each colony will arise from one cell
Pour - plate method (Dilution – plate method)
Materials:
vSterile dilution fluid (water, water peptone 0.1%,
saline 0.85% NaCl) in tubes or bottles.
vSterile pipettes.
vSterile Petri plates.
vSterile suitable solid medium.
Procedure:
The Experiment must be performed
under aseptic conditions
1.a. Shaking the liquid sample to mix the contents.
1.b. Addition of definite weight of the solid sample to
definite volume of the dilution fluid to prepare the
initial dilution and then shaking the suspension to
mix the content.
2. Preparing serial decimal dilutions (10-1, 10-2……)
according to the probable microbial load of the
sample.
3. Transferring 1 ml of the suitable dilutions to 2 or 3
plates for each dilution.
1 g or 1 ml 1 ml 1 ml 1 ml
9 9 9 9
ml ml ml ml
1 ml 1 ml
1 ml 1 ml
10-1 10-2 10-3 10-4
10-1 10-2 10-3 10-4
10-1 10-2 10-3 10-4
4. Melting the medium at 100°c using a water bath.
5. Cooling the melted medium to 50°c.
6. Pouring the medium into the plates.
7. Mixing the medium and inoculum by a circular
movement of the plates.
8. Allowing the plates to set.
9. Incubating the plates in the inverted position at the
appropriate temperature. 10. Counting the colonies in
suitable plates containing from 30 to 300 bacterial
colonies and from 15 to 150 fungal colonies. All colonies
must be counted, including pinpoint colonies.
Calculation:
N = the average of colonies count in plates of the chosen
dilution X the reciprocal of the chosen dilution
N is measured by:
c.f.u / g solid sample
c.f.u / ml liquid sample
c.f.u / cm2 surface area
CFU Colony Forming Unit
Expression of results
ü Rounding off the calculated result to two
significant figures (putting the decimal point
between the first and second digits).
ü If the third digit is less than 5, the preceding
figure will not be modified.
ü If the third figure is greater than or equal to 5,
the preceding figure must be increased by one
unit.
For example:
vNumber of 258963 is expressed as 2.6 x 105
vNumber of 3315895 is expressed as 3.3 x 106
Original Sample
water
Most Probable Number (MPN)
This method is used for estimating the number
of living microorganisms that:
vPresent in very low count in the sample.
vPrefer the liquid media for growth.
In this method, the multiple cultures (3 or 5 liquid
cultures for each chosen dilution) are prepared from 3
serial dilutions. The microbial load is concluded by
determining the number of + cultures (growth) and –
cultures (no growth) for each dilution. Sometimes, the
count is based on the production of a certain reaction in
the medium.
Original
sample
Calculating MPN / unit of the sample (g, ml, cm2) using
MPN table (probability table) as follows:
MPN / unit of the sample = MPN of microorganisms per
1 ml of the first or middle dilution X the reciprocal of
the dilution.
The count is measured by:
MPN / g solid sample
MPN / ml liquid sample
MPN / cm2 surface area
In the dilution plate count, …………………….…….
must be used for determination of specific microbial
group.
In the dilution plate count, the selective media must
be used for determination of specific microbial group.
Lab session
Determination of the total viable count in water
sample
Materials:
Water sample
2 tubes containing 9 ml of sterile water.
3 sterile pipettes.
3 sterile Petri plates.
3 sterile nutrient agar medium.
Determination of the total viable count in water
sample
1 ml 1 ml
1 ml 9
ml
1 ml 9
ml
1 ml
Water sample 10-2
10-1
original 10-1 10-2
üMelting the nutrient agar medium at 100°c
using a water bath.
üCooling the melted medium to 50°c.
üPouring the medium into the plates.
üMixing the medium and inoculum by a circular
movement of the plates.
üAllowing the plates to set.
üIncubating the plates in the inverted position at
the 30°C / 48 – 72 h.
üCounting the colonies in suitable plates
containing from 30 to 300 colonies.
