ENUMERATION OF MICROORGANISMS
 There are numerous occasions when it is necessary to either
estimate or determine the number of bacterial cells.
 Determination of cell numbers can be accomplished by a
number of direct or indirect methods. The methods include:
1- The standard plate count.
2- Turbidity.
3- Direct microscopic counts.
I-Standard Plate Count (Viable Counts)
 The number of bacteria in a given sample is usually too great to
be counted directly. However, if the sample is serially diluted
single isolated bacteria will form visible isolated
colonies.
 We are determining the number of Colony-Forming Units
(CFUs) in that known dilution.
 The number of colonies can be used as a measure of the
number of viable (living) cells in that known dilution.
 A viable cell: Is defined as a cell which is able to divide and form
a population (or colony).
 Indirect viable cell counts, also called plate counts:
1- A viable cell count is usually done by diluting the original sample.
2- Plating aliquots of the dilutions onto an appropriate culture
medium.
3- Then incubating the plates under proper conditions so that
colonies are formed.
4- After incubation, the colonies are counted and from a knowledge
of the dilution used, the original number of viable cells can be
calculated.
Materials
1- 6 tubes each containing
9.0 ml sterile saline.
2- 3 plates of suitable media.
3- 2 sterile 1.0 ml pipettes.
4- Pipette filler.
5- Turntable and bent glass
rod.
6- Dish of alcohol.
1.0 Milliliter (ml) Pipette
Procedure
A- Dilution of bacterial sample:
1. Flame the sample flask.
2. Insert the pipette to the bottom of the flask, and withdraw 1.0
ml of the sample.
3. Flame the first dilution tube.
4. Dispense the 1.0 ml of sample into the tube.
5. Draw the liquid up and down in the pipette several times to
rinse the pipette and to properly mix.
6. Re-flame and cap the tube.
7. Mix the tube thoroughly by holding the tube in one
hand and vigorously tapping the bottom with the other
hand.
8. Using the same procedure, aseptically withdraw 1.0 ml of
the sample from the first dilution tube and dispense into
the second dilution tube. Continue doing this from tube to
tube.
B- Plate out on agar surface:
1. Aseptically transfer 0.1ml from each of last 3 dilution tubes onto
the surface of the corresponding plates.
2. Note that since only 0.1 ml of the bacterial dilution is placed on
the plate, the bacterial dilution on the plate is 1/10 the dilution
of the tube from which it came.
3. Place the bent portion of the glass rod on the agar surface and
start to turn the plate at complete 360o.
4. Repeat for each of the 3 plates.
5. Incubate the 3 agar plates.
1- Using a Pipette to Remove
Bacteria from a Tube.
2- Using a Vortex Mixer to
Mix Bacteria Throughout
a Tube.
3- Using a Pipette to Transfer
Bacteria to an Agar Plate.
4- Using a Bent Glass Rod and a
Turntable to Spread a
Bacterial Sample.
C- Results after incubation (Counting):
1- Choose a plate that appears to have between 30
and 300 colonies.
 Sample 1/100,000 dilution plate (Figure a).
 Sample 1/1,000,000 dilution plate (Figure b).
 Sample 1/10,000,000 dilution plate (Figure c).
2- Count the exact number of colonies on that plate
using the colony counter .
3- Calculate the number of CFUs per ml of original
sample.
Results
(Figure a)
(Figure b)
(Figure c)
 For accurate determination of the total number of viable
cells:
 The total number of viable cells is usually reported as
Colony-Forming Units (CFUs) rather than cell numbers.
 A plate having 30-300 colonies is chosen because this range
is considered statistically significant.
 If there are less than 30 colonies on the plate: small errors in
dilution technique or the presence of a few contaminants will
have a drastic effect on the final count.
 Likewise, if there are more than 300 colonies on the plate:
there will be poor isolation and colonies will have grown
together.
•To determine the number of CFUs per milliliter (ml) of sample:
The number of CFUs per ml of sample =
The number of colonies (30-300 plate) X The dilution factor of the plate
counted
Advantage:
This method of enumeration is relatively easy to perform and is
much more sensitive than turbidimetric measurement.
Disadvantages:
•A major disadvantage, however, is the time necessary for
dilutions, platings and incubations, as well as the time needed
for media preparation.
•Only living cells develop colonies that are counted.
•Clumps or chains of cells develop into a single colony.
•Colonies develop only from those organisms for which the
cultural conditions are suitable for growth.
 A sample of E.coli diluted according to the above diagram. The
number of colonies that grew is indicated on the petri plates.
 How many CFUs are there per ml in the original sample?
II- Turbidity
 When you mix the bacteria growing in a liquid medium,
the culture appears turbid.
 This is because a bacterial culture acts as a colloidal
suspension that blocks and reflects light passing through
the culture.
 The instrument used to measure turbidity is
spectrophotometer.
 The ability of the culture to block the light can be expressed
as either percent of light transmitted through the tube or the
amount of light absorbed in the tube .
 The percent of light transmitted is inversely
proportional to the bacterial concentration (The
greater the percent transmittance, the lower the
number of bacteria).
 The absorbance (or optical density) is directly
proportional to the cell concentration (The greater
the absorbance, the greater the number of
bacteria.)
III- Direct Microscopic Count
 Petroff-Hausser counting chambers can be used as a direct method
to determine the number of bacterial cells in a culture or liquid
medium.
 It has squares 1/20 of a mm by 1/20 of a mm and is 1/50 of a mm
deep.
 The volume of one square therefore is 1/20,000 of a cubic mm or
1/20,000,000 of a cubic centimeter (cc).
 In this procedure, the number of cells in a given volume of culture
liquid is counted directly in 10-20 microscope fields.
 The average number of cells per field is calculated and the number of
bacterial cells ml-1 of original sample can then be computed.
 Procedure:
 Count the number of bacteria in five large
double-lined squares.
 Divide by five to get the average
number of bacteria per large square.
 This number is then multiplied by
20,000,000, since the square holds a
volume of 1/20,000,000 cc, to find the
total number of organisms per cc in
the original sample.
 If the bacteria are diluted (such as by
mixing with dye) before being placed in the
counting chamber, then this dilution must
also be considered in the final calculations.
Counting:
number of bacteria per cc =
the average number of bacteria per large double-lined
square
X
the dilution factor of the large square (20,000,000)
X
the dilution factor of any dilutions made prior to placing
the sample in the counting chamber
Advantage of direct counts:
 Is the speed at which results are obtained.
Disadvantage:
 Since it is often not possible to distinguish living from dead
cells, the direct microscopic count method is not very
useful for determining the number of viable cells in a
culture.