Transcript The Serial Dilution Method of Bacteria Enumeration

Introduction
Many studies require the quantitative determination of
bacterial populations. The two most widely used methods for
determining bacterial numbers are:
I. The standard plate count method.
II. Spectrophotometer (turbid metric) analysis.
The standard plate count method is an indirect measurement
of cell density ( live bacteria).
The spectrophotometer analysis is based on turbidity and
indirectly measures all bacteria (cell biomass), dead and alive.
The Plate Count (Viable Count)
The number of bacteria in a given sample is usually too great
to be counted directly.
However, if the sample is serially
diluted and then plated out on an
agar surface in such a manner that
single isolated bacteria form visible
isolated colonies, the number of
colonies can be used as a measure of
the number of viable (living) cells in
that known dilution.
Keep in mind that if the organism normally forms
multiple cell arrangements, such as chains, the
colony-forming unit may consist of a chain of
bacteria rather than a single bacterium.
In addition, some of the bacteria may be clumped together.
Therefore, when doing the plate count technique, we generally
say we are determining the number of Colony-Forming Units
(CFUs) in that known dilution.
By extrapolation, this number can in turn be used to calculate
the number of CFUs in the original sample.
bacterial counts by these methods are usually expressed as
colony forming units per milliliter (CFU/mL).
Normally, the bacterial sample is diluted by factors
of 10 and plated on agar.
After incubation, the number of colonies on a
dilution plate showing between 30 and 300 colonies
is determined.
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. (too few to count (TFTC).
 Likewise, if there are more than 300 colonies on the plate, there
will be poor isolation and colonies will have grown together. (too
numerous to count (TNTC).
Procedure
1) Using sterile technique, transfer 1 mL sample to the first
dilution blank. Mix the bottle by inverting it 20 times.
Label the bottle "10-1."
2) Using a fresh pipette, transfer 1 mL from the first
blank to the second blank. Mix as before. Label the
second bottle "10-2."
3) Using a fresh pipette, transfer 1 mL from the first blank to the
second blank. Mix as before. Label the second bottle "10-2“.
4) Using a fresh pipette, transfer 1 mL from the first blank to
the third blank. Mix as before. Label the second bottle "10-3“.
5) Using a fresh pipette, transfer 1 mL from the first blank to the
forth blank. Mix as before. Label the second bottle "10-4“.
6) Using a fresh pipette, transfer 1 mL from the first blank to the
second blank. Mix as before. Label the fifth bottle "10-5“.
7) Label the Petri dishes: 10-2, 10-3, 10-4, 10-5, and 10-6,
respectively.
8) transfer liquid from the dilution blanks to the Petri
dishes. Use a separate pipette for each blank, not
for each plate (i.e. if more than one plate uses
liquid from a single blank, a single pipette may be
used for that blank).
9) One at a time, add a tube of molten nutrient agar to each
Petri dish. After adding the agar, gently swirl the dishes in
pattern for 30 seconds to mix the bacteria with the agar.
10)After the agar has thoroughly solidified, incubate the plates
at 37°C for 24 to 48 hours.
11) Count the number of colonies on a plate that has between 30
and 200 colonies. Any plate which has more than 200
colonies is designated as "too numerous to count" (TNTC).
Plates with fewer than 30 colonies do not have enough
individuals to be statistically acceptable.
Colonies Forming Units {CFU}
Calculate the number of bacteria (CFU) per milliliter or gram
of sample by dividing the number of colonies by the dilution
factor multiplied by the amount of specimen added to agar
plate.
To compute the number of CFU/mL, use the formula:
 c = concentration, CFU/mL
 n = number of colonies
 d = dilution blank factor
 s = volume transferred to plate.
CFU Calculation Example
 You count 46 colonies on your plate
 You put 1 ml of bacterial culture into 99 ml of saline and plated
0.1 ml
 Dilution 1/100
CFU= 46
1/100 * 0.1
= 46 * 100 * 10 =46000 CFU/ml
END OF LECTURE