Quantitative Analysis of Microbial Populations through Standard

Viable Plate Count Methods

Damiles, Johmar, De Vera, Alyssa Gail, Elio, Charlene Lorraine, Fernandez, Gino
Miguel*
2BIOLOGY6, Department of Biology, College of Science, UST, Manila
*fginomiguel@yahoo.com
Abstract
Microorganisms are within nature, they occur and are studied within the living environment or inside a laboratory, they
are not isolated then studied as different and separate organisms, but as a whole, as populations within nature, either
in colonies or as dispersed units suspended in a medium or in a suspended animation. Pour plate and Spread Plate
method are the two most common methods used in the isolation of microorganisms from their natural habitat.
Additionally, these two processes give the most approximate or estimate number of organisms present in a
population of microorganisms.
Keywords: Spread Plate Method, Pour Plate Method, Isolation, Suspended in a Liquid Medium

Introduction
Microorganisms are within nature, they occur
and are studied within the living environment or
inside a laboratory, they are not isolated then
studied as different and separate organisms, but
as a whole, as populations within nature, either in
colonies or as dispersed units suspended in a
medium or in a suspended animation. Pour plate
and Spread Plate method are the two most
common methods used in the isolation of
microorganisms from their natural habitat.
Additionally, these two processes give the most
approximate or estimate number of organisms
present in a population of microorganisms. The
pour plate technique can be used to determine the
number of microbes/mL or microbes/gram in a
specimen. It has the advantage of not requiring
previously prepared plates, and is often used to
assay bacterial contamination of foodstuffs. The
principle steps are to 1) prepare/dilute the sample
2) place an aliquot of the diluted sample in an
empty sterile plate 3) pour in 15 mL of melted agar
which has been cooled to 45 o C, swirl to mix well
4) let cool undisturbed to solidify on a flat table top
5) invert and incubate to develop colonies. Each
colony represents a "colony forming unit" (CFU).
For optimum accuracy of a count, the

preferred range for total CFU/plate is between 30

to 300 colonies/plate. One disadvantage of pour
plates is that embedded colonies will be much
smaller than those which happen to be on the
surface, and must be carefully scored so that
none are overlooked. Also, obligate aerobes may
grow poorly if deeply embedded in the agar. On
the other hand, spread plate method can
determine the number of bacteria in a solution that
can be readily quantified. In this technique, the
sample is appropriately diluted and a small aliquot
transferred to an agar plate. The bacteria are then
distributed evenly over the surface by a special
streaking technique. After colonies are grown,
they are counted and the number of bacteria in
the original sample calculated. The end point of
our analysis is the number of colony forming units
per mL (CFU/mL) since we are counting the
number of colonies rather than the actual number
of bacteria. We are assuming that the each viable
bacteria in the suspension will form an individual
colony, which is a valid assumption if we do all the
techniques properly. CFU/mL is actually a more
useful determination than counting all the bacteria
under a microscope because in many bacterial
populations some significant number will be dead
cells and thus of no interest.

Methodology

Pour Plate Technique

The researchers prepared 3 test tubes of
diluent containing 9.0 ml each of 1.0% peptone
broth or 0.85% saline (NaCl) solution. Label each
tube as your groups assigned set of numbers and
their respective coefficients. For the researchers,
the sample was labeled as 10-2, 10-3, and 10-4.
Using a rubber bulb aspirator, the researchers
aseptically pipet 1.0 ml from a 24 hour old culture
broth into the first dilution blank. The contents of
the first dilution bank were then mixed by rotating
the tube between the palms of the researchers
hands. The used pipettes were then discarded or
dipped in a beaker of 0.5% bleach disinfectant. A
second 1.0-ml serological pipette was used to
transfer 1.0 ml from the first dilution bank to the
second one. The contents were mixed once
again. The same pipette was used to transfer the
contents of the first dilution bank to two plates
marked as the same with the first dilution bank.
The pipette was then discarded in the disinfectant.
A third serological pipette was then used to
transfer the contents of the second dilution bank
to the third. The same serological was used to
deposit the contents of the second dilution bank to
two plates labeled the same as the second
dilution bank. Discard the pipette in the
disinfectant. 15-20 ml of molten, sterile PCA was
poured aseptically into each of the plates. The
plates were then gently swirled by touching the
plate with the researchers pointing and middle
finger and moving it back and forth, right-left
direction to mix. The agar was allowed to harden.
The plates were then wrapped in newspaper and
enclosed in plastic bags for incubation for 24
hours at room temperature. After incubation, the
colonies in the plate were then counted with 30300 colonies using a permanent marker to prevent
counting of a colony twice. The average number
of colonies was taken. The average number of
colony forming units per ml was taken.
Spread Plate Technique
A 150 ml PCA sample was prepared
according to instructing and was sterilized via the
autoclave machine. The sterilized sample was
then taken out of the autoclave machine and was
allowed to cool for a bit. It was then poured
individually into 6 sterile petri dishes and was
allowed to solidify. These PCA plates were labeled
accordingly. A 10 fold serial dilution of the 24 hour
broth culture was then prepared likewise with the
pour plate method. Once again, the researchers

had 3 test tubes containing 9.0 ml of sterile 0.85%

saline. A 1.0 ml of the 24 hour broth culture was
aseptically withdrawn and introduced to the first
test tube that was labeled accordingly to the
assigned dilutions to each of the researchers. The
content was then thoroughly mixed. A sterile 1.0
ml serological pipette was used to pipette out 1.0
ml from the first test tube and then introduced to
the second test tube labeled accordingly to
assigned dilution to the respective researchers.
The same serological pipette was then used to
introduce 0.1 ml from the first test tube to two of
the PCA plates that was previously prepared and
labeled accordingly. The 0.1 ml was dropped in
the center of the PCA plates. Unlike with the pour
plate method, only 0.1 ml was introduced to the
plates. The used serological pipette was then
disinfected in a beaker with 0.5% bleach or
disinfectant. Another serological pipette was
obtained and 1.0 ml was withdrawn from the
second test tube and was added to the third test
tube labeled accordingly. The content of the third
test tube was then mixed. The same serological
pipette was used to withdraw 0.1 ml from the
second test tube and introduced to two PCA
plates. Then, another 0.1 ml serological pipette
was obtained, this was used to pipette out 0.1 ml
from the third test tube and was then introduced to
the last two petri dishes. An L-shaped glass rod
was dipped in 95% ethanol in a beaker and
withdrawn. The L-shaped rod was then flamed
over. The flame was left to burn and disappear. A
PCA plate was then obtained and the L-shaped
rod was used to spread the previously dropped
sample all over the PCA plate. This process was
repeated for all PCA dishes. The plates were then
wrapped in a newspaper, and enclosed in plastic
bags. These were then incubated for 24 hours.
The CFU/ml formula was then computed, similar
with the pour plate method, but the volume plated
that is substituted in the formula should be 0.1 ml.

Results and Discussion

Pour Plate Technique
The inverted plate cultures were taken out after
incubation for at least 24 hours. The plates
exhibited proper distinction prior to the dilution of
the sample. Colonies no matter how small has to
be counted as they are considered to form larger
and bigger colonies as it ages. The least diluted
plate 10-6 had the most number of colonies
formed, to the point that one plate exceeded more

than 300 colonies and was assessed with too

many to count, even the dilution of 10 -6 had one
trial assessed with too many to count. The most
diluted samples on the other hand, had a number
that would be proportional to its dilution. Due to
the agar being deposited after the sample has
been placed in the plate, instead of colonies
having to settle only on the surface of the agar,
there were some which settled in the middle of the
agar medium, thus, making it more difficult to
count the colonies unlike the spread plate
technique which had its colonies settle only on the
surface of the medium. This technique may also
have killed or stunted the growth of obligate
aerobic microorganisms because of their
settlement in the middle of the medium. The three
plates with reasonable colony count was
averaged by mathematical calculations and
resulted to a concentration of 4.071x10 8 cells per
mL and was a reasonable for sewage water
sample.

organisms.

Spread Plate Technique

The inverted plate cultures were taken out after
incubation for at least 24 hours. The plates
exhibited proper distinction prior to the dilution of
the sample. Colonies no matter how small has to
be counted as they are considered to form larger
and bigger colonies as it ages. The least diluted
plate, 10-2 had the most number of colonies.
Because, spread plate method was used, the
colonies were almost evenly spread throughout
the plate and making it easier to count. The
colonies in this method were settled only on top of
the solidified agar. The three plates with their
respective colony counts was averaged and
resulted to a concentration of 5.72x10 -2 cells per
ml and this was the result for the researchers
group for pond water.

Conclusion
From the experiment, the researchers have
inferred that the best way of quantitative analysis
in microbial populations would be the spread plate
technique. Spread plate technique is a better way
of determining the amount of colonies in the
sample because it does not damage the colonies
in any way, and the colonies would be almost
evenly distributed through the plate thus making it
easier to count. Pour plate could kill some of the
microorganisms because the researchers would
have to pour molten agar into their diluted sample,
and the heat might have killed off some of the

References

[1] http://www2.hendrix.edu/biology/CellWeb/Techniques/microspread.htm
[2] http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Plate.html
[3] Laboratory Manual in General Microbiology. (2014). Manila: University of Santo Tomas.