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Quantitative Analysis 1

Quantitative Analysis
of Microbial Populations through
Standard Viable Plate Count Methods
Dulguime, Pol Vincent
Dumatol, Jose Primo
Echon, Angelique Mae
Garcia, Patricia
University of Santo Tomas

Quantitative Analysis

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of Microbial Populations through
Standard Viable Plate Count Methods
Today to assure consumer's safety, FDA required food manufacturers to monitor the
number and type of bacteria in their products. By doing so, the number of "harmless" bacteria
have greatly reduced. Monitoring is also done on the production of dairy products, beer and
wine, and most importantly on water ways to inspect the growth rate of bacteria.
Apart from the chemicals present in our water ways which may be toxic like Mercury
that forms no known functions in living organisms (Beldowska & Falkowska, 2015), microbial
growth is also apparent in our water system. Microbes are present in the water we drink and most
especially in non-potable water like rain water, gray water and reclaimed or recycled water. In
spite of the widespread practice of disinfection techniques, biofilm formation still occurs because
of the continuous flow of the aqueous medium which acts as a nutrient feeding solution to
microorganisms (Friedman, Harif, Herzberg & Mamane, 2015). Despite current prevention and
treatment methods, human populations are still impacted by many water pathogens, so providing
pathogen-free drinking water represents a key priority of the European Drinking Water Directive
(Kerambrun, 2015). Now it was concluded that safe drinking water for all is one of the major
challenges of the 21st century, now must be the right time that microbial control in drinking
water should be the norm everywhere (Cabral, 2010). Methods used to quantify microbial water
quality vary but the most commonly employed approach uses nutrient rich media to culture fecal
indicator organisms (Oliver,2015). Aside from monitoring drinking water for microbial
contamination, farmers are required under the proposed Product Safety Rule of the FSMA to
monitor and assess the microbial quality of agricultural water such as irrigation water and take

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corrective actions, if needed, to ensure the safety of fresh produce (Li, 2015). Though some
microbes are harmful there are also beneficial microbes that promote growth of agricultural and
horticultural crops as they act as bio fertilizers, bio stimulants of plant growth or as bio control
agents for plant pathogens or parasites (Swaminathan, 2015). Conducting a colony count of the
microbes present in marine or fresh water also provides a way to measure the quality of aquatic
life (Harry, Ameh, Coulon & Nocker, 2015).
Truly, aside from knowing what kind of microorganisms there are in a sample, their
quantities also matter and quantitative analysis of microbial growth are done in a variety of ways.
Total population count by direct and indirect method, viable count, turbidity measurement, and
plate count procedure are used but for this activity, the researchers focused on "Plate Count
Procedure". One method in plate count would be "Pour Plate Method" wherein the sample is
suspended in molten agar that is just barely warm enough to keep the agar form setting up, this
technique make the colonies stay small and compact, however students encounter difficulty in
keeping the agar hot enough to prevent it from setting up and at the same time cool enough to not
kill the microorganisms present in the sample. Another plate count method called "Spread Plate
Method" is also widely used. Here a student pipettes a small volume (around 0.1 mL in the
activity) of the sample on to the plates and then evenly spreads it out around the surface of the
plates. This is expected to give reliable and consistent results. According to the comparison done
by Hoben and Somasegaran (1982), there are three methods used in plate count namely Pour,
spread and drop plate method which are interchangeable, however the drop plate method was
preferred because of its economy in materials and labor. In this study, only the Pour plate method
and spread plate method are discussed.

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Before doing these plate count procedures, serial dilution is the first step to be done as
contaminated samples might contain billions of microbial cells and serial dilution reduces a
dense culture of cell to a more usable concentration. In the end, we can determine how many
colonies of bacteria there are in the original sample.
In the case of the researchers, they were assigned to obtain a sample of the UST Field
recycled water and perform serial dilution using the spread plate method. The spread plate
method is reliable and tested as evidenced by another study conducted by Gibbs and Hayes
(2008) which also made use of the spread plate method to enumerate the heterotrophic bacteria
in water, but in the potable kind. After incubating the plates, the viable cell number of culture
was computed by manually counting the colonies and computing for colony forming units (CFU)
per mL if applicable.
Method
Materials
The materials used were a 200 mL erlenmeyer flask, 3 regular sized test tubes that were
labelled 10-1, 10-2 and 10-3, and 6 sterile petri dishes. Two of the petri dishes were labelled 10-2,
another two were labelled 10-3 and lastly, two were labelled 10-4. Four 1 mL sterile serological
pipettes, two 10 mL serological pipettes, two L-shaped rods, a test tube rack, two 100 mL
graduated cylinders, cotton, foil, plastic, newspapers, alcohol lamp and a rubber aspirator were
also used. Plate Count Agar, 0.85% saline solution, a beaker of 0.5% bleach disinfectant, a
beaker of 95% ethanol, distilled water and recycled water were prepared for the experiment. The
apparatus used include an autoclave, a vortex mixer and a triple beam balance.

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Procedure
Recycled water was first obtained from the faucet near the soccer field.
9 milliliters of saline solution were added to each of the three test tubes using a 10 mL
serological pipette. The test tubes were then placed aside on a test tube rack. The PCA solution
was prepared by dissolving 3.102 g of PCA powder, which was weighed using a triple beam
balance, into 132 mL of distilled water placed in a 200 mL erlenmeyer flask. The flask was
covered with cotton plug, foil and plastic and then placed on the autoclave at 121 C for 15-20
minutes. After autoclaving, the PCA solution was allowed to cool, after which 20 mL of the
solution was aseptically poured to each of the 6 sterile petri dishes with the use of a serological
pipette. The solution was then allowed to solidify.
A 10 fold serial dilution of the recycled water was prepared by introducing 1.0 mL of the
sample to the test tube labelled 10-1 using a 1.0 mL serological pipette. The content was mixed
using a vortex mixer and the serological pipette was placed on the beaker of disinfectant.
Another 1.0 mL serological pipette was used to transfer 1.0 mL of liquid from the first test tube
to the test tube labelled 10-2. The same pipette was used to transfer 0.1 mL from the test tube
labelled 10-1 to each of the two sterile plates labelled 10-2. The contents of the test tube labelled
10-2 were mixed using the vortex mixer and the second pipette was placed on the beaker of
disinfectant. A third 1.0 mL serological pipette was used to convey 1 mL of liquid from the
second test tube to the test tube labelled 10-3. The same pipette was used to convey 0.1 mL from
the test tube labelled 10-2 to each of the 10-3 sterile plates. The contents of the 10-3 test tube were
mixed using the vortex mixer and the third serological pipette was placed on the beaker of
disinfectant. Using a fourth 1.0 mL serological pipette, 0.1 mL of the solution from the test tube

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labelled 10-3 was dispensed to each of the petri dishes labelled 10-4. The fourth pipette was placed
on the beaker of disinfectant.
An L- shaped glass rod was dipped into the beaker of 95% ethanol and then withdrawn,
letting the rod touch the walls of the beaker to remove excess alcohol. The rod was flamed over
an alcohol lamp until the flame burnt and disappeared. Aseptically, the bent glass rod was
allowed to touch the agar on each of the PCA plates, moving back and forth across the agar. This
procedure was done three times, rotating the plates 30 degrees before each spreading.
The plates were wrapped with newspapers inverted and enclosed tightly in plastic bags.
They were incubated at 35C and then the colonies were counted after with the aid of a Colony
counter machine. The CFU/mL was obtained using the following formula:
CFU/mL = (Average number of colonies x Inverse of dilution level) / Volume of cell
suspension plated
Data from other groups were gathered for comparison.
Results
In computing for the number of colony-forming units (CFU) per milliliter, we multiply
the average number of colonies to the inverse of the dilution level where the colony is observed,
the result is divided to the volume of cell suspension plated. A limit of 30-300 colonies is
followed when computing for the CFU. An excess (termed as TNTC), or shortage in the number
of colonies relative to the limit would void the data for computation.
Following the results observed in our microbial culture using recycled water as our
source, the average number of colonies formed in the plates were invalid, thus computing for the

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CFU is not allowed (See Table no 1. ). Among all the samples wherein a colony count was
performed by specific groups of the class, only the samples of S.marcescens,tap water, pond
water and barbecue sauce exhibited more than 30 colonies and hence only the CFU/mL of those
samples were computed. (Refer to Table no. 1).
Discussion
Before the growth of microorganisms was observed, our group hypothesized that the
plates with the highest dilution level of broth culture would display higher number of colonies
formed. The number of colonies formed goes down as the dilution level decreases, thus
displaying a direct relationship between the two variables. This idea was hypothesized due to the
reason that a broth culture, when diluted in a saline solution, would allow the colonies of
microorganisms to spread all over the solution. Extracting a certain amount (in this case, 1.0 mL
and 0.1 mL) from that solution would render a decreased amount of colonies, since not all would
be concentrated in a particular spot in the solution.
The results of our experiment matched with the hypothesis given prior the
experimentation. The plate with 10-2 dilution level has 76, up to a numerous (TNTC) number of
colonies; the one with 10-3 dilution level has 13, to 15 colonies; and the one with 10-4 dilution
level has no colonies present. There is a noticeable decrease of colonies as the dilution level goes
down. This pattern of colony growth should be observed in all n-fold serial dilutions, regardless
of the method used (spread plate, or pour plate) because, as stated beforehand, several microbial
colonies remain on the saline solution, and are not extracted. Deviations from the expected
pattern (e.g. higher number of colonies present on lower dilution level vs. higher) may be caused
by the following reasons: wrong/unequal amount of solution transferred on the succeeding test

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tube, wrong usage of serological pipettes (unaware when to discard it), error on the calculations
for the n-fold serial dilutions, improper incubation of plates, etc. These reasons were mentioned
as some groups displayed results that deviate from the expected pattern of microbial colony
growth.
Overall, this experiment proves that quantitative analysis of microbial colonies is possible
through the use of serial dilutions, accompanied by any of the plate methods. This is an effective
way of approximating the number of colonies present in a volume of a culture. Although
effective, it does not give the exact amount of colonies formed, nor the nature of the
microorganisms isolated in the culture medium. This method should be used when one needs to
quantify a target number of microbial colonies, and if such amount exists in the culture they are
working on.

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References
Beldowska, M., & Falkowska, L. (2015). Mercury in marine fish, mammals,
seabirds, and human hair in the coastal zone of the southern Baltic. Water,
Air, Soil Pollution, 227, 52. doi: 10.1007/211270-015-2735-5
Cabral, J. P. (2010). Water microbiology. bacterial Pathogens andwater.
International Journal of Environmental Research and Public Heath, 7, 36573703. doi:10.3390/ijerph7103657
Cleary, D. F., De Voogd, N. J., Polnia, A. R., Freitas, R., & Gomes. N. C.
(2015). Composition and predictive functional analysis of bacterial
communities in seawater, sediment and sponges in the spermonde
archipelago, Indonesia. Microbial Ecology, 70, 889-903. DOI
10.1007/s00248-015-0632-5
Friedman L., Harif, T., Herzberg, M., & Mamane, H. (2015). Mitigation of
biofilm colonization on various surfaces in a model water flow system by
use of UV treatment. Water, Air, Soil Pollution, 227, 43.doi: 10.1007/s11270015-2732-8
Gibbs, R.A., & Hayes, C.R. (2008). The use of R2A medium and the spread
plate method for the enumeration of heterotrophic bacteria in drinking
water. Letters in Applied Microbiology,6,19-21.
Harry, I., Ameh, E., Coulon, F., & Nocker, A. (2015). Impact of treated sewage
effluent on the microbiology of a small brook using flow cytometry as a
diagnostic tool. Water, Air, Soil Pollution, 227, 57.doi: 10.1007/s11270-0152723-9
Hoben, H.J., & Somasegaran, P. (1982). Comparison of the pour, spread, and
drop plate methods for enumeration of Rhizobium spp. in inoculants made
from presterilized peat. Apllied and Environmental Microbiology, 44,12461247.
Kerambrun, E., Ladeiro, M. P., Clivot, A., Geffard, O., Dupuis, E., Villena,
I., . . . Geffard, A. (2015). Zebra mussel as a new tool to show evidence of
freshwater contamination by waterborne Toxoplasma gondii. Journal of
Applied Microbiology,120, 498-508. doi:10.1111/jam.12999
Kimmitt, P. T., & Redway, K. F. (2015). Evaluation of the potential for virus
dispersal during hand drying: a comparison of three methods. Journal of
Applied Microbiology,120, 478-486. doi:10.1111/jam.13014
Li, B., Jackson, S. A., Gangiredla, J., Wang, W., Liu, H., Tall, B. D., . . . Elkins,

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C. A. (2015). Genomic evidence reveals numerous Salmonella enterica
serovar newport reintroduction events in Suwannee watershed irrigation
ponds. Applied and Environmental Microbiology, 81, 24.
doi:10.1128/AEM.02179-15
Oliver, D. M., Hanley, N. D., Van Niekerk, M., Kay, D., Heathwaite, A. L.,
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doi:10.1111/jam.13006

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Appendix
10-fold serial dilution of Recycled water

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Table 1
Colony Count and CFU/mL of the Different Samples Used by 2BIO4
Sample
Taho
Fishball sauce
Dirty ice cream
S. Marcescens
Recycled water
(Field)
Pond water
Tap water (Biology

Group
1
2
3
4
5

Colony Count
19/8 0/0
0/0
1/0
25/0 1/1
2/0
0
0
TNTC/TNTC 72/43 21/4
TNTC/76
13/15 0

Dilution Level
10-3, 10-4, 10-5
10-2, 10-3, 10-4
10-5,10-6, 10-7
10-6,10-7,10-8
10-2,10-3,10-4

CFU/mL
X
X
X
1.00 x107 CFU/mL
X

6
7

TNTC/TNTC 100/7 24/20


1/14 74/7 3/12

10-2,10-3,10-4
10-2,10-3,10-4

5.35 x104 CFU/mL


4.1 x105 CFU/mL

8
9
10

4/0
1/1
0
TNTC/TNTC 36/42 89/42
0
0
0

10-2, 10-3, 10-4


10-3, 10-4, 10-5
10-6,10-7,10-8

X
3.9 x10 CFU/mL
X

department Faculty
room)
Drinking water
Barbecue sauce
Soil

Figure Caption
Figure 1. Colony count of Recycled water at 10-2 dilution level.

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Figure 2. Colony count of Recycled water at 10-3 dilution level.
Figure 3. Colony count of Recycle water at 10-4 dilution level.