Quantitative Analysis of Select Microbial Populations through Standard Pour Plate

Method and Spread Plate Method

Dones, Maria Ronalee, Enriquez, Camille, Enriquez, Vergel Jigs B. and Figueroa, Annielle
Cyreen D.
Department of Biological Sciences, University of Santo Tomas, Manila, Philippines

Abstract

The experiment aims to isolate and determine the viable cell number of select microbial
populations through quantitative analysis namely pour plate method and spread plate method.
Select microbial populations were obtained from samples namely soil, rice milk, pork tocino and
fishball sauce as well as grown cultures of Micrococcus luteus and Serratia marcescens.
Results were computed to acquire the number of colony-forming units of each sample for
spread plate technique showed: 9.10 x , CFU/mL for Fish ball sauce sample, 1.97 x
-4
CFU/mL for Rice milk, 4.9 x 10 CFU/mL for Soy Milk (Taho), 6.08 x CFU/mL for Serratia
marcescens, and 3.16 x CFU/mL for Micrococcus luteus. Remaining samples for spread
plate technique were not viable for CFU/mL computation. Pour plate technique resulted in 1.35
x for Group fish ball sauce, 4.45 x for rice milk and 2.41 x for Micrococcus
luteus. Likewise, remaining samples for pour plate technique were not viable for CFU/mL
computation.

Introduction

In numerous microbiological research studies, whether it be the inspection or

examination of water, food, milk or even urine and other human waste materials, it is essential
to gain information about the bacterial count present in the sample. Through this,
microbiologists are able to analyze in-depth microbial traits ranging from their capacity to form
colonies to their growth in feasible media. The number of cells in a specific volume of material
capable of dividing on or in solid agar medium is referred to as the viable number. However, the
viable number is more commonly expressed as colony-forming units (CFUs). With regards to
this, the CFUs of bacterial suspension can be determined by the use of the spread plate
technique or the pour plate technique (Wistreich, 2003).

As stated previously, the spread plate and pour plate technique are quantitative methods
to determine the number of bacteria in a sample. However, due to the fact that samples contain
several hundreds or even thousands of cells, serial dilutions must be performed before the
plating technique. A serial dilution procedure is a method of sequentially diluting a culture
sample through a series of sterile dilution blanks (Johnson & Case, 2010). In the spread plate
technique, a small volume of dilute bacterial mixture containing 100 to 300 cells or less is
transferred to the center of the agar plate which is spread evenly over the surface using a
sterile, L- shaped glass rod. The glass rod is sterilized by means of dipping it in alcohol and then
flaming it off to burn the excess. After incubation, isolated colonies are to be seen. A colony is
an aggregation of bacterial cells seen as discrete entities by the naked eye (Harley, 2011). On
the other hand, the pour plate technique also involves serial dilutions yet within tubes of liquid
nutrient agar. After dilution, the medium is then poured into petri dishes for incubation. It is
within the medium as well on its surface are the appearance of the bacterial colonies. It is a
precaution that once the technique has started, it must be performed without delay because of
the quick solidification of the agar medium (Pommerville, 2007).
 With regards to the samples, rice milk is a starch suspension acquired through either
draining boiled rice or boiling water until completely dissolved. Secondly, fishball sauce is a type
of dipping sauce, liquid or sometimes semi-solid in consistency used to add flavor and/or texture
to certain foods. Third, pork tocino or simply tocino is a cured meat product native to the
Philippines. It is made from pork and the preparation is quite similar to ham and bacon although
beef and chicken can sometimes be used as alternatives. Fourth, Taho or soy milk is a Filipino
snack food high in protein and low in fats basically made of fresh tofu, brown sugar or vanilla
syrup and pearl sago. Lastly, soil is primarily attributed as the top layer of the earths crust. It is
formed by mineral particles, organic matter, water, air and living organisms thus making it an
extremely complex, variable and living medium. For the given bacterial cultures, Micrococcus
luteus is a catalase gram positive bacteria included in the microflora of the skin occurring in
pairs, tetrads and irregular clusters. On the other hand, Serratia marcescens is a gram negative
rod shaped bacteria often associated with various hospital-acquired infections (HAIs).

Taking this to account, the experiment aims to isolate and determine the viable cell
number of select microbial populations from samples namely soil, rice milk, pork tocino, soy
milk, and fishball sauce as well as grown cultures of Micrococcus luteus and Serratia
marcescens through quantitative analysis specifically pour plate method and spread plate
method.

Methodology

For the pour plate method, eight test tubes of diluent containing 0.85% saline (NaCl)
solution were prepared and labeled with 10-1,10-2,10-3,10-4,10-5,10-6 and 10-7, respectively.
Likewise, six sterile plates were labeled but only with 10-5, 10-6 and 10-7 respectively. Next, 1.0
ml from the provided 24 hour old broth culture containing Micrococcus luteus was aseptically
pipetted using an aspirator into the dilution blank marked 10-1. Then contents were then mixed
using a vortex mixer. The used pipettes were discarded in a beaker of 0.5% bleach disinfectant.
Afterwards, a second serological pipette was used to transfer 1.0 ml from the 10-1 to the 10-2
tube. The contents were mixed again via vortex mixer. The same pipette was then used to
deposit 1.0 ml from the 10-1 to each of the two plates marked 10-1. The said pipette was then
discarded in the disinfectant. Then, a third pipette was used to transfer 1.0 ml from the 10 -2 to
the 10-3 tube. The contents were mixed again via vortex mixer. The same pipette was then used
to deposit 1.0 ml from the 10-2 to each of the two plates marked 10-2. The said pipette was then
discarded in the disinfectant. This particular process was repeated until the 10-7 tube and the
two 10-7 plates. Soon after, 15ml of molten Potato Dextrose Agar (PDA) was aseptically poured
into each plate. The plates were then swirled in a gentle motion so as to evenly distribute the
PDA. The PDA plates were then allowed to harden and then wrapped with newspapers in an
inverted manner to prevent fruit fly infestation. Afterwards, they were stored and left for
incubation at 37oC for 24 hours. After incubation, the colonies of plates that ranged from 30 to
300 were counted. Moreover, the average number of colonies in duplicate plates was obtained.
After acquiring said data, the colony-forming units per ml of 24 hour old broth culture were
computed. Overall process was simultaneously done with other samples namely soil, rice milk,
pork tocino, soy milk (taho) and fishball sauce as well as grown cultures of Serratia marcescens

On the other hand, the spread plate technique utilized the same eight test tubes used in
the pour plate technique. Likewise, six agar plates were also prepared and labelled with 10-5,10-
6
and 10-,7 respectively but contained Tryptic Soy Agar (TSA). Next, from the 10-4 test tube, 0.1
ml was aseptically pipetted and deposited on the center of the 10-5 TSA plate and the process
was repeated for the duplicate TSA plate. Following this, an L- shaped glass rod was utilized to
evenly spread the inoculum on the surface of the said TSA plate. The L-shaped glass rod was
then disinfected by dipping it in 95% ethanol and then passing it through a flame to burn off the
excess ethanol. Then, from the 10-5 test tube, 0.1 ml was aseptically pipetted and deposited on
the center of the 10-6 TSA plate and the process was repeated for the duplicate TSA plate. The
L-shaped glass rod was then used again for even distribution in the TSA plates which was
sterilized in the manner stated before. This was process was repeated for the 10-6 test tube and
10-7 TSA plates. Similarly, the plates were then also stored and left for incubation at 37 oC for 24
hours. After incubation, the colonies of plates that ranged from 30 to 300 were counted.
Moreover, the average numbers of colonies in duplicate plates were obtained. After acquiring
said data, the colony-forming units per ml of 24 hour old broth culture were computed. Overall
process was simultaneously done with other samples namely soil, rice milk, pork tocino, soy
milk (taho) and fishball sauce as well as grown cultures of Serratia marcescens

Results

PLATE 1 PLATE 2

Figure 1. Micrococcus luteus PDA plate - Pour Plate Technique

PLATE 1 PLATE 2

Figure 2. Micrococcus luteus PDA plate - Pour Plate Technique

 PLATE 1 PLATE 2

Figure 3. Micrococcus luteus PDA plate - Pour Plate Technique

PLATE 1 PLATE 2

Figure 4. Micrococcus luteus TSA plate - Spread Plate Technique

PLATE 1 PLATE 2

Figure 5. Micrococcus luteus TSA plate - Spread Plate Technique

 PLATE 1 PLATE 2

Figure 6. Micrococcus luteus TSA plate - Spread Plate Technique

Table 1. Data of Colony-Forming Units per milliliter

Spread Plate Technique

Group 1 Soil sample

Plate 1 0 0 0
Plate 2 1 0 0
CFU/ml N/A N/A N/A
Average CFU/ml N/A
Group 2 Serratia marcescens sample

Plate 1 TNTC TNTC 77

Plate 2 TNTC TNTC 76
CFU/ml N/A N/A 7.65 x
Average CFU/ml 7.65 x
Group 3 Fish ball sauce sample

Plate 1 218 132 72

Plate 2 178 112 54
CFU/ml 1.98 x 1.22 x 6.3 x
Average CFU/ml 9.10 x
Group 4 Micrococcus luteus sample

Plate 1 288 59 13
Plate 2 166 99 41
CFU/ml 2.27 x 7.9 x 4.1 x
Average CFU/ml 3.53 x
Group 5 Tocino sample

Plate 1 10 4 0
Plate 2 13 4 0
CFU/ml N/A N/A N/A
Average CFU/ml N/A
Group 6 Serratia marcescens sample

Plate 1 TNTC 270 80

Plate 2 TNTC 230 50
CFU/ml N/A 2.5 x 6.5 x
Average CFU/ml 4.5 x
Group 7 Rice milk

Plate 1 23 TNTC 1
Plate 2 39 7 37
CFU/ml 3.9 x N/A 3.7 x
Average CFU/ml 1.97 x
Group 8 Micrococcus luteus sample

Plate 1 120 48 11
Plate 2 TNTC 44 4
CFU/ml 1.2 x 4.6 x N/A
Average CFU/ml 2.9 x
Group 9 Soy Milk (Taho)

Plate 1 48 27 5
Plate 2 50 33 4
CFU/ml 4.9 N/A N/A
Average CFU/ml 4.9 x 10-4
Average CFU/mL of Bacterial Cultures from select groups
Serratia marcescens 6.08 x
Micrococcus luteus 3.16 x

Pour Plate Technique

Group 1 Soil sample

Plate 1 15 5 1
Plate 2 13 7 2
CFU/ml N/A N/A N/A
Average CFU/ml N/A
Group 2 Serratia marcescens sample

Plate 1 TNTC TNTC TNTC

Plate 2 TNTC TNTC TNTC
CFU/ml N/A N/A N/A
Average CFU/ml N/A
Group 3 Fish ball sauce sample

Plate 1 TNTC 267 162

Plate 2 TNTC 243 137
 CFU/ml N/A 2.55 x 1.495 x
Average CFU/ml 1.35 x
Group 4 Micrococcus luteus sample

Plate 1 TNTC 111 3

Plate 2 TNTC 131 17
CFU/ml N/A 1.21 x N/A
Average CFU/ml 1.21 x
Group 5 Tocino sample

Plate 1 5 0 0
Plate 2 0 0 0
CFU/ml N/A N/A N/A
Average CFU/ml N/A
Group 6 Serratia marcescens sample

Plate 1 TNTC TNTC TNTC

Plate 2 TNTC TNTC TNTC
CFU/ml N/A N/A N/A
Average CFU/ml N/A
Group 7 Rice milk

Plate 1 91 85 7
Plate 2 70 23 6
CFU/ml 8.05 x 8.5 x N/A
Average CFU/ml 4.45 x
Group 8 Micrococcus luteus sample

Plate 1 TNTC 6 0
Plate 2 TNTC 47 3
CFU/ml N/A 4.7 x N/A
Average CFU/ml 4.7 x
Group 9 Soy Milk (Taho)

Plate 1 TNTC 7 4
Plate 2 TNTC 9 TNTC
CFU/ml N/A N/A N/A
Average CFU/ml N/A
Average CFU/mL of Bacterial Cultures from select groups
Serratia N/A
marcescens
Micrococcus luteus 2.41 x

Discussion

As stated previously, serial dilutions were done prior to plating technique so as to

minimize the bacterial count thus gaining viable colony forming units. Moreover, different
serological pipettes were utilized for each dilution and transfer to prevent contamination.
Furthermore, two quantitative methods of microbial analysis was employed namely spread plate
technique and pour plate technique. Spread plate technique employed 0.1 ml of diluted samples
which were then aseptically poured on prepared Tryptic Soy agar plates and the evenly spread
L-shaped glass rods. On the other hand, pour plate technique utilized 1 ml of diluted samples
that were introduced to the plate prior to pouring the plate count agar thus being embedded in
the media. Furthermore, results were consolidated and computed for the colony-forming units
(CFU/mL). This was acquired using the formula:

Given this formula, only colonies that showed statistical feasibility specifically those that ranged
from 30-300 were taken into account. Data that went beyond given range was termed as Too
Numerous To Count (TNTC). Moreover, duplicate plates within given range were averaged so
as to be used in the formula. For the spread plate technique, only seven groups among nine
groups were able to compute for the CFU/mL. It is as follows: 7.65 x CFU/mL for Group
two, 9.10 x CFU/mL for Group three, 3.53 x CFU/mL for Group four, 4.5 x
CFU/mL for Group six, 1.97 x CFU/mL for Group seven, 2.9 x CFU/mL for Group
eight , 4.9 x 10-4 CFU/mL for Group nine. CFU/mL of bacterial cultures Micrococcus luteus and
Serratia marcescens were averaged that showed 3.16 x and 6.08 x , respectively.
Among these, group nine that sampled soy milk attained the highest CFU/mL. On the other
hand, for the pour plate technique, only four groups out of nine were able to compute for
CFU/mL. It is as follows: 1.35 x for Group three, 1.21 x for Group four, 4.45 x for
Group seven and 4.7 x Group eight. As with the previous method, CFU/mL of bacterial
cultures Micrococcus luteus and Serratia marcescens were averaged that showed 2.41 x
for the former and nonviable count for the latter. Among these, group seven that sampled rice
milk acquired the highest CFU/mL. Albeit these results, errors may have arisen due to a number
of factors. These factors may likely be improper aseptic transfer of culture, wrong dilution of
given cultures and improper execution of plating techniques. Moreiver, inferring from these
results, colony forming unit are more likely to arise in homogeneous samples such as those of
rice milk and soy milk. Futhermore, based on the acquired CFU/mL of each technique, spread
plate technique is likely more advantageous than pour plate technique given that it provided
more viable data for CFU/mL computation.

Conclusion

With the given results and discussion, select microbial populations from samples
particularly soil, rice milk, pork tocino, soy milk, and fishball sauce as well as grown cultures of
Micrococcus luteus and Serratia marcescens were isolated and underwent quantitative analysis
specifically pour plate method and spread plate method. Acquired data was computed to obtain
the colony-forming units (CFU/mL). Among given samples, soy milk sample for spread plate
technique produced highest CFU/mL whereas rice milk sample for pour plate technique
produced highest CFU/mL.
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