1

Spread Plate Technique

Conclusion

A measured quantity, that is 0.1 mL of microorganisms from an environmental

sample, was used to inoculate a Petri dish. A sterile L-spreader was used to distribute

the liquid across the surface of the media, which was incubated for 48 hours.

Subsequently, the diversity and number of microbial colonies visible on the plate were

counted. This number was used to compute the number of microorganisms that were

present in the original sample. Overall, this experiment has demonstrated that HPC,

which is based on discrete colony formations, can be used to isolate microorganisms

distributed over the surface of a plate of agar.

Discussion Questions

1. What does it mean if a sterile dilution water spread plate has growth on

it? Did any of your sterile dilution water plates show any growth?

It means that the sample tested contains isolated and / or visible a colonies of

microbial flora or bacteria. The final plate, which was inoculated with sterile dilution

water only, did not show any visible signs of growth as the microbial plate count was

zero (Table 1). In essence, it served as a control for the experiment.

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2. What are your estimated microbial concentration values in each of your

samples, and how do these values compare with typical values reported

in literature?

Table Coloni (CFU/

1: Bacterial es mL)
Plate Count
after 48 hours
of
incubationXS
ample
Soap 10 10000
Soap 11 11000
Dirty 10 10000
Hands
Dirty 9 9000
Hands
Wash 5 5000
ed
Wash 35 35000
ed
0
1/100 6 6000
1/100 0 0
1/1,00 0 0
0
1/1,00 0 0
0
1/10,0 0 0
00
1/10,0 0 0
00
Contr 0 0
ol

Consistent with theoretical predictions, the colonies were embedded on the agar

plate in all the samples (Miller p.3). The bacterial plate count increased when the

incubation period was sparingly extended beyond 48 hours (Table 2).

 3

Table 2: Microbial concentration of the samples after a 48-hour incubation period

SAMPLE COUNT COLOR DIAMETE (CFU/mL

R (cm) )
Soap 15 5 Yellow, 10 White 0.5 15,000
Soap 24 8 Yellow, others 0.1 - 0.3 24,000

White
Dirty 13 6 Yellow 0.05 - 0.2 13,000

Hands
Dirty 10 1 Yellow 0.05 - 0.2 10,000

Hands
Washed 16 4 Yellow, and others 0.1 - 1.3 16,000

white
Washed 56 13 Yellow and others white 0.1 - 0.5 56,000
5 cm of bacteria/ unknown

present
Control 22 1 Yellow 0.005 - 0.3 22,000
1/100 23 18 Yellow 0.001 - 0.3 23,000
1/100 80 24 Yellow 0.001 - 0.4 80,000
1/1,000 1 Yellow 0.001 1,000
1/1,000 3 White 0.1 3,000
1/10,00 0 0

0
1/10,00 2 White 0.1 2,000

0
Control 0 0
 4

Heterotopic Plate Count (HPC):

1 1 CFU
HPC=X ÷ × ( )
100 0.1 mL mL

Where CFU denotes the Colony Forming Units, and X is the Spread Plate Colony

Count.

3. Provide a table of all your spread plates from the swab tests, arranged

in order from the highest diversity to the lowest diversity. If a

monoculture is observed (only one type of colony), does it mean that no

other organisms are present? Why?

Evidently, the most diluted concentration (1/10,000) displayed the least microbial

diversity while the most concentrated solution (1/100) displayed the highest HPC as

shown in Table 1.

Table 3: Diversity of the microbial colonies in decreasing order

Swab Test Colony Color Diameter Diversity

(cm) (CFU/mL
)
1/100 80 24 Yellow 0.001 - 0.4 80,000
1/100 23 18 Yellow 0.001 - 0.3 23,000
Control 22 1 Yellow 0.005 - 0.3 22,000
1/1,000 3 White 0.1 3,000
1/1,000 1 Yellow 0.001 1,000
1/10,00 2 White 0.1 2,000
0
1/10,00 0 0
0
Control 0 0

The spread plate method is selective: only fast growing and motile bacteria can

be detected. Thus, the identification of a monoculture does not mean that no other
 5

organisms are present. Factors, such as growth inhibition (nutrient deficiency) and

substrate completion make it difficult to detect slow growing bacteria with this technique

(Miller p.2).

4. Bar graph illustrating hand-washing techniques with the highest HPC

The HPC increased in the following order: Dirty Hand (13,000), Soap (15,000

CFU/mL), and Washed samples (16, 00)0 CFU/mL) as shown in Exhibit 1.

HPC For Hand-Washing Techniques

18000
16000
16000 15000
14000 13000
12000
HPC (CFU/mL)

10000

8000

6000

4000

2000

0
1 2 3
Hand-washing Techniques

Exhibit 1: HPC for the samples

Upon rearranging the order of observations to correspond to the order of

swabbing (for the hand-washing experiment), the observable effects on the results were

significant. Still, the washed sample displayed the highest HPC (56,000 CFU/mL),

closely followed by the soap (24,000 CFU/mL), and dirty hand (10,000) CFU/mL in their

respective order as shown in Exhibit 2 below.

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SWABBED COLONIES COLOR DIAMETER (CFU/mL)

SAMPLE (cm)
Soap 24 8 Yellow, others 0.1 - 0.3 24,000
White
Dirty Hands 10 1 Yellow, 0.05 - 10,000
0.2
Washed 56 13 Yellow and 0.1 - 0.5 56,000
others white, cm

HPC For SWABBED Samples

60000 56000

50000

40000
HPC (CFU/mL)

30000 24000
20000
10000
10000

0
1 2 3
Hand-washing Techniques

Exhibit 2: HPC for the swabbed samples

There was no correlation between the initial and the swabbed spread plate

colony counts since HPC measurements at any given time are not representative of

specific heterotrophic bacteria. Rather, the tests are a measure of cultural organisms as

a fraction of the total bacteria present in a sample (Sanders p.3). The count is based on

bacterial formations under specific conditions of growth, such as pH, nutrient conditions,

and temperature (ranges from 20 – 40 0C).

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Works Cited

Miller, Morten. Plate Counts and Bactiquant. Technical paper 01-032016, N.p., 2016, p.

1 – 4.

Sanders R. Erin. “Aseptic Laboratory Techniques: Plating Methods.” Journal of

Visualized Experiments, 63, e3064 (2012): pp. 3 -6., doi:10.3791/3064