Practical 3: Determining Numbers of Microorganisms in a sample

Objectives
1.To learn how to determine the number of microorganisms in a sample, a process called
enumeration.
2.To utilize several methods for enumerating microorganisms.

Introduction
Finding the amount of microorganisms in a sample is often essential. A wide range of techniques
have been devised to count microorganisms. These techniques assess cell counts, mass, or
proportionately related cell components. These methodological strategies all have certain
advantages as well as disadvantages. This is important for a number of applications in research,
clinical diagnostics, food safety, and environmental monitoring.
The following four broad methods are commonly employed to estimate the magnitude of microbial
populations:

1. Direct counts of microbial biomass or cell populations; an electronic particle counter or a

microscope are used to count the cells.
2. Direct determination of microbial biomass: By weighing whole cells, cell mass is ascertained;
biomass and cell counts can be connected using a standard curve.
3. Indirect counts of cell numbers: After microorganisms in a sample are concentrated or diluted
and grown on a suitable medium, the number of microorganisms in the original sample is estimated
based on the growth of the growing microorganisms, such as colony formation on agar plates.

4. Indirect estimates of microbial biomass: turbidity can be used to estimate microbial biomass
indirectly. Indirect estimates of microbial biomass can be correlated with cell number using a
standard curve. Microbial biomass is estimated by measuring relatively constant biochemical
components of microbial cells, such as protein, adenosine triphosphate, lipopolysaccharide,
murein, and chlorophyll.

The viable plate count method is a highly dependable methodology for estimating microbial
population sizes. It calculates the quantity of living microorganisms that may form colonies under
specific conditions. Because of its precision and capacity to discern between living and dead cells,
this approach is preferred. This technique involves plating successive dilutions of a sample
containing live microorganisms onto an appropriate growth medium. The suspension is combined
with liquid agar and either distributed onto the surfaces of agar plates (spread plate method) or
poured onto Petri plates and let to harden (pour plate method). After that, the plates are incubated
in a setting that encourages microbial reproduction, allowing colonies to grow and be observed
without a microscope. Every bacterial colony is thought to have originated from a single cell that
underwent cell division. Therefore, the quantity of bacteria in the original sample can be
determined by counting the colonies and taking the original dilution factor into consideration.

Pour plate method and spread plate method are two main techniques that are part of the plate count
method. The viable population in a bacterial sample can be determined using the plate count
method. A small portion of the specimen is added to a sterile water dilution tube to achieve
dilutions. The dilution of 1:100 (10-2) after 1 mL of a sample is introduced to 99 mL of sterile
water. As seen in the above image, greater dilutions can be obtained by progressively diluting the
sample in series. Counting of the colonies occurs after incubation. The concentration of bacteria is
determined by counting the number of colonies on the plates, which range from 30 to 300. Too
many to count is indicated by the usual notation "TNTC" (more than 300 colonies). In this case,
the number of bacteria in the initial water sample would be determined using the plate containing
61 colonies. 6.1 x 105 (61 x 10,000) bacteria/mL were found in the original sample because 61
colonies formed on a plate that had 1 mL of a 1:10,000 dilution added to it.

In the pour plate method, melted agar is combined with a measured volume of a sample, then the
mixture is poured onto a petri dish and left to harden to facilitate microbial growth. The plates are
then incubated such that individual bacterial cells multiply to form colonies, and the number of
colonies that form is counted. It is nearly always required to set up a dilution series in order to
guarantee that you will acquire a dilution having a reasonable amount of bacteria to count, as most
of the time you have no idea how many germs are in a sample. By using this method, bacteria
imbedded in the agar can be counted, offering a three-dimensional growing habitat. Samples that
may be oxygen-sensitive or have a high concentration of microorganisms benefit greatly from it.

Dilutions in the range of 10-1 (1/10) to 10-8 (1/100,000,000) are typically used for the enumeration
of bacteria, however, the range of dilutions may be limited with specific types of samples. For
instance, the maximum dilution required for non-turbid water samples is approximately 10-6
(1/1,000,000) because we know that the water would be turbid if there were 107 or more bacteria
per milliliter. In the pour plate approach, liquefied agar is combined with microbial inoculums to
distribute the bacteria somewhat equally throughout the medium upon the agar's solidification.
The fact that some bacteria cannot withstand exposure to 45°C poses a constraint to this process.

The bacteria were isolated and counted using the pour plate technique. After the agar medium has
been liquefied and chilled to between 45° and 50°C, a sample of known dilution is combined with
it and put into a sterile Petri plate. Give the agar time to set. The number of colonies that grow
during incubation is counted, and the concentration of microorganisms in the initial suspension is
computed.

In contrast, the spread plate method uses a sterile spreader to equally distribute a determined
volume of the sample across the surface of an agar plate that has solidified. The spread plate
method is another option to carry out a standard plate count. Agar plates are covered with repeated
dilutions of a sample, as the name suggests. Using a sterilized glass rod, 0.1 to 1 mL is typically
spread over the surface of a solid nutritional growth media. After that, the plates are incubated,
and the number of colonies that grow is counted. This method makes it easier to observe and count
colonies by isolating and counting the microorganisms growing on the agar surface. The spread
plate approach has the advantages of being straightforward, simple to apply, and efficient at
recovering a wide range of microorganisms.
The spread plate method of isolating and counting microorganisms. An agar medium is pipetted
with a sample aseptically. An alcohol dip and flame sterilize a glass spreading rod.Next, the
suspension is dispersed across the agar's surface using the sterile rod.

Both approaches are chosen according to the particular needs of the experiment, the characteristics
of the sample, and the type of microorganisms under investigation. In microbiology, the viable
plate count is still a fundamental method that offers important information about the dynamics of
the microbial population, the degree of contamination, and the efficacy of antimicrobial treatments.
Materials
A.1 Pour Plate Method
Tap water or pond water sample
Nutrient agar pours (15 mL/tube)

Sterile dilution water blanks (99 mL)

Sterile Petri plates

Pipettes (1 mL, sterile)

Pipette bulb or mechanical pump

Marking pen
Water bath at 50°C

A.2 Spread Plate Method

Tap water or pond water sample

Nutrient agar plates

Dilution water blanks (9 mL)

Pipettes (1 mL, sterile)

Pipette bulb or mechanical pump

Glass spreaders
Alcohol (in a beaker)

Vortex mixer
Marking pen
Procedures
A.1 Pour Plate Method
1) Six nutrient agar pours were melted in a boiling water bath. After they liquefied, they were
placed in a 50°C water bath until ready for use.

2) Three of the 99-mL dilution blanks were labeled 10⁻², 10⁻⁴, and 10⁻⁶, respectively. The six Petri
plates were labeled 10⁻² through 10⁻⁷.

3) The unknown sample was shaken to ensure an even distribution of microorganisms (generally
shaking side to side 25 times). Aseptically, 1 mL of the sample was removed with a sterile pipette
and transferred to the 10⁻² dilution blank.

4) The dilution blank was shaken vigorously to distribute the bacteria evenly.

5) Using a new sterile pipette, 0.1 mL and 1.0 mL from the 10⁻² dilution were aseptically
transferred to the agar plates labeled 10⁻³ (0.1 mL) and 10⁻² (1 mL), respectively. With the same
pipette, an additional 1.0 mL was removed from the 10⁻² dilution blank and transferred to the 10⁻⁴
dilution blank. The original 1 mL of the sample had now been diluted 1 part in a total of 10,000
parts.

6) The shaking procedure was repeated for the 10⁻⁴ blank, and 0.1 mL and 1.0 mL portions from
this dilution bottle were transferred with a new pipette to the plates labeled 10⁻⁵ (0.1 mL) and 10⁻⁴
(1 mL).

7) This same procedure was repeated to form a 10⁻⁶ dilution blank from which 10⁻⁷ (0.1 mL) and
10⁻⁶ (1.0 mL) plates were established.

8) Aseptically, a tube of melted agar (50°C) was poured into each Petri plate to which a dilution
of the sample had already been added. The plate was swirled to mix the sample with the agar,
ensuring that the agar did not run over the edges of the plate. The lid was replaced, and the agar
was allowed to cool and solidify.

9) The inverted plates were incubated at 30°C.

10) After incubation, the colonies on each plate were counted. Both the colonies on the agar
surface and the colonies growing within the agar were counted. The colonies were counted by
marking their position on the back of the Petri plates with a marking pen to aid in keeping track of
those colonies previously counted and avoid recounts. Some counters were electronic and
automatically tallied the count each time a colony was touched with the probe. The counts were
recorded. If a plate had more than 300 colonies, it was recorded as TNTC (too numerous to count).

11) From the plate-count data, the concentration of bacteria in the original sample was calculated.
For statistical reasons, only plates with between 30 and 300 colonies were used in this calculation.
Each colony-forming unit (CFU) represented the progeny of a single cell. Therefore, the number
of bacterial cells in the original sample was determined by multiplying the number of colonies on
a dilution plate by the corresponding dilution factor. For example, if 200 colonies were counted
on the 10⁻⁴ plate, 200 x 10,000 = 2,000,000 colonies or 2 x 10⁶ viable cells/mL were present in the
original sample. Generally, replicates of each dilution were plated, and the mean count recorded.

A.2 Spread Plate Method

1) Three of the 9 mL dilution water blanks were labeled 10⁻¹, 10⁻², and 10⁻³, respectively. Four
nutrient agar plates were labeled 10⁻¹ through 10⁻⁴.

2) Aseptically, 1.0 mL of the water sample was pipetted into the 10⁻¹ dilution tube. The 10⁻¹
dilution tube was mixed thoroughly by vortexing or vigorous shaking. The concentration of
bacteria in this tube was 1/10 of the original sample.

3) Using a new pipette, 1 mL from the 10⁻¹ dilution tube was aseptically transferred to the 10⁻²
dilution tube. The sample was vortexed. The concentration of bacteria in this tube was 1/100 of
the original sample.

4) Using this same procedure, 1 mL from the 10⁻² dilution tube was transferred to the 10⁻³ dilution.
The sample was vortexed. The concentration of bacteria in this tube was 1/1000 of the original
sample.

5) Using a new sterile pipette, 0.1 mL from each of the dilution tubes and the original sample was
transferred to agar plates. Because only 0.1 mL was transferred, the sample was effectively diluted
by another factor of ten. Therefore, the greatest dilution plate was 10⁻⁴. Starting with the greatest
dilution, the tube was vortexed and 0.1 mL was pipetted onto its respective agar plate. This was
done for each tube and the original sample so that plates ranging from 1/10 to 1/10,000 of the
original concentration were prepared. Because the procedure started with the greatest dilution, the
same pipette was used for each of the tubes.

6) The glass spreader was sterilized by dipping it in alcohol and flaming it in a Bunsen burner
flame. Caution was taken because alcohol fires can easily result.
7) After the glass rod cooled, the sample was spread over the surface of the plate by touching the
rod to the agar and rotating the plate.

8) This spreading procedure was repeated for each of the plates, ensuring to sterilize and cool the
glass rod before spreading each sample.

9) The inverted plates were incubated at 30°C for 24-48 hours.

10) After incubation, the colonies were counted. As in the pour plate method, plates with a range
of 30-300 colonies were counted. The counts were recorded.

11) The concentration of bacteria in the original sample was calculated.

Results
A.1 Pour Plate Method

10-1 10-2 10-3 10-4

PLATE COUNT:

Dilution Count 1 Count 2 Average

-1
10 12 8 10
-2
10 3 3 3
-3
10 4 4 4
-4
10 3 3 3
CONCENTRATION OF BACTERIA IN THE ORIGINAL SAMPLE:

10 × 10 = 100 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿

3 × 100 = 300 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
4 × 1000 = 4000 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
3 × 10000 = 30000 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿

100+300+ 4000+30000
Average concentration: = 8575 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
4
.
A.2 Spread Plate Method

10-1 10-2 10-3 10-4

PLATE COUNT:

Dilution Count 1 Count 2 Average

-1
10 106 104 105
-2
10 67 65 66
-3
10 93 91 92
-4
10 170 168 169
CONCENTRATION OF BACTERIA IN THE ORIGINAL SAMPLE:

105 × 10 = 1050 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿

66 × 100 = 6600 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
92 × 1000 =92000 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
169 × 10000 = 1690000 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿

1050+6600+ 92000+1690000
Average concentration: = 449912.5. 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
4
.
Question
A.1 Pour Plate Method

1. Why are counts above 300 and below 30 statistically unreliable?

Counts above 300 will make it hard to distinguish the colony and will be counted as TNTC. Count
below 30 is not reliable, it shows that there are very little colonies, meaning that there are error in
dilutions or contaminations presence,

2. Did you enumerate all the bacteria using this method?

No, not all bacteria were enumerated. Heat-sensitive bacteria will not grow using pour plate
method, some bacteria might be killed during the process and some might not be counted
accurately.

3. What factors make the pour plate method selective? How can you reduce the selectivity of
this procedure?
Factors such as the heat of molten agar may inhibit heat-sensitive organisms. Selectivity can be
minimized by allowing the agar to cool somewhat before pouring into the petri dish.

4. How can you achieve a 1/100 dilution other than by adding 1 mL of sample to a 99 mL
dilution blank?
1/100 dilution can be achieved by adding 0.1 mL of sample to 9.9 mL of diluent or by performing
two consecutive 1/10 dilutions.

A.2 Spread Plate Method

1. What does the term colony forming unit mean and how does it differ from a colony?
A colony-forming unit predict the number of live microorganisms in a sample. A colony can
consist of a small number of not visible to naked eyes microorganisms, but a colony forming unit
(CFU) is a visible to naked eyes colony of bacteria that can be formed by many colonies.

2. What types of errors would be introduced by using a single pipette for all operations in
this procedure?
Systematic error will be introduced since it cause cross contamination.

3. What are the advantages and limitations of the spread plate method compared to other
enumeration procedures?
The advantages is this procedure is suitable for the heat-sensitive microorganisms. The limitation
of this procedure is over-crowding might happen thus the enumeration of the microorganisms
might not precise.
4. How could you modify the spread plate procedure to enumerate obligate anaerobes?
Use anaerobic jars to provide an oxygen-free atmosphere after inoculation.

Discussion
The aim of the experiment was to use the Pour Plate Method and the Spread Plate Method to count
the number of microorganisms in a sample. The outcomes gave important information about the
sample's microbial load and how well each method quantified live bacteria.

In the first part of the experiment, the Pour Plate Method was used, the material is combined with
melted agar and then poured into Petri dishes. The average colonies for each dilution is dilution
10⁻¹ 10 colonies, dilution 10⁻² 3 colonies, dilution 10⁻³ 4 colonies, dilution 10⁻⁴ 3 colonies. Since
it permits the growth of both aerobic and anaerobic species inside the medium, this approach may
be beneficial. Limitations in this procedure appear to be indicated by the reduced counts observed
across the dilutions, especially in the higher dilutions. Certain bacteria may become less viable
due to the heat from the agar or the process of embedding cells in it, which could have an impact
on colony growth. This is compatible with research by Peters (2020), who observed that the
recovery of sensitive bacteria can occasionally be adversely affected by the heat from molten agar.

Next, in the second part of the experiment the Spread Plate Method was used, a small volume of
the sample is spread out over the surface of an agar plate. The average colony for each dilution is
dilution 10⁻¹ 105 colonies, dilution 10⁻² 66 colonies, dilution 10⁻³ 92 colonies, dilution 10⁻⁴ 169
colonies. Given that it guarantees oxygen exposure, this method works better for counting aerobic
bacteria in general. This approach's noticeably higher counts compared to the pour plate method
especially at higher dilutions. This indicates a higher success rate in obtaining viable cells. The
higher counts at higher dilutions (10⁻⁴) in contrast to lower dilutions (10⁻³) are unusual, this could
indicate that there are errors in this experiment.

The disparity in colony counts between the two approaches demonstrates how different techniques
may yield different results when assessing live microorganisms. Higher counts were regularly
obtained using the Spread Plate Method, indicating that it might be more reliable for this sample.
This is consistent with findings by Yadav (2021), who discovered that because spread plating
provides greater aeration and less heat stress for microorganisms, it frequently recovers higher
viable counts than pour plating.

Comparing the two methods we could observe that the spread plate method yielded higher colonies
than the pour plate method. This is because while the spread plate method does not harm heat-
sensitive bacteria, the pour plate method might harm the bacteria. Therefore, compared to the
spread plate method, the colonies in the pour plate method are lower.
Recent research supports the findings of this experiment. For instance, Peters (2020) pointed out
that, especially for aerobic bacteria, the Spread Plate Method frequently yields greater counts of
live microorganisms than the Pour Plate Method. Similar to this, a study by Yadav (2021)
underlined the significance of procedure selection based on the sample type and microorganisms
of interest and highlighted how methodological variations can greatly affect the results of
microbial counts.

However, based on the results observed, some error could have been caused by a few factors. First,
variations in the shaking or vortexing of the samples may have caused an uneven distribution of
microbes, which would have affected the accuracy of the dilution. This issue can be resolved by
employing a standard shaking or vortexing procedure for every sample to guarantee thorough and
consistent mixing. Furthermore, the heat from the molten agar may have damaged cells during the
Pour Plate Method, which would have decreased the viable count. By letting the melted agar cool
to an ideal temperature (around 45–50°C) before adding the sample, this problem can be
minimized, and the microorganisms will experience less heat stress. Finally, inconsistent spreading
methods and pipetting errors may have impacted the final colony counts. use calibrated pipettes
and make sure all operators utilize the same procedure to obtain more accuracy. Furthermore,
minimizing variances and enhancing accuracy can be achieved by using the correct pipetting and
spreading techniques.

Conclusion
In summary, this experiment illustrated the process of enumeration, the counting of
microorganisms in a sample. The count of bacterial colonies was done using the spread plate and
pour plate procedures. Microorganisms that are resistant to heat can be counted using the pour
plate method, whereas heat-sensitive microorganisms can be counted using the spread plate
method. All things considered, the experiment enhanced the ability to precisely count
microorganisms.
References

Kleinfeld, H. (January 23, 2024). What are colony-forming units? In Blog. Retrieved May 28,
2024, from https://omnibioticlife.com/blogs/blog/what-are-colony-forming-units

Peters, M. J., Mackay, A., & Smith, R. (2020). Comparison of pour plate and spread plate methods
for the enumeration of bacteria in food samples. Journal of Microbiological Methods.

Yadav, M., Shukla, P., & Sharma, V. (2021). Evaluation of microbiological methods for the
determination of viable counts in environmental samples. Applied Microbiology and
Biotechnology.