NOTE: Students are required to fill

in the followings clearly before

submission.

Lecturer’s Name:
Assoc. Prof. Dr. Chan Hor Kuan
Subject code & name:
MB110/BAB1044 Microbiology

FACULTY OF APPLIED SCIENCES

LABORATORY REPORT SUBMISSION

Experiment Title : Enumeration of Microorganisms

STUDENTS DECLARATION OF WORK

We declare that the work submitted is our own. We confirm that we have read and understood the
University regulations with regard to Plagiarism, Collusion and Cheating.

No. Student ID Group Members Signature

1 1002163489 Yeoh Ying En YingEn
2 1002163556 Chong Kai Xun KaiXun
3 1002162652 Ho Zhuo Xuan ZhuoXuan
1002162776 Clement Chia Qi Le Clement
4 1002268381 Abdulrahman Badreddin Shaibani Shaibani

ASSESSMENT CRITERIA
Criteria Wtg. Mark Awarded Percentage
Introduction and aims 5 Marks: Gained:
Methods 10
Results 20
Discussion 40 15%
100
Questions 10
Conclusions 10
References 5
(100)
TOTAL:

Remarks/Comments (if applicable):

1
Introduction
The enumeration technique is commonly used in bacteriological analysis to determine the concentration of a
specific bacteria present in the culture media (Gill, 2017). To put it in simpler words, enumeration is the
process of determining the number of a specific microbe, such as bacteria, in a sample. The enumeration of
microorganisms can be done using different methods and techniques, which are viable counts, total cell
count using Haemocytometer, as well as turbidimetry. Since most bacteria are too tiny to be visualized,
bacterial colonies instead of single bacterial cells are counted to reflect the viable count. In this experiment,
a series of cultures of different concentrations (10-1, 10-2, 10-3, 10-4, 10-5) would be prepared by using the
serial dilution technique prior to the enumeration of microorganisms.

Under the viable count technique, the spread plate method involves a transfer of a small amount (e.g. 0.1ml)
of a dilution onto the center of a NA plate. Secondly, the Most Probably Number (MPN technique) is often
used when the bacteria cannot be cultured using solid medium. The principle of this technique is to dilute a
sample until each aliquot is estimated to contain one viable cell. Besides the 3 tube method that is being used
in this experiment, the 5 tube method can also be carried out. If diluted sufficiently, at the end of the
incubation, some tubes would turn turbid, indicating that growth has occurred (Erkmen, 2022). The tubes
that show growth are considered “positive” while the others are “negative”. The most probable numbers of
bacterial samples are then obtained by referring to the MPN table [Table 3.1].

[Table 3.1 Table of Most Probable Number (MPN) for 3 Tubes Method]

P1 P2
0 0
0 0
0 1
1 0
1 0
1 1
1 1
1 2
2 0
2 0
2 1
2 1
2 2
2 2
3 0
3 0
3 0
3 1
3 1
3 1
3 2
3 2
2
P3 MPN
0 0.03
1 0.03
0 0.03
0 0.04
1 0.07
0 0.07
1 0.11
0 0.11
0 0.09
1 0.14
0 0.15
1 0.20
0 0.21
1 0.28
0 0.23
1 0.39
2 0.64
0 0.43
1 0.75
2 1.20
0 0.93
1 1.50
 3 2
3 3
3 3
3 3
3 3
2 2.10
0 2.40
1 4.60
2 1.100
3 24.00

Furthermore, the total cell count can be enumerated by using a haemocytometer, such as the Neubauer
counter which would be used in this experiment. A haemocytometer is a thick crystal slide, with its size
similar to a glass slide (30 x 70 mm and 4 mm thickness). The enumeration of cells is performed at the
central counting area (Absher et al., 1973). As shown in [Diagram 3.1], each counting area is ruled into 9
large squares, each with 1 mm2 of area. The squares at the four corners are further divided into 16 squares,
each occupying an area of 1/16 mm2. Whereas the central square millimeter is divided into 25 medium
squares, which are each further divided into 16 small squares (each 1/400 mm2 bounded by single lines).

[Diagram 3.1 The Haemocytometer]

Lastly, turbidimetry is an indirect method to enumerate microorganisms. It involves the measure of light
absorbed (loss of light intensity) by a suspension containing microbes to determine the number of cells in
the suspension. Light of a known wavelength passed through a cuvette containing the suspension. The more
the cells suspended in the sample, the more turbid the suspension is, and the greater the scattering effect. In
this experiment, the optical density (OD) of each dilution is measured using a spectrophotometer at 600 nm.
Prior to the measurement of the suspensions containing different culture concentrations, a blank (nutrient
broth without any microorganism) must first be introduced (Omar and Matjafri, 2009).

3
Aims /Objectives
i) To prepare a series of cultures of different concentrations using serial dilution technique.
ii) To investigate and perform the techniques used for enumeration of microorganisms, namely the spread
plate method, MPN method, the use of haemocytometer and turbidimetry.

Apparatus and Materials

A. Serial Dilution of a Culture

Flask containing 12 hr Escherichia coli, five test tubes containing 9.5 ml sterile water, and 11ml pipette
(sterile)

B. Viable Counts

(a) Spread Plate Method (Lawn Culture)

Dilution series prepared in A, sterile 0.1ml pipette, 6 nutrient agar (NA) plates, 1 bent glass spreader
(hockey stick), and 1 beaker of alcohol

b) Most Probable Number (MPN) Technique

Dilution series prepared in A, 1ml sterile pipette, and 12 tubes containing 9ml Nutrient Broth (NB)

C. Total cell count using Haemocytometer

Haemocytometer, microscope, and diluted culture.

D. Turbidimetry

Flask containing 12 hr E. coli culture, 4 test tubes, 1 10ml pipette, flask of Nutrient Broth (NB), and
spectrophotometer

Methods

A. Serial Dilution of a Culture

1. 5 test tubes are labelled 10-1, 10-2, 10-3 etc to 10-5.

2. The culture is rotated to mix, then 1ml of the culture is pipetted into the tube marked 10-1.

3. Ensuring that the content is well-mixed, 1ml of the contents of the tube marked 10-1 is pipetted into the
tube marked 10-2.

4
4. Step 3 is repeated by transferring 1ml of the contents of the tube marked 10-2 into the tube marked 10-3.

5. This procedure (similar to step 4) is repeated several times until a complete dilution series from 10-1 to

10-5 is obtained.

B. Viable Counts

(a) Spread Plate Method (Lawn Culture)

1. The culture labeled 10-5 is mixed gently. By using aseptic technique, 0.1ml of this dilution is transferred
onto the center of a NA plate.
2. The bend end of the glass spreader is dipped in alcohol, then sterilized by flaming and allowed to cool.
Using the glass spreader, the sample is spread evenly over the surface of the plate.
3. This procedure is repeated on a second NA plate. These two plates are labeled 10-5, so a duplicated plate
is prepared for counting.
4. Steps 1, 2 and 3 are repeated for dilutions 10-4 and 10-3 in the same order. At the end of this step, 6 plates
are prepared, with 2 at each of the dilutions 10-5, 10-4 and 10-3.
5. The plates are incubated at 32ºC for about 2 days. The number of colonies on plates is counted.
6. The number of viable cells per ml in the original culture is calculated for each dilution.

b) Most Probable Number (MPN) Technique

1. The diluted sample is gently mixed in the tube labeled 10-5. 1ml of this dilution is transferred aseptically
to one of the tubes of NB. 2 more tubes are repeated in the same way, all the tubes are labeled 10-5.
2. The procedure is repeated for dilutions 10-5, 10-4 and 10-3, making a total of 12 tubes. All these tubes are
labeled appropriately. The tubes are incubated at 32ºC for two days.
3. After incubation, the tubes with growth are checked and recorded for each dilution.
4. Using the table of Most Probable Numbers [Table 3.1], the viable number of E. coli per ml in the original
culture is calculated.

C. Total cell count using Haemocytometer

1. A drop of original culture is placed on the Haemocytometer.

2. A representative sample of cells in the center 25 squares (bounded by 3 lines) is counted. For cells on
lines, only the cells on the right and lower lines are counted. The 16 small squares (bounded by single lines)
are used as a counting aid only.
3. The cell number per ml of the original culture is calculated by using the formulas below:

i) Counting all 25 squares (bounded by 3 lines)

5
Concentration of cells/ml = n x 104

n = visual count

ii) Counting only some of the 25 squares (s)

Concentration of cells/ml = n/s x 25 x 104 x D

D = dilution factor

s = number of 25 squares counted

n = Total visual count

D. Turbidimetry
1. 4 test tubes are set up in the following way:

Test tube 1 2 3 4

NB (ml) 10 8 6 4

Culture (ml) 0 2 4 6

2. The absorbency of the contents of each of the tubes is measured using the spectrophotometer, the machine
is set to “zero” first using NB as the blank.
3. The results are recorded and converted into cell concentration.
4. A graph of Optical Density (OD) versus the numbers of bacteria is plotted.

6
Results
B. Viable Counts
(a) Spread plate method (Lawn Culture)

Figure 3.1 Spread plate method for 10-3 Figure 3.2 Duplicated spread plate method
for 10-3

Figure 3.3 Spread plate method for 10-4 Figure 3.4 Duplicated spread plate method
for 10-4

7
Figure 3.5 Spread plate method for 10-5 Figure 3.6 Duplicated spread plate method
for 10-5

Table 3.0 Spread plate method (The number of viable cells per ml)
Dilution Factor Number of viable cells Average number of Number of viable
1 2 viable cells cells per mL
1×10-3 >300 >300 Too numerous to TNTC
count (TNTC)
1×10-4 >300 >300 TNTC TNTC
1×10-5 >300 >300 TNTC TNTC

8
(b) Most Probable Number (MPN) Technique

Figure 4.1 MPN method for Figure 4.2 MPN method for Figure 4.3 MPN method for
culture 10-3 (P1) culture 10-4 (P2) culture 10-5 (P3)

Table 4.0 Most Probable Number (MPN) Technique

Sample
Presence of growth (Positive/Negative) Total

P1 Positive Positive Positive 3

P2 Positive Positive Positive 3
P3 Positive Positive Positive 3

Table 4.1 Table of Most Probable Number (MPN) for 3 tubes method
P1 P2
0 0
0 0
0 1
1 0
1 0
1 1
1 1
1 2
2 0
2 0
P3 MPN
0 0.03
1 0.03
0 0.03
0 0.04
1 0.07
0 0.07
1 0.11
0 0.11
0 0.09
1 0.14

9
2 1
2 1
2 2
2 2
3 0
3 0
3 0
3 1
3 1
3 1
3 2
3 2
3 2
3 3
3 3
3 3
3 3
P1, P2, and P3 represent sample concentrations of 10-3,10-4,10-5 of the original sample. Based on our results,
P1=3 positive tubes, P2= 3 positive tubes, and P3= 3 positive tubes. Thus, the MPN number is 24.00 as
indicated in table 4.1.
Center of dilution factor =10-4
Viable count
Viable count
cells/ml
0 0.15
1 0.20
0 0.21
1 0.28
0 0.23
1 0.39
2 0.64
0 0.43
1 0.75
2 1.20
0 0.93
1 1.50
2 2.10
0 2.40
1 4.60
2 1.100
3 24.00
10
C. Total cell count using Haemocytometer

Figure 5.0 Cell in haemocytometer (original E. coli)

∴ The number of bacteria observed is too numerous to count.

D. Turbidimetry
Test tube 1 2 3 4
Nutrient Broth (ml) 10 8 6 4
Culture (ml) 0 2 4 6
Cell concentration 0.00 4.00 8.00 12.00
Absorbency 0.057 0.066 0.252 0.327
(optical density)
Test tube 1:
Test tube 2:
Test tube 3:
Test tube 4:

Graph 6.0 Optical Density (OD) versus Number of Cells

11
Discussion

The experiment performed included several common enumeration techniques The enumeration
techniques were performed on culture prepared through serial dilution. Serial dilution allows us to estimate
concentrations of samples by counting the number of colonies formed, this value can then be backtracked to
measure the count of the initial unknown concentration (Ben-David and Davidson, 2014). In this experiment
the dilution was performed 5 times with a dilution factor of 10X, obtaining 5 separate cultures with different
concentrations, they were labeled 10-1, 10-2, 10-3, 10-4, 10-5, and were prepared by first pipetting 1mL of the
stock culture into the test tube labeled 10-1, which contained 9mL of sterile water, then the same procedure
was repeated to obtain the further dilutions.

The first enumeration technique performed was the spread plate technique which involves spreading
of the diluted culture on a nutrient agar plate. This involves pipetting 0.1mL of the culture onto a nutrient
agar (NA) and was spread evenly throughout the nutrient agar with a glass spreader. The NA was then
incubated for 2 days and the number of colonies formed is counted. Diez-Gonzalez, (Diez-Gonzalez, 2014)
has stated that these colonies would appear as white or cream-coloured ellipses on the surface of the NA or
are trapped inside the NA. This technique was only chosen to be performed with the cultures from 10-3, 10-4,
10-5 dilutions as any concentration higher would result in formation of colonies that are too numerous to
count. However, there was confluent growth of E.coli even when the 10-3, 10-4, 10-5 dilutions are used. The
confluent growth of the E.coli on the NA plates could be caused by a mistake during transferring the culture
broth onto the NA plate causing excess of the sample to be transferred and cultivated.

The next technique performed was the Most Probable Number (MPN) technique which is a statistical
method for the estimation of viable bacteria in a sample in an inoculating broth based on the principle of
extinction dilution (Karunasaga et al., 2018). This is performed by using a liquid media thus could be an
alternative when a solid medium could not be used. The technique involves incubating 3 tubes of the 10-3,
10-4, 10-5 dilutions and recording if the tubes exhibit positive growth of E.coli. The results are then estimated
with the aid of the table seen in Table 3.1. In the results from this experiment, all the incubated tubes
showed increased turbidity suggesting bacterial growth, the unexpected high amounts of growth could be
attributed to an error in the dilution process or the pipette used is not sterilized completely in between
transfers of the dilutions.

The Neubauer counter Haemocytometer was also used to estimate the concentration of bacteria in
this experiment. In this method, a drop of the culture was dropped on the culture chamber of the
Haemocytometer which has a calibrated grid built in and the number of cells per grid square is counted

12
under the microscope. It is suggested that to be statistically reliable, at least 20 grid squares should be
counted and averaged (Liu, 2017).

However, the results obtained from this method were too numerous to count as the sample was not
diluted enough, this could be attributed to the usage of the original culture as the sample observed. The
original culture was used in this case due to a lack of results seen in the attempts made with the other 10-1,
10-2, 10-3, 10-4, 10-5 dilutions. This may be caused by several factors which include mistakes when utilizing
the microscope causing insufficient lighting for the cells to be visualized or during the transferring of the
dilutions onto the Haemocytometer, the cells may be mistakenly eliminated due to inexperienced handling
of the apparatus. Another factor that may cause this discrepancy was that the dilutions have reduced the
bacterial density until no significant activity could be observed under the microscope.

The final enumeration technique performed in this experiment was turbidimetry. Turbidimetry is a
test of the dilution samples by measuring the turbidity of the samples which is done by quantifying the
degree of “attenuation” of a beam of light of known initial intensity by the sample. This could be done by
using a spectrophotometer (Lawler, 2015). As the concentration of samples increases, in this case the
concentration of E.coli, the turbidity of the sample increases as the beam of light is scattered more by the
larger concentration of organisms. This is supported by the results we have obtained with the samples with
larger volume of culture exhibiting a higher optical density (OD) when tested by the spectrophotometer.

Questions
1. Elaborate on the methods available for the enumeration of bacterial viable numbers compare and contrast
on the effectiveness and the shortcoming.

The spread plate method is a common technique used to enumerate viable bacterial numbers. An agar plate's
surface is covered with a known volume of a liquid sample containing microorganisms. To determine the
approximate number of live bacterial cells in the initial sample, count the colonies that have developed on
the agar's surface after incubation.

The spread plate method is efficient for various bacterial species and is quite easy and affordable. Since each
colony on the agar surface represents a single viable bacterial cell from the initial sample, it is also a reliable
way to count the number of viable bacteria. The composition of the agar medium can be modified to
stimulate and encourage the growth of some bacterial species while suppressing the growth of others,
making this technique extremely selective.

13
However, the spread plate method also has some significant drawbacks. The bacterial sample must be
dispersed equally across the agar surface to prevent clumping and colony overlap. Results may be correct if
the sample is distributed uniformly. Moreover, the spread plate approach takes time because the plates need
to be incubated properly, which can vary from a few hours to a few days, depending on the bacterial species
being examined. The spread plate approach is inappropriate for samples with low bacterial counts because
there can be too few colonies for a reliable count. Under certain circumstances, the most probable number
(MPN) approach may be more applicable.

In conclusion, the spread plate method is a popular and highly efficient way to count the number of live
bacteria. Its advantages include being straightforward, accurate, and selective for different bacterial species.
However, it also has several drawbacks, such as the requirement for expert execution, the length of the
process, and its inadequacy for samples with low bacterial counts.

The Most Probable Number (MPN) method is a statistical approach to estimating the number of viable
bacterial cells in a liquid sample. This method involves serial dilutions of the sample into tubes containing
nutritional broth and is frequently used for samples with low bacterial counts. The MPN is determined based
on the number of positive tubes at each dilution level after incubating and checking for bacterial growth.

Comparing the MPN method to the spread plate method, it is a quick and affordable way to count the
number of viable bacteria. Since the results can be reported as colony-forming units (CFUs) per milliliter of
a sample, it is also useful when determining the concentration of viable bacterial cells in a sample.
Furthermore, the MPN method is adaptable and may be used with various bacterial species by adjusting the
composition of the nutrient broth or incubation conditions.

The MPN method does, however, have significant drawbacks. It takes time since the tubes must be
incubated appropriately, which can vary from a few hours to many days, depending on the type of bacterial
species being examined. The MPN method relies on a subjective assessment of growth in each tube, which
makes it less accurate than other methods, like the spread plate approach. Moreover, it can also be subject to
error if the bacterial cells clump together, resulting in false negatives.

The MPN method still has room for development, as samples with high bacterial concentrations are
inappropriate because an accurate count of the positive tubes can become impossible. The spread plate or
pour plate method might be more suitable in these circumstances.

In conclusion, the MPN method provides a practical and affordable method for counting the number of
viable bacteria, especially for samples with low bacterial concentrations. Its advantages include adaptability,
14
simplicity, and the capacity to calculate the quantity of live bacterial cells. However, it has certain
drawbacks, such as the fact that it takes a long time, that its accuracy could be better than that of other
techniques, and that it is unsuitable for samples with high bacterial concentrations.

2. With the total cell count and the viable cell count, find out the % viable cells in the E. coli culture
provided. Comment on the result.

As a result of the sample not being sufficiently diluted and the use of the original culture as the sample, the
results from this technique were too numerous to enumerate. The original culture was used in this instance
because efforts to use the other dilutions of 10-1, 10-2, 10-3, 10-4, and 10-5 failed to produce the desired
results. The cells may not be able to be seen due to errors made when using the microscope, or during the
transfer of the dilutions onto the haemocytometer, the cells may be accidentally eliminated due to
inexperienced manipulation of the equipment. Another factor that may cause this discrepancy was that the
dilutions have reduced the bacterial density until no significant activity could be observed under the
microscope.

3. In what ways is the turbidity measurement of bacterial suspension useful in the enumeration exercise.

Correlation with bacterial cell density: A bacterial suspension's turbidity is inversely correlated with the
amount of bacteria present in the sample. We can therefore determine the density or concentration of
bacterial cells by detecting the turbidity of a sample. Besides that, turbidity measurement is a quick and easy
measurement: Turbidity measurement eliminates the need to count individual bacteria under a microscope,
making it an efficient way to estimate bacterial cell density. Then, turbidity measurement is suitable for high
throughput screening: Since multiple samples can be measured concurrently using a spectrophotometer,
turbidity measurement is perfect for high throughput screening of bacterial cultures. Other than that,
turbidity measurement is a non-destructive technique that does not call for the destruction or disruption of
bacterial cells, enabling the same sample to be used for multiple experiments. Last but not least, monitoring
bacterial growth: Since a rise in turbidity is correlated with an increase in bacterial cell density, measuring
turbidity can be used to track bacterial growth over time.

Conclusion:
In conclusion, this experiment aimed to compare and contrast different enumeration techniques for
estimating the concentration of bacterial cultures. The techniques employed in this experiment included the
spread plate technique, the Most Probable Number (MPN) technique, the Neubauer counter
Haemocytometer, and turbidimetry.

15
 The results obtained from the spread plate technique showed that it were able to form colonies for
determining the concentration of bacteria in the 10-3, 10-4, and 10-5 dilutions, but the colonies were too
numerous to count. The MPN technique was also effective in determining bacterial concentration, but the
results were unexpectedly high, possibly due to errors in the dilution process or pipette sterilization.

The Neubauer counter Haemocytometer was less effective in this experiment, as the results were too
numerous to count. This may have been due to insufficient dilution of the original culture or errors in
handling the Haemocytometer. Finally, turbidimetry proved to be an efficient and reliable technique for
estimating bacterial concentration, with the optical density measurements correlating with the concentration
of bacterial cells.

Overall, this experiment demonstrated the importance of choosing the appropriate enumeration
technique based on the concentration of the bacterial culture and the nature of the sample. Each technique
has its strengths and limitations, and it is important to consider these factors when selecting a method for
quantifying bacterial populations.

16
References

Absher, M., Kruse, P. F., Patterson, M. K., 1973. Chapter 1 - Hemocytometer Counting, Tissue Culture.
Academic Press, pp. 395-397.

Ben-David, A., & Davidson, C.E., 2014. Estimation method of serial dilution experiments. Journal of
Microbiological Methods, 107, 214-221.

Diez-Gonzalez, F., 2014. TOTAL VIABLE COUNTS | Specific Techniques. In: Batt, C.A., & Tortorello,
M.L., eds. Encyclopedia of Food Microbiology. Academic Press, pp. 630–635.

Erkmen, O., 2022. Isolation and Councting of Bacillus cereus: 19.2.3.2 Most Probable Number Counting
Technique. Microbiological Analysis of Foods and Food Processing Environments.

Gill, A., 2017. The Importance of Bacterial Culture to Food Microbiology in the Age of Genomics.
Frontiers in microbiology, 8, pp. 777.

Karunasagar, I., Maiti, B., & Kumar, B. K., 2018. Molecular Methods to Study Vibrio parahaemolyticus and
Vibrio vulnificus From Atypical Environments. In: Gurtler, V., & Trevors, J.T., eds. Microbiology in
Atypical Environments. Methods of Microbiology, Vol 48. Elsevier, pp. 287-417.

Lawler, D. M., 2015. SPECTROPHOTOMETRY | Turbidimetry and Nephelometry. In: Worsfold, P.,
Townshend, A., & Poole, C., eds. Encyclopedia of Analytical Science. Elsevier, pp. 343–351.

Liu, S., 2017. How Cells Grow. Bioprocess Engineering, Kinetics, Sustainability, and Reactor Design. 2nd
Ed. Elsevier, pp. 629–697.

Omar, A. F., Matjafri, M. Z., 2009. Turbidimeter Design and Analysis: A Review on Optical Fiber Sensors
for the Measurement of Water Turbidity. Sensors (Basel, Switzerland), 9(10), pp. 8311-8335.

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