SBT 1064

TROUBLE SHOOTING IN BIOLOGICAL TECHNIQUES

EXPERIMENT 2
TECHNIQUES IN MICROBIOLOGY

Date and Time of Practical Class

21 JUNE 2023 , 10:00 AM

Bil. Name Student ID Lecture Group

1 MASALIYAHTUL SAFA BINTI E20221030623 A

ZANAABIDIN
EXPERIMENT 2

TECHNIQUES IN MICROBIOLOGY

OBJECTIVES
1. To optimize serial dilution method in counting the number of microorganism
present in a culture.

INTRODUCTION
As the name suggests, serial dilution is a series of sequential dilutions which are
carried out to turn a dense solution into a more manageable concentration. Bacterial
populations are usually very large, hence in serial dilution, the density of the cells is
decreased at each step, so that the concentration of the cells in the original solution
can be measured more easily by measuring the total dilution over the whole series.
Incubated culture plates with an easily countable number of colonies (around 30-100)
can be obtained by diluting a sample in a controlled manner and the number of
microbes present in the sample can be determined.

MATERIALS

Escherichia coli cultures in nutrient broth

1.5 ml sterile tubes (14 tubes)
Micropipettes
Micropipette’s tips
14 Nutrient agar plates
Inoculation loop
Vortex mixer
Bunsen burner
Ethyl alcohol

METHODS :

I) Serial dilution Method 1

1. 1.5 ml tubes containing distilled water was labelled from 10-1 to 10-7 .
2. 0.9 ml sterile dH2O was placed into each labelled tube.
3. The bacterial culture was vortexed to homogenize it.
4. Using a micropipette, 0.1 ml of bacterial culture aseptically was transferred to the
first tube labeled 10-1.With this transfer you have diluted the bacterial culture by 10
times (1/10=10- 1) . Pipette tips was replaced after each transferred.
5. This test tube was vortexed before use a new tip to transfer 0.1 ml of diluents into
the next test tube labelled 10-2.
6. This process was continued until test tube labelled 10-7.

II) Serial dilution Method 2

1. The 1.5 ml tubes containing distilled water was labelled from 10-1 to 10-7.
2. 0.9 ml sterile dH2O was placed into each labelled tube.
3. The bacterial culture was mixed gently to homogenize it.
4. Using a micropipette, 0.1 ml of bacterial culture aseptically was transferred to the
first tube labelled 10-1.With this transfer the bacterial culture was diluted by 10 times
(1/10=10- 1) .The same pipette tips was used for each transferred.
5. This test tube was mixed gently before used a new tip to transfer 0.1 ml of diluents
into the next test tube labelled 10-2.
6. This process was continued until test tube labelled 10-7.

III) Spread plate method

1. The diluted bacterial culture was used, 0.1 ml of the 10-5 dilution was transferred
aseptically to the middle of the nutrient agar plate.
2. The glass hockey stick was sterilized over a Bunsen burner. The stick was cooled
before used it to spread the culture.
3. The lid of the Petri dish was opened and the glass hockey stick was used to spread
the 0.1 ml of the diluted culture immediately.
4. Step (1) was repeated for all the remaining dilutions from 10-6 and 10-7 .
5. The spread culture was allowed to absorb into the gel and dried up before inverting
the plates and incubated them at 30oC for 48 hours. 

OBSERVATION:

1. Observe the number of colonies from every dilution.

2. Determine the number of bacterial cells in 1ml of the original sample.

 Bacteria per 0.1ml = Number of colonies
Dilution
Bacteria per 1ml = Number of colonies x 10
Dilution

To determine the number of microbes in the original sample, plates should contain 25
to 250 bacterial colonies. Plates containing less than 25 colonies is taken as inaccurate
representation of brial population while colonies more than 250 is too many to count
(TMTC).

Method Method Bacterial per 0.1 ml = Bacterial per 1 ml =

Number of colonies Number of colonies x 10
1 2
Dilution Dilution
Method 1 Method 2 Method 1 Method 2

10-5 217 TMTL 21 700 000 TMTL 217 000 000 TMTL

10-6 No 260 0 260 000 000 0 2600 000 000

Distinct
colonies
10-7 0 239 0 2390 000 000 0 2.39x1010

DISCUSSION

1. List experimental setup conditions for each serial dilution method.

I) Serial dilution method 1

1. Obtain the 1.5 ml tubes containing distilled water was labelled from 10-1 to 10-7 .
2. Using a micropipette, 0.1 ml of bacterial culture aseptically was transferred to the
first tube labeled 10-1.
3. Pipet 1 mL of “solution0” into the test tube labeled 10-1. Vortex 10-1.
4. Remove 1 mL from test tube 10-1 and add it to test tube 10-2. Vortex 10-2.
5. Remove 1 mL from test tube 10-2 and add it to test tube 10-3. Vortex 10-3.
6. Remove 1 mL from test tube 10-3 and add it to test tube 10-4. Vortex 10-4.
7. Remove 1 mL from test tube 10-4 and add it to test tube 10-5. Vortex 10-5.
8. Remove 1 mL from test tube 10-5 and add it to test tube 10-6. Vortex 10-6.
9. Remove 1 mL from test tube 10-6 and add it to test tube 10-7. Vortex 10-7.

II) Serial dilution Method 2

1. Obtain the 1.5 ml tubes containing distilled water was labelled from 10-1 to 10-7 .
2. Using a micropipette, 0.1 ml of bacterial culture aseptically was transferred to the
first tube labeled 10-1.
3. Pipet 1 mL of “solution0” into the test tube labeled 10-1. Mix 10-1.
4. Remove 1 mL from test tube 10-1 and add it to test tube 10-2. Mix 10-2.
5. Remove 1 mL from test tube 10-2 and add it to test tube 10-3. Mix 10-3.
6. Remove 1 mL from test tube 10-3 and add it to test tube 10-4. Mix 10-4.
7. Remove 1 mL from test tube 10-4 and add it to test tube 10-5. Mix 10-5.
8. Remove 1 mL from test tube 10-5 and add it to test tube 10-6. Mix 10-6.
9. Remove 1 mL from test tube 10-6 and add it to test tube 10-7. Mix 10-7.

-Serial dilution method 1

The bacterial culture was vortex to homogenize it in 1.5 ml tubes labeled 10-1 to 10-7
.0.9 ml of sterile dH20 was added to each tube. The culture was transferred aseptically
to the first tube labeled 10-1, diluted by 10 times (1/10). The culture was then re-
transferred to the next tube labeled 10-2 .The process continued until the final tube was
labeled 10-7.

-Serial dilution method 2

The bacterial culture was mixed gently to homogenize it in 1.5 ml tubes labeled 10-1
to 10-7 . 0.9 ml of sterile dH20 was added to each tube, and 0.1 ml was transferred
aseptically to the first tube labeled 10-1 . The culture was diluted by 10 times using the
same pipette tips. The next tube labeled 10-2 was then mixed with 0.1 ml of diluents,
and the process continued until the last tube labeled 10-7.

2. Calculate and report the number of colonies in CFU, and briefly explain the
outcome.

Number of colonies Number of colonies in CFU 3. S

tate
Method 1 Method 2 Method 1 Method 2

10-5 217 TMTL CFU= 0 x 10 TMTL

10-5
=217 000 000
-6
10 No 260 CFU= 0 x 10 CFU = 260
Distinct 10-6 10
colonies =0 =260 000 0000
10-7 0 239 CFU= 0 x 10 CFU= 239 x 10
10-7 10-7
=0 =2.39x1010
which experimental setup gives you the best results.
Serial dilution method 2.

4. State the problem observed for each serial dilution method.

 Serial dilution Method 1
-Don't know the time required to vortex the test tubes.

Serial Dilution Method 2

-The way to mix the culture in the test tubes is different.

3. It is performed in a stepwise manner, therefore it needs a more long period of time

which restricts the capability of the method.

4.This technique only reduces the counts of bacteria/viable cells but not separate them
like in other methods such as flow cytometry.

5. List all the possible causal factors or explanations.

1. Dilution Factor

2. Volume of Sample: The initial volume of the sample is crucial for calculating the
dilution factor. It determines the amount of the original sample used in each dilution
step. Precision in pipetting the sample volume is necessary to achieve accurate
dilutions.

3. Number of Dilutions: The number of serial dilutions determines the final

concentration of the microorganisms in the diluted solution. Each dilution step
reduces the concentration by a known factor. The number of dilutions depends on
the starting concentration of the sample and the desired concentration for analysis.

4. Mixing and Uniformity: Proper mixing of the samples is essential to ensure

homogeneity. Vigorous and consistent mixing techniques, such as vortexing or
pipetting up and down, should be used to distribute microorganisms evenly
throughout the solution.

5. Aseptic Technique: Maintaining aseptic conditions throughout the serial dilution

process is crucial to prevent contamination. Using sterile equipment, working in a
clean environment, and avoiding cross-contamination are essential steps to ensure
accurate results.

6. Highlight the most probable causal factor listed in question 5, at the same time,
eliminate nonpossible explanations.

Mixing and Uniformity: Proper mixing of the samples is essential to ensure

homogeneity. Vigorous and consistent mixing techniques, such as vortexing or
pipetting up and down, should be used to distribute microorganisms evenly
throughout the solution.

7. Check again the causal factors listed in question 6, with your experimental results
(refer again with results obtained in question 2).
 Based on the results , serial dilution in method 1 which is vortex didn’t give the best
results. It is because the consistent of vortex the sample. However , serial dilution in
method 2 which is mix gently the test tubes gives the balance and best results for this
experiment.

8. Identify the main causal factor/s.

The main causal factor is the way of mixing and uniformity the mixtures.

9. Make a conclusion for each experimental setup.

-Serial dilution Method 1

This method not give the best results because the mixtures not well homogenized and
the number of microorganism is not present in large numbers.

-Serial dilution Method 2

This method gives the best results because the mixture is well homogenized which is
give large number of colonies.

CONCLUSION

We can determine the number of bacteria in a sample by enumeration which can be

done by spread plate method. Serial dilution is really beneficial as it makes it easy to
easily count the colonies of bacteria in a more manageable concentration. The
experiment has proven that each dilution will decrease the number of bacteria (around
30 -100) and number of cells per mL, and the more set of streaks we conduct, the
more isolated colonies we achieve.

REFERENCE

Aryal, S., & Aneeba. (2019, August 15). Spread Plate Technique- Principle,
Procedure and Uses. Retrieved December 02, 2020, from
https://microbiologyinfo.com/spread-plate-technique-principle-procedure-and-uses/

Boundless. (n.d.). Boundless Microbiology. Retrieved December 09, 2020, from

https://courses.lumenlearning.com/boundless-microbiology/chapter/counting-bacteria/

Libretexts. (2020, July 21). 1.8: Serial Dilutions and Standard Curve. Retrieved
December 02, 2020, from
https://bio.libretexts.org/Bookshelves/Biotechnology/
Lab_Manual:_Introduction_to_Biotechnology/01:_Techniques/
1.08:_Serial_Dilutions_and_Standard_Curve
Sapkota, A., Ferahtia_fs, Singh, D., Sawaira, Romano, D., Vishwathi, . . .
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dilution/

Basic Practical Microbiology-Manual. The society of General Microbiology.

Retrieved from
https://microbiologyonline.org/file/7926d7789d8a2f7b2075109f68c3175e.pd