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12 views4 pages

Exp 1

Uploaded by

ifty0907
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Summer, 2024

North South University


Department of pharmaceutical Sciences
Course: PHR223L (Pharmaceutical Microbiology-II Laboratory)

[Exp-01] Title of the Experiment: Serial Dilution for Colony Counting


__________________________________________________________________________________
Objective: The objective of this experiment is to perform decimal or sequential dilutions (which is
used to reduce a dense culture of cells to a more usable concentration). Each dilution will reduce the
concentration of bacteria by a specific amount.

Principle: In quantitative Microbiology we are concerned with determining the concentration of


Colony -Forming Units (CFU) in our sample (i.e. the number of CFU per ml or per gram of the
sample). More realistically the concentration of CFUs in the sample could have been considerably
greater. Counting the colonies on the plate inoculated with 1 ml of sample may be impossible in
many cases. We would like to have ‘countable plates’ containing between 30-300 colonies on a 10
cm dish. If fewer than 30, we may run into greater statistical inaccuracy, if greater than 300, the
colonies would be tedious to count and also would tend to run together.

In practice, more than a single ten-fold dilution is required to obtain a countable number of bacterial
colonies. Cultures routinely contain millions of living bacteria per milliliter (mL). So, a culture may
need to be diluted millions of times and this can be achieved by applying serial dilution. Example;
when 1 ml cultured sample is added to 9 ml of sterilized liquid, it gives a 10-fold dilution; whereas
addition of 1 ml sample in to 99mL gives a 100-fold dilution.

When fixed amount of this dilution series is spread over an appropriate agar plate and incubated,
then different numbers of colonies will be obtained. By working back from an easily counted plate
and using the appropriate dilution factor, the number of micro-organisms in the original source
culture can be calculated.]

Materials required:

1. Sterile test tubes 7. 10 cm petri-dishes


2. Micropipette with sterile tips 8. Conical flask
2. Sterile distilled water 9. Labeling tape
4. Sterile nutrient broth 10. Incubator
5. Sterile nutrient agar 11. Colony counter
6. 70% alcohol 12. Sample (can be solid or liquid, example: tap
water, blood, soil, foods, sausages etc.)

Page 1 of 4
Summer, 2024

North South University


Department of pharmaceutical Sciences
Course: PHR223L (Pharmaceutical Microbiology-II Laboratory)

[Exp-01] Title of the Experiment: Serial Dilution for Colony Counting


__________________________________________________________________________________
Procedure: There are four major steps in procedure-

1) Preparation for serial dilution

2) Spreading the sample onto the agar plate

3) Counting the Bacterial colony

4) Calculation of total numbers of viable bacteria in the original sample from those counts.

Preparation of Serial dilution:

1) Collect the sample in a sterile container, if it is solid then dissolve it with appropriate

amount of sterile distilled water.

2) Take five test tubes and label them 10-1 to 10-5.

3) Pipette 9 ml of distilled water or nutrient broth into each of the tubes.

4) Pipette 1 ml of undiluted sample into the tube marked 10-1 and mixes it properly.

5) Discard the tips and take a new tip and transfer 1 ml from the 10-1 to 10-2.

6) Again, change the tips and continue transferring 1 ml sample from 10-2 to 10-3, 10-3 to 10-

4
, 10-4 to 10-5.

7) Now take 0.1 ml (100µl) sample from each test tubes on to the 10 cm nutrient agar

plate.

8) With the help of sterile spreader, spread the sample all over the petri-dish.

9) Incubate all the plates at 37˚C for 12~24 hours.

10) Observe the growth of the bacteria on the petri-dish and count the colony by colony

counter.

Page 2 of 4
Summer, 2024

North South University


Department of pharmaceutical Sciences
Course: PHR223L (Pharmaceutical Microbiology-II Laboratory)

[Exp-01] Title of the Experiment: Serial Dilution for Colony Counting


__________________________________________________________________________________

Figure-2: Colony found on plate according to Dilution and calculation

 You now have the following dilutions of the original sample--

When 1mL is spread When 0.1mL is spread


Test Tubes Dilution Factor Dilution Dilution Factor Dilution
Original sample 100 1 100 1
Test tube-1 101 1/10 102 1/100
Test tube-2 102 1/100 103 1/1000
Test tube-3 103 1/1000 104 1/10,000
Test tube-4 104 1/10000 105 1/100,000
Test tube-5 105 1/100000 106 1/100000

Page 3 of 4
Summer, 2024

North South University


Department of pharmaceutical Sciences
Course: PHR223L (Pharmaceutical Microbiology-II Laboratory)

[Exp-01] Title of the Experiment: Serial Dilution for Colony Counting


__________________________________________________________________________________
Calculation: Back calculation should be carried out for counting the actual number of bacteria into
the original sample. The CFU/ml can be calculated using the formula:

No. of bacteria per mL sample = (Total no. of colonies on the plate x dilution factor) CFU/mL

= _ _ _ _ _ CFU/mL

Result: The actual bacterial count of the original sample is _ _ _ _ _ _ _ _ CFU/mL.

Precautions:

1) Wear apron, gloves and mask.


2) Make sure that all glass apparatus, nutrient broth or distilled water has been
autoclaved properly.
3) Never pipette cell suspensions or medium by mouth.
4) Before and after each lab period wipe your bench top down with disinfectant and
wash your hands.
5) Change the tips each and every time of dilution.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Report:

Write down the laboratory report for this experiment and submit it one week after the experiment
to the lab instructor. Remember to include the followings in your report:

1) Title of the experiment


2) Purpose/objective
3) Materials required
4) Procedure
5) Result
6) Importance of Serial dilution and colony counting
7) What you have learned from this experiment
8) Precaution

Page 4 of 4

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