Serial Dilution Lab Report PDF

Preview

Summary

Serial Dilution Lab Report...

Description

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 [ CITATION Sap20 \l 17417 ]. The most frequently method of measuring bacterial populations is the plate count. We could either perform the pour plate method or spread plate method. All observable colonies are measured and interpreted as colony forming units (CFU) [ CITATION Bou \l 17417 ]. Then, the CFU is then multiplied with the corresponding dilution factor. An important advantage of this method is that it measures the number of viable cells. The disadvantage is it takes some time for visible colonies to form, usually 24 hours or more [ CITATION Tor19 \l 17417 ].

Apparatus Bunsen burner, test tubes, pipette, spreader, colony counter

Materials Bacterial cells, distilled water, nutrient agar plates, disinfectant

Procedure 1. The work space is disinfected and aseptic technique is applied. 2. Five test tubes are filled with 9 ml of distilled water each by using a sterile pipette. 3. 1 ml of bacterial cells is drawn into another sterile pipette. 4. The sample is then added to the first tube to make the total volume of 10 ml. This provides an initial dilution of 10-1 and is thoroughly mixed.

5. The pipette tip is discarded, and a new pipette tip is attached to the pipette. 6. Now, 1 ml of mixture is taken from the 10 -1 dilution and is emptied into the second tube. The second tube now has a total dilution factor of 10-2. 7. The same process is then repeated for the remaining tube, taking 1 ml from the previous tube and adding it to the next 9 ml diluents. 8. As five tubes are used, the final dilution for the bacterial cells will be 10-5. 9. Using a new sterile pipette, 0.1 ml of each dilutions is transferred into five nutrient agar plates respectively. 10. The sample is spread over the surface of the plate by touching the spreader to the agar while the plate is rotated. 11. The spreading procedure is repeated for each plate and the apparatus is sterilized before spreading each sample. 12. A range of 30 – 300 colonies are required to count plates. A counter is used and each colony is counted by marking each colony with a dot at the bottom of the plate. 13. The plates are placed in the autoclave to kill any bacteria present at the end of the experiment.

Results

Dilution 10-1 10-2 10-3 10-4 10-5

Colonies Counted TNTC TNTC TNTC 67 5

Discussion It is very difficult to determine the actual number of bacteria in a population. Thus, we calculate the number of bacteria using serial dilution that convert a dense solution into a less concentration of bacteria which simplify the counting. Viable plate count is the method being used most frequently in serial dilution. Pour plate method and streak plate method can be used for viable plate count [ CITATION Tor19 \l 17417 ]. Colony forming unit (CFU) is introduced in viable plate count for enumeration. Even so, there are several problems in viable plate count which are it takes some time for the colony to form and too many colonies may cause error in the count. Hence, to overcome the overcrowded, dilution is used. The spread plate method allows the bacteria to grow on the surface. The bacteria are poured on the agar and is spread over using a sterile rod. By referring to the experiment result, the spread plate method shows that there are more than 300 colonies and TNTC is used in the dilution 10-1, 10-2 and 10-3. This is because there are a lot of bacteria and overcrowded occurred, thus less visible colonies can be grown due to the competition among others. Thus, in dilution 10-4, 67 CFU in the dilution and in 10-5, 5 CFU is shown with the mean of 585000 viable cells/mL. This overall result show that the more we dilute the original culture, the less grown bacteria colonies can be observed on the plate and is easy to obtain a countable number of colonies ranging around 30 – 300.

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. After conducting five fold of serial dilution, we achieved 585000 viable cells/mL indicating the number of bacterial cells in the original culture. We can observe how serial dilution affects the colonies of bacteria in each dilution by looking into the results of the experiment.

References Aryal, S., & Aneeba. (2019, August 15). Spread Plate Technique- Principle, Procedure and Uses. Retrieved December 02, 2020, from https://microbiologyinfo.com/spread-platetechnique-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_Biotech nology/01:_Techniques/1.08:_Serial_Dilutions_and_Standard_Curve Rijal, N., Tankeshwar, A., Says:, T., Says:, S., Says:, I., Says:, K., . . . Says:, M. (2019, August 26). Spread Plate Technique: Principle, Procedure and Results. Retrieved December 02, 2020, from https://microbeonline.com/spread-plate-technique-principle-procedure-results/ Sapkota, A., Ferahtia_fs, Singh, D., Sawaira, Romano, D., Vishwathi, . . . Nwachukwu. (2020, May 27). Serial dilution- definition, formula, calculator, procedure, uses. Retrieved December 02, 2020, from https://microbenotes.com/serial-dilution/ Tortora, G. J., Funke, B. R., & Case, C. L. (2019). Microbiology: An introduction. Boston: Pearson.

Questions Determine the number of bacterial cells in the original culture. Show your calculations. Number of bacterial cells in the original culture: 5 x 105 = 500000 viable cells/mL 67x 104 = 670000 viable cells/mL Mean = 500000 + 670000 = 585000 viable cells/mL 2...