Viable Plate Count

Laboratory Exercise No. 10 VIABLE PLATE COUNTS RESULTS AND DISCUSSION Table 1. Summary of Results dilution Trial 1

Trial 2

Trial 3

134

TNTC

TNTC

Contaminated with fungal growth

Contaminated with fungal growth

TFTC

TFTC

Contaminated with fungal growth

TFTC

10-6

10-7

10-8

Bacterial numbers in a broth culture of E. coli were determined using the viable plate method. This method is an indirect measure of cell density for live bacteria. The bacteria was inoculated in the plates using the spread plate technique which the bacteria are evenly spread onto the surface of the medium by means of a spreader. The plates were incubated for 24h and the number of colonies were counted. In this procedure, each colony counted is assumed to arise from one bacterial cell. The results of the experiment (Table 1) did not show the correlation of the serial dilution and the number of colonies present in the agar plates. Theoretically, as the dilution proceeds further, the number of bacterial colonies decreases. Only dilution 10-6 Trial 1 has a countable bacterial colonies. From which, the CFU ml -1 of the

original broth culture containing E. coli was calculated to be 1.3x108. Other results are labelled to be TFTC or Too Few To Count (#colonies < 25), Too Many To Count (#colonies >250). Some plates were contaminated with fungal growth characterized by the large to moderate sized filamentous growth present in the plates. Errors may be due to unsuitable culture conditions which may include inappropriate use of medium, inadequate environment to facilitate growth (Temperature and other conditions). Error may also arise from procedural errors such as inaccurate transfer or dilutions and inadequate mixing of culture before inoculation. Cell clumping may also overestimate the number of bacteria present. STUDY QUESTIONS: 1. Why do you think it is important to be able to quantify the number of viable bacteria in a sample? Quantification of the number of viable bacteria in a sample is one of the methods of measuring microbial growth which is essential clinically, particularly in the potency, mechanism and diagnosis of infectious diseases. Also, the total number of viable counts are important in dairy microbiology, food microbiology, and water microbiology. 2.

What is a CFU? Colony forming unit or CFU refers to each colony that can be counted in a plate. In addition, each viable cell can yield one colony. 3.

Why is the viable plate count technique considered to be an indirect measurement of cell density? Viable plate count technique is done indirectly, in a series of dilution, that is, the sample is serially diluted then plated out on an agar surface isolating visible colonies. This is done because most bacterial populations are very large to be counted directly. Thus, serial dilution can readily estimate the number of bacteria in the original sample and reveals only information about live bacteria (Tortora, 2010). 4.

Give an example of an industrial setting where quantifying viable bacteria would be a useful tool. Heterotrophic plate count (HPC) tests play an important role in water microbiology. HPC measurements are used: to indicate the effectiveness of a water treatment process; as a measure of numbers of microorganisms that may have a sanitary significance; and, as measure of possible interference with coliform measurements in lactose-based culture methods (Allen et al., 2002). 5.

Describe turbidimetric method to quantify the number of bacteria in a given sample. In this method, turbidity is estimated as a practical way of monitoring bacterial growth because as bacteria multiply in liquid, the liquid becomes turbid as cells multiply in the medium. The turbidity is measured by a spectrophotometer or colorimeter (Tortora, 2010).

REFERENCES

Bartram, J. (Ed.). (n.d.). Heterotrophic Plate Counts and Drinking-water Safety. Retrieved October 14, 2016, from http://www.who.int/water_sanitation_health/dwq/HPCFull.pdf Tortora, G. J., Funke, B. R., & Case, C. L. (2014). Microbiology: An Introduction (10th ed.). San Francisco, CA: Pearson Education, Inc. Bartram, J. (Ed.). (n.d.). Heterotrophic Plate Counts and Drinking-water Safety. Retrieved October 14, 2016, from http://www.who.int/water_sanitation_health/dwq/HPCFull.pdf Counting Bacteria. Retrieved October 14, 2016, http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/counts.pdf

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APPENDIX Calculation of CFU ml-1 of original broth culture *Only for the dilution 10-6 Trial 1 can be calculated because the other results is TNTC, TFTC or contaminated with fungal growth.

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CFU ml 1  134 1 6  1.3 108 10