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Direct cell count via hemocytometer

A Neubauer chamber (also known as a haemocytometer) was used to conduct a direct cell count. In your lab, 15 squares are typically counted. Initially we you tried to count the number of cells per square there were too many to count accurately. So you conduct a 10-fold dilution of the sample and then do a direct cel count. In the diluted sample you counted the following number of bacteria per square:
21, 21,20, 25, 31, 13, 22, 21, 22, 14, 17, 20, 18, 20, 19.


Using the information provided in the appendix, conduct the calculations to determine the total number of bacteria present in the water sample. Report your results. Total Heterotrophic Plate Count: A serial dilution was conducted using standard methods, then selected dilutions were used to set up pour plates with total plate count agar, and plates
incubated for 48 – 72 hours. Each dilution was plated in duplicate, with 1.0 ml of inoculum placed into Petri plates, then pour plates prepared. After incubation, you counted the following:
10-3 dilution: Too many to count on both plates.
10-4 dilution: 332 colonies; 327 colonies.
10-5 dilution: 31 colonies; 38 colonies.
10-6 dilution: 2 colonies; 5 colonies.


Select the appropriate dilution and use that dilution to calculate the number of colony forming units per ml of sample. Remember to take into account the volume of liquid used to inoculate the agar plates.Enumeration of Escherichia coli and Total Coliforms by Plate Count: Chromogenic agar was used to enumerate E. coli and total coliforms. There are various formulations of chromogenic agar, which can be used for different target organisms. The chromogenic agar
used in this analysis is selective for the growth of coliform bacteria. It is differential in that E. coli appears purple, and other coliforms appear pink (see Figure 2 in Appendix). You used the same dilution series as above to prepare spread plates, with 0.1 ml of inoculum placed onto each plate. After incubation, you counted the following number of colonies:
10-3 dilution: Plate 1: 52 pink, 98 purple. Plate 2: 48 pink, 104 purple.
10-4 dilution: Plate 1: 4 pink, 10 purple. Plate 2: 5 pink, 11 purple.
10-5 dilution: Plate 1: 2 pink, 2 purple. Plate 2: 0 pink, 0 purple.
10-6 dilution: Plate 1: 0 pink, 0 purple. Plate 2: 0 pink, 0 purple.


Select the appropriate dilution and use to calculate the number of colony forming units per ml of sample for both E. coli and all coliforms. Remember to take into account the volume of liquid used to inoculate the agar plates. Most Probable Number (MPN): The MPN method is widely used in microbial laboratories as a method of enumerating bacteria. An overview of the MPN method is provided in the appendix.In your laboratory you conduct an MPN for thermotolerant coliforms and E. coli based on the relevant Australian Standard AS 5013.15-2006 (you can find this standard through the Australian Standards link in the database section of the Federation University library if you are
interested).

Using the dilution series previously prepared you inoculate lauryl sulphate broth and incubate appropriately. Each dilution is prepared in triplicate, so you end up with 12 tubes in total (3 tubes each with 1 ml of 10-3 dilution; 3 tubes each with 1 ml of 10-4 dilution; 3 tubes each with 1 ml of 10-5 dilution; 3 tubes each with 1 ml of 10-6 dilution). After incubation at 37°C for 48 hours, all 12 tubes are positive, so you use the 10-4 to 10-6 tubes to inoculate a tube of EC broth (thus resulting in nine tubes of EC broth), which are incubated at 44°C for 48 hours. The results of the EC broth are listed.

Direct cell count via hemocytometer

Results and Calculations

Direct cell count via hemocytometer

Average cell count per square- 304/15= 20.26

Each square has a volume of 6.25 × 10-3µl =0.00625 µl

Therefore, the total number of bacterial cells per ml of liquid is =

= average cells/square X 1000            = 20.26      X 1000 = 3241600 = 3.2 × 106 cells/ml

0.00625   0.00625

Direct cell count via heterotropic cell count

Average colonies on plate- 34.5 colonies

Dilution factor- 105

Volume of sample plated- 1.0 ml

Total no. of CFU/ml of sample is =

= (average no. of colonies X total dilution factor)/ volume plated

= (34.5 X 105)/ 1.0

= 34.5 X 105 CFU/ml =3.45 X 106 CFU/ml

Plate count of coliforms

Average colonies of coliform on plate- 50

Average colonies of E. coli on plate- 101

Dilution factor- 103

Volume of sample plated- 0.1 ml

Total no. of CFU/ml of sample (E. coli) is =

= (average no. of colonies X total dilution factor)/ volume plated

= (101 X 103)/0.1

=10 X 105 CFU/ml

Total no. of CFU/ml of sample (coliform) is =

= (50 X 103)/0.1

= 5 X 105 CFU/ml

Total no. of CFU/ml of sample (all coliform) is =

= (151 X 103)/0.1

= 15 X 105 CFU/ml

Most Probable number (MPN)

  1. coli- 23 X 105

Thermotolerant coliform - 93 X 105

Discussion questions

Answer 1

  1. Both the hemocytometer cell count and heterotropic plate count are categorized as direct cell count methods. Both the methods yielded approximately similar results. For instance, in the hemocytometer count revealed a cell count of 3.2 × 106cells/ml and the heterotropic cell count revealed 3.45 X 106CFU/ml. However, both the methods revealed similar results. Within these two methods, the heterotropic cell count method revealed higher count. Although both of the cell counting procedures is evaluating the direct cell count, the hemocytometer counts both viable and dead cell, i.e. the total cell count on the plate. On the other hand, in case of heterotropic cell count, only the viable cells will develop visible colonies, which are then counted (McFeters, 2013). Thus, it can be said that the plate cell count method is more suitable to determine the accurate cell count in a sample.
  2. According to the expected results, the direct cell count by hemocytometer should be 40-50 times higher compared to the plating method or viable cell count, as the non-viable cells are being excluded from the sample in case of plating method, i.e. heterotropic plate count (Bauman, Machunis-Masuoka & Cosby, 2012). However, no such results were revealed in the current context. Thus, the results are not justified according to the standard number of colonies in standard water sample.

Thus, it can be interpreted that the viable cell count method is more suitable for checking the water quality in Alice’s laboratory. It has been revealed from Canadian Drinking Water Quality Guidelines that no MAC is specific for HPC bacteria in water supplied by semi public, public and private drinking water system (Sen & Ashbolt, 2011). Increase in HPC concentrations more than baseline levels are undesirable conditions. The ideal HPC count is less than 10 CFU/ml. However, the count in Alice’s laboratory water is significantly higher than the ideal level.

  1. The indicator organisms are microorganisms, presence of which in water indicates the presence of pathogens and probability of contamination. These organisms have significant use in the water and food industry to monitor the environment, ecosystem, habitat or consumer product. In this context, certain fungi and bacteria are used as indicator organism. In case of indicator bacteria, the indicator organisms are usually found in human faeces, known as coliform bacteria, indicating faecal contamination.
  2. E. coli is the standard indicator organisms, whose presence in water indicates water contamination, which should not persist in the environment for long periods of time following efflux from the intestine (Odonkor & Ampofo, 2013). The indicator bacteria are used for detecting and estimating the level of faecal contamination of water. However, these are not dangerous to the human health; rather these are used for indicating the presence of health risk upon consuming the contaminated water. It has been estimated that each gram of human faeces contains approximately 100 billion bacteria (Da Silva et al., 2013). These bacteria include pathogenic bacteria including Salmonella or Campylobacter sp. E. coli and Enterococci.

Criteria for indicator organism

  • Organism should be present whenever enteric pathogens are present
  • Organism should have a longer survival time than the hardiest enteric pathogen
  • Organism should have a longer survival time than the hardiest enteric pathogen
  • Organism should be found in warm-blooded animals’ intestines
  • Organism should not grow in water
  • Organism should be facultative anaerobe
  • Organism should be gram negative in nature

There is zero tolerance for faecal coliforms in treated drinking water and presence of these organisms above the level makes the water unacceptable for drinking. Thus, water industry uses these organisms as an indicator of health risk and use for monitoring the safety level of drinking water (Gunnarsdottir et al., 2012).

  1. Coliform bacteria are rod shaped gram negative, motile or non-motile, non-spore forming, lactose fermenting and acid producing organism. These organisms are used as an indicator of sanitary quality of foods and water. However, the thermotolerant coliform bacteria are specifically identified with the ability of producing gas and lactose at 44.5ºC in 48 hours. For instance, E. coli is the thermotolerant coliform (Bain et al., 2012).

The thermotolerant coliform, i.e. E. coli is a better indicator of faecal contamination. Indicator value Escherichia coli is considered the most suitable index of faecal contamination (Hammes et al., 2012). As these organisms are more heat tolerant, thus are more resistant to the harsh environment, which indicates that their  presence in water is more dangerous compared to the other coliforms, which can grow at 35 ºC -37 ºC.

  1. Two methods have been conducted for enumerating the total number of coliforms in the plate, including the plate count and most probable number. The most probable number yielded higher number of coliforms compared to the most probable number. For instance, the most probable number yielded 23 X 105 and 93 X 105E. coliand other coliforms respectively, whereas plate count yielded 10 X 105 CFU/ml and 5 X 105 CFU/ml E. coli and other coliforms respectively. Thus, there is a significant difference between results yielded from two methods. MPN yielded a significant higher count (Yates et al., 2016).

In both of the colony counting methods, the coliform and thermotolerant coliform count have been done. However, comparison is justified, as it is helping in identifying the level of health risk upon consuming the contaminated water sample. Coliforms are the bacteria, which may harmful to the human body; however, the thermotolerat conliforms are more harmful as they can survive at high temperature, which enhances the chance of this organism to contaminate treated water in water treatment plant.

Direct cell count via heterotropic cell count

Thus, it is appropriate to compare the coliform and thermotolerant coliform count via two different methods. In layman’s terms, the experiments are done for comparing between apples to apple, not apple to orange (Payment & Locas, 2011). This is because, the characteristics of both the organisms are similar except some key difference including the ability of thermocoliform bacteria to ferment lactose and produce carbon dioxide within 48 hours at high temperature; tthereby indicating the importance of counting two different cell counts for these two sub categories of indicator organism.

For Alice’s future plan of water treatment, the quality of water needs to be assured, which can be done through various procedures including sampling, filtration, culturing and incubation. Filtration is a key step for maximizing recovery of microorganisms while avoiding exogenous contamination, which is performed by passing a known volume of water through a sterile membrane filter with a pore size small enough to retain bacterial cells, which is then transferred to an agar plate and allowed to develop into colonies.

Culture media includes the use of selective bacteria for detecting indicator organism, which have been earlier. In addition, the rapid methods can also be implemented; which include quantitative population. Biofilm is another potential process for testing the quality of water sample being tested in the laboratory. Biofilms are now recognised as complex microbial communities, forming on surfaces (Odonkor & Ampofo, 2013). 

It has been revealed that most of the bacteria in drinking water distribution systems are present within biofilms rather than free living in the water itself.  Salmonella Typhimurium, Campylobacter, Pseudomonas aeruginosa and Aeromonas hydrophila are the organisms identified from biofilms, which are potentially pathogens for human. High heterotropic plate count is suitable to identify the pool of microbial population in biofilms, thus it is a suitable method for testing water sample. Finally, the IMViC test is a significant microbial testing for testing the quality of water in laboratory.

It is a series of tests for identifying and confirming the presence of coliform group of bacteria in water. These include indole test, methyl red test, voges proskauer test and citrate test. In the first two tests, the faecal coliform like E. coli gives positive responses, whereas in last two tests, nonfaecal coliforms like Enterococcus gives positive results (Sen & Ashbolt, 2011). FLowcytometer is an advanced method for testing the quality of water also, which Alice can also use.

Reference List

Bain, R., Bartram, J., Elliott, M., Matthews, R., McMahan, L., Tung, R., ... & Gundry, S. (2012). A summary catalogue of microbial drinking water tests for low and medium resource settings. International journal of environmental research and public health, 9(5), 1609-1625.

Bauman, R. W., Machunis-Masuoka, E., & Cosby, C. D. (2012). Microbiology: With diseases by body system. Benjamin Cummings.

Da Silva, N., Taniwaki, M. H., Junqueira, V. C. A., Silveira, N., & GOMES, R. A. R. (2013). Microbiological Examination Methods of Food and Water. CRC Press.

Gunnarsdottir, M. J., Gardarsson, S. M., Elliott, M., Sigmundsdottir, G., & Bartram, J. (2012). Benefits of water safety plans: microbiology, compliance, and public health. Environmental Science & Technology, 46(14), 7782-7789.

Hammes, F., Broger, T., Weilenmann, H. U., Vital, M., Helbing, J., Bosshart, U., ... & Sonnleitner, B. (2012). Development and laboratory?scale testing of a fully automated online flow cytometer for drinking water analysis. Cytometry Part A, 81(6), 508-516.

McFeters, G. A. (Ed.). (2013). Drinking water microbiology: progress and recent developments. Springer Science & Business Media.

Odonkor, S. T., & Ampofo, J. K. (2013). Escherichia coli as an indicator of bacteriological quality of water: an overview. Microbiology research, 4(1).

Payment, P., & Locas, A. (2011). Pathogens in water: value and limits of correlation with microbial indicators. Ground Water, 49(1), 4-11.

Sen, K., & Ashbolt, N. J. (2011). Environmental microbiology: current technology and water applications. Horizon Scientific Press.

Yates, M. V., Nakatsu, C. H., Miller, R. V., & Pillai, S. D. (2016). Manual of environmental microbiology (No. Ed. 4). American Society for Microbiology (ASM).

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