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SCMIC2001 General Microbiology

tag 0 Download 8 Pages / 1,773 Words tag 15-07-2021

Question:

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.
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