16. Discard the flow through from the collection tube as described above. 17. Ce

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16. Discard the flow through from the collection tube as described above. 17. Centrifuge the spin filter at 10,000 xg for I minute at room temperature to remove any residual liquid from the filter or the sides of the filter. 18. Carefully place the dry spin filter in a clean, labeled, 2 ml collection tube. Avoid getting any residual C5 solution on the filter insert. 19. Add 100 1 of solution C6 to the center of the white filter membrane. 20. Centrifuge at room temperature for 30 seconds at 10,000 x g. 21. Discard the spin filter. The DNA is in the collection tube is now ready for downstream applications. 22. Store your DNA in the collection tube at 4°C. Part 4: Bacteria Final Viability Using the lid of a sterile 1.7 ml microcentrifuge tube, transfer 2 lid full of soil from the treatment container into a test tube with 9mL of sterile water& appropriately labeled for each treatment. Flick tube vigorously to mix. The soil will settle to the bottom. Perform a 1:10 dilution for cach treatment tube: Transfer 1 ml of "dirty water from the first test tube to a second test tube of 9 ml of sterile water (labeled "treatment letter"&1:10). Flick tube vigorously to mix. 1. a. Repeat for each treatment: A, B, & C b. A sterile microcentrifuge tube should be used for each treatment 2. Spread plate 1001 (0.1 ml) from cach ofyour 6 tubes (A, A10, B, Bi:10. C. 3. Spread plate 100(0.1 ml) from each of your 6 tubes (A,A1:10,BBI:10, C, 4. (optional) Spread plate 100 ul (0.1 ml) from each of your 6 tubes (A, Al:10, B. c. A total of 6 test tubes will be used CI:10) on to a labeled Enterobacteraceae enrichment agar plate. CI:10) on to a labeled MSA agar plate. B1:10, C, C1:10) on to a labeled R2A agar plate. Questions 1) What is your hypothesis for the bacterial growth on each agar plate? Why are multiple, different agars used for culturing bacteria from all the experimental treatments? 2) 46

Explanation / Answer

1. What is your hypothesis for the bacterial growth on each agar plate?

In the given experiment three different growth media have been used to study the bacterial viability of "A", "B" and "C" soil samples.

Sample A is a fresh soil which is an untreated control.

Sample B is a spiked soil with E. coli and S. epidermidis.

Sample C is an autoclaved soil.

Enterobacteriaceae enrichment agar: This medium is used for the specific isolation of pathogenic bacteria such as E. coli, Salmonella, Shigella, and other enteric pathogens.

Mannitol salt agar: This medium is used for the isolation of Staphylococcus epidermidis and S. aureus.

R2A agar: This medium used for the identification of heterotrophic counts and for the isolation of stressed bacteria.

Now let us write the hypothesis for bacterial growth on each agar plate:

1. Enterobacteriaceae enrichment agar plate: This is a selective media. Hence, bacteria belonging to Enterobacteriaceae such as E. coli, Salmonella, Shigella, and other enteric pathogens must grow. We have to find out the bacterial growth from each sample. Expected results for this agar plate are shown below:

2. Mannitol salt agar plate: The MSA agar is specific for S. epidermidis. So, we must expect the growth of S. epidermidis or species belonging Staphylococcus only in this agar plate. Expected results for this agar plate for each sample are shown below:

3. R2A agar plate: This medium will help us in finding the overall count of the colonies in a given sample. Following results can be expected for each sample:

2. Why are multiple. different agars used for culturing bacteria from all the experimental treatments?

As explained in the previous answer, the characteristics of each sample used in the experiment are unique.

Sample A represents an undisturbed microbial community. So, it consists of a plethora of bacteria belonging to the different family. In contrast to sample A, sample B is an induced microbial community or an artificial microbial community, wherein E. coli and S. epidermidis were introduced to study their interaction in the soil environment. Sample C is an autoclaved soil wherein there will be no bacteria.

Use of different agars helps us to specifically isolate the bacteria and to study the cooperation or competition of microbial communities in the soil environment. From the above-mentioned tables, it is very clear that each media is highly specific in nature and thus use of different media help us for the effective study of microbial communities.

What are your control treatments?

In an experiment, we must keep a positive control and a negative control.

In the given experiment, we are studying the interaction of E. coli and S. epidermidis in a healthy soil.

So, we must use healthy soil alone as a positive control.

For negative control, we have to use autoclaved soil. This soil does not contain any bacteria and thus help us in testing the contamination free experimental conditions.

Sample Growth of bacteria belonging to Enterobacteriaceae No growth at all A Bacteria belonging to Enterobacteriaceae will show growth. The number of colonies will be more as it is undiluted A1:10 Bacteria belonging to Enterobacteriaceae will show growth. The number of colonies will be less due to serial dilution. B Only E. coli will show growth as it belongs to Enterobacteriaceae. The number of colonies will be more as it is undiluted. B1:10 Only E. coli will show growth as it belongs to Enterobacteriaceae. However, the number of colonies will be reduced due to serial dilution. C Yes (Since it is an autoclaved sample, there should not be any growth) C1:10 Yes (Since it is an autoclaved sample, there should not be any growth)

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