Test of Seawater for Fecal Bacteria

We will be collecting seawater from Ventura beaches and testing it for bacteria indicative of fecal contamination. We will compare the results of the different water samples collected at the different Ventura beaches. When we chose the sites what differences do you expect (hypothesize)?

Many actual disease organisms are hard to work with but Escherichia coli (E. coli) is easy to grow, simple to test for, and considered a good indicator of fecal contamination. E. coli is a type of coliform, but there are other bacteria which are coliforms that are not E. coli and do not provide an indication of sewage so it is important to distinguish E. coli from other coliforms.

You may have noticed from your syllabus that this lab spans three weeks. That’s because it has multiple parts and because we have to wait while the bacteria grow.

In the first test, the Presumptive test, we will do two things. One, we will test for the presence of bacteria which can use lactose and produce gas and acid. If they do that (are +) we presume they are coliforms. But remember that doesn’t make them E. coli. Secondly we get a quantitative estimate called a Most Probable Number of bacteria/ coliforms. (MPN)

How do we do this test? While part of each team collects water samples the remainder of each team attends a lecture on the method for the presumptive test. They will learn how we inoculate lactose broth (SSLB & DSLB) containing a pH indicator and Durham tubes in a series to provide qualitative and quantitative information. The home portion of each team will prepare the outline of the Material and Methods section of your scientific paper and explain the procedure to the collectors when they return. Remember that the Materials and Methods section of a paper should contain enough information to enable the reader to duplicate the experiment.

I will incubate the tubes at 35 °C for 24 hours then refrigerate them until next week.

Next week you will check the tubes for color (red vs. yellow) and the presence of a gas bubble in the Durham tube. You will record how many of each trio of tubes are + for both acid (yellow) and gas. Note: + means the tube is BOTH yellow and has a bubble, not just one or the other. These results will go in your report. You will use these data to get a MPN from the MPN table on my desk. Ask me to help you interpret it. Be sure to include the MPN in your report. It estimates how many coliforms were living in each ml of your original sample of seawater—the stuff people would have been swimming in. Notice, and include in your report, the 95% confidence limits. These mean that the MPN is a best guess, but that the statistician is 95% sure that the actual number is not less than the lower number and not higher than the upper number.

We will also do a second test, the Confirmed Test. You will take your most + tube and streak liquid from the tube onto Petri plates of special agar. EMB media grows coliforms as colonies with dark centers. E. coli has a very distinct greenish metallic sheen. Enterobacter aerogenes is an example of a common soil coliform, not indicative of fecal contamination. It would test + in the presumptive test. On EMB it does not have the green sheen. Why is the second test called the Confirmed Test?

In your report consider both your qualitative and quantitative results. Use the data from the entire class so you can compare the different sites. How do results compare to your hypotheses?