BIOL 206 Microbiology

Lab 8 Fecal Contamination of Food and Water

Background:

Read the section of your text that discusses drinking water. Review the significance of water and food borne human diseases in the appropriate sections of your text. Also see excercises found in the lab manuals (lower drawer in instructors desk) on analysis of water, milk, and foods. Note the rationale for and the significance of the procedures used to examine these materials. The question you will be asked at the beginning of lab is, "What materials do you need in lab today and how will you use them?"

Also take a look at Identibacter Interactus on the BioLab1 Mac. Take time to become familiar with the information supplied and the process for working with virtual unknowns. Understanding the strategy for working with these virtual unknowns will help you prepare for working with laboratory unknowns later.

Bacteriologic Analysis of Water: The principle means through which pathogenic microorganisms reach water supplies is fecal contamination. Methods for bacteriologic examination of water are designed to provide an index of fecal contamination. Escherichia coli and other coliforms are used as indicators of fecal contamination. A presumptive test for coliforms is performed by inoculating a sample of water into tubes of lactose broth. Gaseous fermentation of lactose provides presumptive evidence of coliforms. This test must be confirmed. A confirmed test is done by plating a sample of the positive lactose broth onto EMB media on which the coliforms show a deep purple with a coppery, metalic sheen to confirm the presumptive test. A completed test requires inoculation of another lactose broth and an agar slant with isolated colonies from EMB media. Gas formation and microscopic demonstration of gram-negative rods on the agar slant are complete evidence for coliforms in the original sample. As you can tell the complete analysis requires several days. Plan accordingly.

1. Inoculate 1.0 ml of each water sample into separate tubes of lactose broth which have complete labels.
2. Incubate for 24-48 hr and examine for gas production (+ or -).
3. For gas + tubes inoculate EMB media to obtain single colonies, incubate for 24-48 hr, and examine for coliforms.
4. Pick a coliform and a noncoliform colony to inoculate into separate tubes of lactose broth and onto agar slants.
5. Incubate for 24-48 hr, examine for gas production, and prepare a gram stain from each slant culture.
6. Record the results and discuss the implications of each test in your notebook.

Conclusions

Discuss in your lab notebook the implications of the results you observe.

Questions

1. What is the bacteriologic standard for potable water? Citation?
2. How could one determine this value for water samples? Citation?
3. Why not test for pathogens like Salmonella directly rather than coliforms? Citation?
4. Why isn't a presumptive test sufficient to indicate that water is unsafe to drink? Citation?
5. List 5 waterborne infectious diseases and their causal agents. To what major taxonomic group does each agent belong? If Eubacteria, which Section? Site your source(s).
6. How many organisms are included in the list from Identibacter Interactus? How many tests are available?
7. What were the key tests that allowed you to identify your virtual unknown?

Use "Identibacter Interactus", a simulation of bacterial identification installed on the BioLab1 Mac, to identify a virtual unknown. You may need to spend some time with background information on species and tests provided in the program prior to your actual work with an unknown. Record or print the steps you took and the results of your virtual investigations. Please include your reflections and conclusions on the simulated and the virtual identification activities in your lab book.