Cholera -- samples and results

Vibrio cholerae outbreak in an urban neighborhood

We used a non-pathogenic Vibrio strain to spike any positive samples.

For all student designed experiments, we tried to encourage students in the right direction. For example, groups were discouraged from testing four very similar organisms for the Western blot and we encouraged them to be as specific as possible (about antigens, samples, etc.) when designing the ELISA.

Cultured blood and urine samples were negative. Due to the time constraints of this course, we chose to give the students stool samples already streaked on LB agar plates, rather than simulate samples for them to plate from. This plate contained only the non-pathogenic Vibrio in the interest of simplicity and time. However, for a longer course, it would be possible to give both Vibrio and a normal flora species in this sample, allowing for practice in distinguishing mixed cultures. Samples from this plate were appropriate to the Vibrio species.

For all of the students' ouchterlony experiments, we used the reagents provided in a Carolina Biologicals' ouchterlony kit. However, samples were relabeld as fungal, viral and bacterial extracts and a serum sample from one case patient. In this case, a positive reaction from the patient was seen only for the "bacterial extract." This experiment was used more to get across the concept of antibody-antigen reactions and to prepare them for the Western blot and ELISA experiments than to help them distinguish their particular outbreak. However, some groups did use this experiment as further confirmation of their results.

Antibiotic susceptibility results can just be done for whatever organism is actually provided to the students as long as one avoids antibiotics to which the actual outbreak organism is known to be resistant.

For the Western blot, students were asked to give us four candidate organisms against which they wished to test one patient serum. In the interest of time, we explained the concept of the Western blot to the students and then ran the gel and transferred it for them. Students were only involved in the probing of these blots. Blots can be done with any readily available samples and antibodies that will provide the appropriate pattern of positive and negative results. Additionally, groups that named two or more closely related organisms did get back false positives from cross-reacting antigens.

At the point in time in which we did the ELISA, the students understood basic immunology and their own cases well enough that we allowed them to design their own experiments. Samples, antibodies, etc. were obtained from a Carolina Biologicals ELISA kit and positive or negative samples were simply labelled appropriately for each group. Most groups used this to confirm organisms they were almost certain were causing their outbreak. Students were pushed to focus on including appropriate controls in their design of this experiment. THIS EXERCISE REQUIRES EXTENSIVE PRE-LAB PREPARATION FOR THE INSTRUCTOR.