Subj.: Progress report on NSF-AIRE Course Development Grant

Date: 9/9/99

This is a report on progress to date on the development of three research-oriented projects that are to be implemented into my Distributional course, BI 133 Microorganisms and Society. As outlined in my original proposal to the NSF-AIRE Steering Committee, these projects will be introduced beginning in the Spring semester 2000.

A time period that totaled an equivalent of four weeks was devoted to the development of these laboratory exercises that will serve to augment subject material presented in the lecture aspect of the BI 133 course. I will describe progress made in each of the three projects outlined below. Finally, I will present my proposed strategy regarding the implementation of these projects.

Project 1: The isolation and characterization of toxic heavy metal tolerant bacterial strains from the environment.

Samples of soil from various environments were collected and assayed for toxic metal tolerant bacteria on heavy metal-amended solid-plating media. The highest concentration of tolerant bacterial isolates was obtained from samples taken from sites known to be contaminated with toxic heavy metals, e.g., Hawk Ridge Composting Facility in Unity, ME, and as a positive control, from the Los Alamos National Laboratory Testing Grounds, NM. The highest concentrations of isolate populations were found to be tolerant to the metals, Chromium III, Lead II, Copper II, and Zinc II, and were not tolerant to high levels of Chromium VI.

Negative control samples, i.e., sites not known to be contaminated with heavy metals such as garden soil, yielded low concentrations of metal-tolerant bacterial isolates. It should be noted that ICP analysis of heavy metal content and levels on all soil samples collected by the students will be performed. With heavy metal concentration ICP data, students can then make inferences regarding the natural selection of heavy metal tolerant strains in those environments contaminated with high levels of specific metals.

As manifested by colloidal deposition of the elemental form of the metal on colonial growth, reductive mechanisms of metal resistance was observed almost exclusively on those isolates tolerant of Lead II. Chromium III reduction was also observed on approximately half of the Hawk Ridge samples tested. Recall from my NSF-AIRE proposal that toxic metal reduction capability by specific strains of bacteria could ultimately lead to bioremediation applications. Upon microscopic examination, those isolates capable of metal reduction were found to be primarily Gram negative, oxidase-positive bacilli. Gram-positive spore-forming bacilli were also prevalent morphological types.

After performing the study outlined above, I am confident that my BI 133 students should also obtain meaningful data regarding the isolation of metal-tolerant bacteria from environmental samples. These data should allow the students to make clear inferences between their findings and material covered in lecture regarding environmental and public health, and bioremediation.

Project 2: The isolation of antibiotic-producing and antibiotic- resistant strains of microorganisms from environmental samples.

Soil samples from fertile and non-fertile sites were used as potential sources of antibiotic-producing strains of microorganisms. The soil was serially diluted, plated by the spread plate technique, and the cultures were incubated for several days. Immediately it was observed that the soil samples used would produce fungal growth that would essentially take over the entire culture plate. This would make the examination and isolation of both eukaryotic and prokaryotic antibiotic-producing strains impossible, or at the very least, difficult for students. Therefore, the antibiotic cycloheximide was incorporated into the plating media to inhibit the growth of the eukaryotic fungi, but would still allow prokaryotic bacterial strains to grow in the presence of cycloheximide. This approach worked, because streptomycete bacteria, which are notorious antibiotic producers, grew and were observed to exhibit antibiosis on neighboring bacterial colonies. Microscopic examination confirmed the streptomycete characteristics of the antibiotic producing strains, i.e., long filamentous cellular morphological types.

As expected from past experience, the isolation and confirmation of antibiotic-resistant strains was simple and direct. The diluted soil samples used in the study above were used as the source from which purified strains of bacteria were obtained. These pure bacterial cultures were used as inocula for standard antibiotic susceptibility assays using the sensitivity disk method. Antibiotic resistant strains were isolated by transferring onto fresh plating media those colonies that were growing within the zone of inhibition of a specific antibiotic. These cultures were used as inocula for the gradient plate assay to determine the isolated strain's level of resistance to that specific antibiotic.

As stated in the proposal, considerable treatment of antibiotic drug discovery, and the relatively recent development of antibiotic-resistant infectious disease agents in our society, is presented in the lecture component of BI 133. The data obtained outlined above would allow the students to observe first-hand how readily bacterial mutations can occur that would lead to the selection of antibiotic resistant strains. These same mechanisms are operational when infectious disease agents become resistant to antibiotics as a result of chemotherapeutic misuse of the drugs by humans.

Project 3: Water quality analysis: Detection and quantification of coliform bacteria in potable water and in local lake and river water systems.

Water samples were obtained from various sources including the potable water supply in Arey, and from the Kennebec and Messalonskee Rivers. Samples were subjected to the coliform assay using the standard membrane filtration procedure. As expected, the potable water yielded no coliform bacteria, however both river water samples produced significant numbers of coliforms. The presence of coliforms indicates that fecal contamination of the water supply is probable, and that pathogenic strains of fecal-borne bacteria may be present. I did not perform the Enterotube II assays because of the cost of the assay system, and I did not have any of these kits on hand. However, this type of bacterial identification system is used routinely in my BI 238 Bacteriology course, and I foresee no problems with its implementation in this application.

Immediately, students should make the connection between the source of water used by humans, and the incidence of transmission of enteric pathogens by the fecal to oral route. As mentioned in the proposal, this subject material is covered in depth in lecture because of its important implications in the areas of public health especially in developing countries.

Implementation of the three projects and pedagogy.

Because of the length that each of these projects would require in a non-science majors laboratory situation, I anticipate that only one of these projects would be implemented per semester. It is most likely that I will incorporate each of the three projects, in the order presented, over the next three years that the BI 133 course is offered. Students will work in teams of four students each. Thus, there will be a maximum of 6 teams per section. This would allow for multiple samples to be run for any one project in each laboratory section.

Students would be required to write a scientific paper based on their results, and to present their findings orally to their laboratory section of 24 students during a laboratory colloquium at the end of the semester. Hand-outs to accompany these projects are forthcoming.

Thank you for granting me this opportunity to incorporate the three described research-oriented projects in my course, BI 133 Microorganisms and Society. I am grateful for the course development support of funds from the Colby College NSF-AIRE grant.


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