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Testing bacteria for the production of hydrogen sulfide (H2S) is a standard laboratory exercise in many introductory microbiology courses and becomes part of the student arsenal of tests that form the “unknown” laboratory in which students have to identify bacteria from a culture given to them by the course instructor. These “unknown” labs are critical for assessing both laboratory skills and critical thinking. Historically, one organism that has served as a positive control for H2S production is Proteus vulgaris (1, 2). However, P. vulgaris has a biosafety level (BSL) 2 classification, and not all introductory microbiological labs are equipped, nor is training provided, to allow students to work with BSL2 organisms.
The first objective of this project was to identify a BSL 1 organism to serve as an alternative to P. vulgaris. We tested Citrobacter freundii (1) for this purpose, using P. vulgaris as a positive control and Enterobacter aerogenes (1) as a negative control. We next determined an optimal detection method, which included two media alternatives (peptone iron agar and triple sugar iron agar) as well as lead acetate strips. Because it is typical in introductory microbiology labs for there to be a week between the time a test is conducted and when it is evaluated, we evaluated these tests over two time points (48 hours and one week). Finally, we implemented this process in an introductory microbiology laboratory.
Cultures of C. freundii (Ward’s catalog 851003), P. vulgaris (Ward’s catalog 851172), and E. aerogenes (Ward’s catalog 850033) were grown on nutrient agar slants and incubated at 37°C. P. vulgaris was cultured in a biological safety cabinet under sterile conditions.
Triple sugar iron (TSI) agar, Kligler iron agar, sulfide indole motility (SIM) agar, and peptone iron agar were each mixed in flasks following directions provided by the manufacturer. Media were covered with aluminum foil and heated until boiling while stirred. Then, 3 mL of media was pipetted into 20 test tubes (16 mm × 125 mm), making a total of 80 test tubes for the four different media. Tubes were autoclaved and then put on slants for the media to solidify.
TSI agar, Kligler iron agar, SIM agar, and peptone iron agar were inoculated with each type of bacteria with a sterile inoculating loop, either streaked on the surface of the slant or as a stab. Five trials were performed for each of the three bacteria tested on the different types of media. Finally, 3-cm lead acetate strips from Key Scientific were taped to the inside of the tubes, and the tubes were capped. Cultures were checked, and media and strips were analyzed at 48 hours and at one week. Positive tests for the media were indicated by a black precipitate.
Students in our introductory Microbiology laboratory subsequently carried out these experiments, using TSI agar and peptone iron agar as described above, along with the lead acetate strips. They evaluated test results after one week of incubation.
Standard laboratory safety procedures were used and students were trained in the use of BSL 1 organisms. In addition, P. vulgaris was cultured in a biological safety cabinet and using BSL 2 guidelines. BSL 1 and BSL 2 guidelines are provided here (https://www.asm.org/images/asm_biosafety_guidelines-FINAL.pdf). Briefly, these guidelines include training of students in handling BSL2 organisms, having students acknowledge this training by signing a safety agreement, conducting the experiments in a designated laboratory, wearing latex/nitrile gloves, wearing lab coats, disinfecting all surfaces before and after use, and disposing of all cultures by autoclaving after use.
Our work indicates that C. freundii serves as an excellent BSL 1 alternative to P. vulgaris in tests for H2S production. With regard to media, we found that the use of media alone was not sufficient for detecting H2S production, although some lead acetate precipitate was noticed in some of the stab cultures in each media. However, using TSI along with the lead acetate strips provided results using C. freundii that were consistent with those of P. vulgaris (Fig. 1). These results were consistent over evaluation at both 48 hours and one week (data not shown).
Peptone iron was also effective along with lead acetate strips in distinguishing C. freundii and P. vulgaris as H2S producers over the negative control, but the results were not as definitive as with TSI (Fig. 1). Kligler iron agar and SIM media were also tested but were not as effective in these tests (data not shown).
This project was originally investigated as part of undergraduate research projects (NR and CN) but was also tested in an undergraduate microbiology lab with similar results. For the purpose of quantitating the data in a microbiology lab, the darkness of lead acetate strips was analyzed using a 16-step grayscale (such as https://c1.staticflickr.com/5/4040/4487679731_1f7c8d5c99_z.jpg).
The authors gratefully acknowledge the work of Drs. J.R. Kerfoot, Andy Madison, Michael Schiebout, and William Thierfelder in help with statistical analysis and experimental design of the original student research data. We also appreciate the assistance of Dr. Kerfoot with photography. We furthermore acknowledge the assistance of Chance Mattox and appreciate the help of Frances Lancaster and Steven David Goforth. NR and CN presented some of the work in this manuscript as part of the Union University Scholarship Symposium.
Support was provided by Union University through the Undergraduate Research Grant program and Biology Department. The authors declare that there are no conflicts of interest.