In this experiment, we experienced many difficult challenges that first being with the serial dilutions. This is why we used two different techniques. Switching to the second squeezing technique allowed us to extract of liquid from the without the addition of granular material found in the contents. The liquid allowed us to perform a better serial dilution that may have eliminated contaminants.
After incubation, the plates showed an enormous amount of growth. This made it difficult to isolate one particular organism. The first streak for isolation failed to isolate any pure colonies. Many of those colonies obtained showed multiple organisms in gram stains and therefore required further streaks for isolation. It was not until the research project shifted from strict anaerobes to facultative anaerobes that we were able to obtain pure cultures.
Upon obtaining the five pure cultures, we commenced with biochemical testing and found that the genus of these microorganisms are possibly Micrococcus (culture A), Staphylococcus (culture B), Bacillus (culture C), Brochothrix (culture D), and Listeria (culture E). All the organisms were positive for containing the enzyme catalase. This is most likely due to the facultative nature of the organisms. They all grow well on the nutrient agar but not on the cellulose. This would suggest that they require nutrients other then cellulose. This is surprising because the organisms in the rumen are there to break down cellulose. Our cellulose media may not have been the right mix.
In order to determine the genus of the organisms obtained, we used the manual Bergeys manual of determinative Bacteriology, 9th Ed (1994). This book in conjunction with the tests performed suggested that culture A could belong to the Micrococcus group of organisms. Culture B was determined to be Staphylococcus epidermidis by the publication provided with the API test strip. There was a 98% chance that the organism belonged to the species group. Culture C only differed from the other gram-positive rods in size and the formation of endospores. The Bergeys manual suggested that this organism belonged to the Bacillus genus due to the test results. With respects to cultures D and E, the biochemical tests and the colony morphologies suggested two possibilities of genus types for these organisms. The Bergeys manual suggested the two possibilities Brochothrix and/or Listeria. The determining factor between these two geniuses is motility. Several attempts were made to test motility, however these tests were considered undeterminable due to unfamiliar results obtained. Culture D was labeled Brochothrix due to no motility, even though there was very small spreading in the SIMS test. Culture E was labeled Listeria due to more spreading, but still small, in the SIMS test.
The first trial run was extremely difficult due to lack of equipment and techniques. The plastic anaerobic hood used in this experiment was difficult to use because of the space constraints and the inability to maintain aseptic techniques required for isolating pure cultures. The requirement of duct tape to seal it off was not efficient enough to maintain an anaerobe environment. Leaking nitrogen gas that escaped through these holes would suggest that oxygen could possibly sneak in, and effectively kill all strict anaerobic microorganisms. It was also difficult to store supplies required for various task, including serial dilutions and streaks for isolation because of the space constraints. This lack of space also posed a problem with garbage accumulation.
All the faults of the anaerobic hood may have contributed to the increase possibility for contamination. The gloves were extremely difficult to maneuver and when performing serial dilutions the gloves remained contaminated because of the inability to disinfect them. When streaks for isolations were performed the gloves hindered the ability to follow aseptic techniques. This contamination may have contributed to the isolation of our five facultative microorganisms.
The sterilized inoculating needles that were provided to carry out streaks for isolation were possibly contaminated prior to use. Removing the sterilized needles from the tube was extremely difficult and they were hard to handle. When trying to remove one needle a few would touch the glove, which could possibly contribute to contamination of needles.
Based on our findings, this research project was a valuable learning experience. Things to be aware of for future experiments should include having adequate equipment and tools that would allow one to obtain pure cultures of strict and facultative anaerobes. A proper anaerobic chamber with the ability to inoculate equipment should be a primary piece of equipment and should be used if possible. Using individually prepackaged sterile inoculating needles would be a preferred tool in order to eliminate possible contaminants.
This experiment requires a lot of time and patience in order to obtain complete knowledge of all the organisms in the rumen of caribou.
In order to determine the best possible diet for ruminant animals, this experiment will need to incorporate diet changes and further evaluation of rumen microorganisms. An experiment like this could potentially take years of research, money, and manpower. If we could successfully identify the best diet that is economically feasible and lowers the methane being produced this could potentially reduce the effects of global warming. Although this would help reduce global warming this is not the only source of greenhouse gases. The need to educate people about pollutants and their part of global warming is still a necessity.
