In this experiment the purpose is to seek understanding between an organism and its use of oxygen. Oxygen is a very important part of the organisms’ survival. Oxygen has a tendency to form very reactive by-products inside a cell. These by-products create havoc by reacting with protein and DNA, thus inactivating them. Cells that are able to live in the presence of oxygen have evolved enzymes to cope with H2O2 and O2- and thus are not inhibited by O2. Also many anaerobes have oxygen labile Fe-S centers and no cellular machinery to protect them from the oxidizing power of oxygen.
Organisms that cannot deal with the problems presented by oxygen cannot survive in air and are killed (Anaerobic Organisms Wikipedia). On the basis of oxygen tolerance, microorganisms can be placed into four classes. Strict aerobes cannot survive in the absence of oxygen and produce energy only by oxidative phosphorylation. Strict anaerobes, in many cases, generate energy by fermentation or by anaerobic respiration and are killed in the presence of oxygen Aerotolerant anaerobes generate ATP only by fermentation, but have mechanisms to protect themselves from oxygen.
Faculatative anaerobes prefer to grow in the presence of oxygen, using oxidative phosphorylation, but can grow in an anaerobic environment using fermentation. (Fundementals of Microbiology p. 105) Oxygen utilization is a primary diagnostic tool when identifying microorganisms. Special media has been developed for the purposes of determining the oxygen relationship and method of fermentation vs. respiration of microorganisms. One such medium, Thioglycollate Broth is useful for determining the oxygen relationship of a microorganism. The medium contains thioglycollic acid, cystine and agar, among other things. Anaerobic Organisms Wikipedia). The thioglycollic acid and agar prevent oxygen from entering the entire medium. The medium used was a plate of TSA +glucose, one incubated anaerobically in a Gaspack jar and another incubated aerobically. When the anaerobic jar had completely depleted the oxygen the indicator strip turns from blue to white. (Fundementals of Microbiology p. 106) After preparation, Thioglycollate Agar will develop a stable oxygen gradient, with high concentrations of oxygen near the surface of the agar and no oxygen near the bottom. Microbes will display different growth patters depending upon their oxygen relationship.
Microaerophiles will only grow near the surface of the agar (Micrococcus luteus. ). Facultative anaerobes and fermentative organisms will grow throughout the tube, but will display more growth near the top of the tube (Escherichia coli and Enterococcus faecalis). Strict aerobes will only grow in the presence of oxygen, at the top of the tube (Pseudomonas aeruginosa). Strict anaerobes will only grow in the bottom of the tube where oxygen is absent (Clostridium sporogenes). (Fundementals of Microbiology p105) The results of the thiglycollate broth tubes showed to be pretty accurate with only a few disagreements.
Clostridium sporogenes which as actually an anaerobic organisms appeared to be facultatively anaerobic in our tube. In addition to this Pseudomonas aeruginosa which is a strict aerobe appeared to be fermentative. Some possible reasoning behind these errors is simply due to procedure and experimental error. When one is inoculating tubes it is very important that the instruments are properly burned under the fire. More importantly than this, it is imperative to remember to not shake the tubes to avoid introduction to air.
The results of the TSA +glucose plates, both aerobically and anaerobically incubated, had extremely inaccurate results. It is to be expected that in a plate that is aerobically incubated, anaerobic organisms would not be able to grow, and in a plate that is aerobically incubated an anaerobic organism would not be able to grow. Strict aerobes cannot grow in the absence of oxygen, while strict anaerobes cannot grow in the presence of oxygen. This would mean that Clostridium sporogenes would not grow in the anaerobic plate while Pseudomonas aeruginosa would not grow in the anaerobic plate.
Unfortunately both of these organisms grew, showing that out plates were not accurate. There are many possible errors that lead to this inaccuracy. It is possible that the plates, when inoculated were not inoculated properly. It is very important that the 5 sections are not running into each other and contaminating each other. It is also important that between inoculating different organisms, excess organisms are properly burned off from the loop. It is possible that certain organisms grew under anaerobic conditions due to residual oxygen present in the anaerobe jar.
“Anaerobic Organism.” Wikipedia, the Free Encyclopedia. 3 June 2010. Web. 22
Gino Intrieri. The Fundementals of Microbiology. CT: University Of Connecticut, 2011. Print.
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