The ability to identify organisms is a crucial technique for anyone working in a microbiology lab. Researchers have at their disposal a plethora of techniques and media to make a positive identification of organisms. Through the use of various types of media, they have almost an infinite number of characteristics to test for. Whether particular metabolic pathways are chosen for identification, or if identification can be made by using a simple stain to view the shape of the microbe, tests are often down in combinations to narrow down the broad field of potential microbes to make a positive identification.
Materials and Methods
To help narrow the field of potential microbes, a dichotomous key was created. The flow chart created a systematic approach to eliminating microbes according to biochemical test results. It is assumed that the single microbe left after conducting all necessary tests in the dichotomous is the correct microbe in question and a positive identification can be made.
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To start, a dichotomous key was created based on preexisting data about known organisms, particularly metabolic characteristics. A copy of the created key can be seen attached to the end of the report. The tests performed came from a list of known characteristics of several different types of bacteria. The tests were aligned onto the dichotomous key in a way that each test would divide the remaining group in bacteria in half. A complete list of the tests performed, as well as, basic information about each test can be seen below.
Gram Stain- The first test conducted to identify the bacteria. Following protocol already described in previous lab experiments, the specimens were dyed according to procedure. This helped differentiate the bacteria, and ultimately cut the list of potential candidates in half.
Catalase Test- This tests for the bacteria's ability to produce catalase enzymes to combat against potentially hazardous oxygen radicals. To perform the test, a swab of culture is placed onto a slide. A few drops of hydrogen peroxide are then placed on the slide. If bubbles are noted then the bacteria is positive for catalase production.
Oxidase Test- This tests for the bacteria's presence of cytochrome C oxidase. To test the bacteria, either a swab or test strip (Oxy-Swab) is placed in contact with the bacteria. If a purple color change is noted on the swab or test strip, then the bacteria is positive of containing cytochrome C oxidase.
Glucose Fermentation- Like the name suggests, this experiment tests for the bacteria's ability to ferment glucose as a carbon source. To test the bacteria, a sample of culture is placed into the test tube and then incubated. A positive test result is evident by a color change from red to yellow. Any gas production will be evident by gas bubbles being noted in the small, inserted tube.
Lactose Fermentation- Again like the glucose fermentation test, lactose fermentation tests for the bacteria's ability to ferment lactose. The culture is placed into the liquid media and then incubated. A positive test result is evident by a color change in the media.
Casein Test- This test tests for the ability of bacteria to break down casein, a protein found in milk. The culture is simply inoculated onto a plate containing casein. Following incubation, if the plate shows any clearing around where the plate was inoculated then the bacteria is positive for its ability to break down casein.
Citrate Test- This test tests for the bacteria's ability to utilize citrate as a carbon source. Cultures are inoculated onto the slant and incubated. If the test is positive then the slant will turn from blue to green, indicating citrate utilization.
Mixture of Two Unknowns
Initially, a mixture of 2 unknown microorganisms was obtained. The ultimate goal was to first separate the 2 into pure cultures, and then by following the flow chart on the dichotomous key eliminate microorganisms until ultimately 2 microbes were left.
To start, the unknown vial #5 was obtained. The mixture was streaked onto a TSA media plate, in order to obtain a pure culture. Following a 48 hour incubation period, two distinct types of colonies were noted on the plate. Organism A had a distinct yellow color to the colonies, while organism B was noted as having a red coloration.
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Next, in keeping in following with the dichotomous key, a Gram stain was performed on both organisms. After several repeated Gram stains, it was ultimately decided that both Organism A and Organism B were Gram negative bacteria.
Next, according to the dichotomous key, a lactose fermentation test was performed to test for the bacteria's ability to ferment lactose, a key metabolic process. The test was conducted in accordance with the procedure described above. In addition to the procedure, a tube was inserted into each of the test tube to test for gas production. Upon reviewing the test results, Organism A was positive for lactose fermentation as evident by the change in media from red to yellow. However, the organism was negative for gas production, with no air bubbles being seen in the smaller, inserted tube. Organism B was negative for lactose fermentation, with no change in the coloration of the initially red media.
The next step to indentify Organism A was to conduct a citrate test. Again, according to protocol, the citrate test was conducted. After a 48 hour incubation period it was shown that the test was negative for citrate utilization, as evident by the failure of the media to turn from the initial blue color. Based on this it was ultimately determined that Organism A was E. coli.
To continue the identification of Organism B an oxidase test was performed. In accordance with laboratory protocol, the test strip was inserted into the plate, and allowed time to change color. Ultimately, it was shown that the bacteria were oxidase positive, as evident by the purple coloration seen on the test strip. Based on the test results, it was concluded that Organism B was P. capacia.
Identification of Second Unknown
The main difference between the identification of the first unknown and the second unknown was the necessity for the first unknown to isolate 2 distinct colonies. For the identification of the second unknown, there was only 1 bacteria present, therefore no isolation was needed. Still, initially the unknown (unknown # 111) was streaked unto a TSA plate to obtain a pure culture.
Like in the first portion of the lab, a Gram stain was performed to help distinguish the bacteria. Following completion of the Gram stain, the bacteria was observed to be Gram positive rods.
Following the flow chart, the next test to be performed for Gram positive bacteria was the glucose fermentation test. The bacteria tested positive for glucose fermentation, with gas production being noted.
The catalase test was then performed next on the bacteria. The bacteria tested positive for catalase as evident by the formation of bubbles after hydrogen peroxide was applied to a sample of the bacteria. Based on the results found after this test, it was ultimately concluded that the unknown bacteria was B. subtilis.
It must be noted that in identifying the first set of unknown organisms, human error occurred during the Gram staining process. It was later discovered that the decolorizer had been applied for too long and could have resulted in a false negative result. Nevertheless, the data obtained from Gram stain was initially treated as being accurate
All three of the identified bacteria are relatively common bacteria which inhabit a wide variety of environments. They are all fairly harmless unless they come into contact with an immunocompromised individual, in which they can prove to be fatal.
Bacillus subtilis is a relatively common bacteria that has been isolated from numerous sources, including soil, water, and air. The bacteria have the ability to produce endospores which aid in protection when adverse conditions arise. The endospores allow the bacteria to revert to a hibernation-like state, in which the bacteria remain, until conditions improve, and they resort back to a vegetative state.
Escherichia coli is a Gram negative rod bacteria. This common bacteria has been found to inhabit numerous environments. It is a common bacteria of the human intestinal tract, and is important in food digestion. While most strains pose no immediate threat to humans, there are however some strains which pose a significant threat of food poisoning if a large enough quantity of bacteria are ingested.
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Pseudomonas capacia is a Gram negative bacteria that is common throughout the world. It is typically found as part of the normal flora of the skin. It is normally not associated with disease in humans, however, some strains have been noted as beginning to show resistance to antimicrobials. This is of particular concern to immune compromised individuals in which horrible infections can result.