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Unknown Bacteria Microbiology

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Bacteria are microscopic unicellular prokaryotic organisms characterized by the lack of a membrane-bound nucleus and membrane-bound organelles. They are remarkably adaptable to diverse environmental conditions and are found in bodies of all living organisms and on all parts of the earth. The purpose of microbial biochemical tests is to identify the unique traits it yields and with that knowledge we can then categorize them in groups and specify them by scientific name. These experiments included the Triple-sugar iron agar (TSIA), Sulfur Indole Motility (SIM), Methyl Red (MR), Voges-Proskauer (VP), Citrate, Urease, Gelatin, and Oxidase Test. In order for these tests to produce reliable and credible results, the bacterium organism must be grown using strict and meticulous procedure to produce viable colonies of pure culture. Having pure culture is significant to ensure that a single type of bacteria is used for identification without contamination so tests can be run without complications or confusion. Once all these tests are performed, the unknown bacteria in this lab will be one of the following: Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Klebsiella pneumoniae, or Salmonella typhimurium. This report included the results and details to these experiments which are discussed further on.

Gram negative bacteria Unknown #12 was run through an array of tests which produced positive and negative results. The results obtained from the various tests were used for the process of elimination with the use of our Gram Negative Unknown chart to conclude that unknown #12 was Proteus mirabilis. This report will include information regarding the methods and materials used as well as the discussion about the biochemical tests and the results.

Materials and Methods
The first step in the process of identifying the unknown bacteria was to transfer the bacteria from a random unknown broth medium test tube onto a TSA (Trypticase Soy Agar) plate using the T-streak method as well as aseptic methods to check for contamination and colony morphology. A TSA slant was also streaked at the top for back-up purposes incase the TSA plate became invalid due to reasons such as contamination of the TSA plate.
The second step was to inoculate the bacteria into each test. After inoculation, all media are placed in the hot room which is approximately 37¢ªC, and if placed it the cold room, the temperature is approximately 4¢ªC. Each test had a different technique of getting the end result and this part of the report identifies each of those methods as well as their inoculation times spent in their designated temperature rooms:
The TSIA Test: The triple-sugar iron agar media was used to indicate which sugar had been fermented by the organism depending on where the color change took place between the slant and the butt. This test also determined if the organism produced hydrogen sulfide and gas as by-products of fermentation. Phenol red was used as an indicator for this experiment and the bacterium is inoculated by the stab-streak method. The notation for stating whether the slant or the butt fermented are K for alkaline (red/pink), and A for acid (yellow). Notation of the change are K/K, K/A, and A/A with the first being the slant and the second being the butt respectively. Incubation will be checked after 6 hours and 24 hours of incubation for changes.
The SIM Test: The Sulfur Indole Motility media was used to conduct three simultaneous tests. The media was inoculated by stabbing the bacteria into the media. These tests were incubated for 24 hours in the hot room.
1) Sulfur reduction test: tested for hydrogen sulfide production. Black meant that the test was positive for hydrogen sulfide production.
2) Indole test: tested for indole production after several drops of Kovak¡¯s reagent was added. The color on top of the media was the color used to determined test result. Red meant it tested positive and brown meant that it was negative.
3) Motility: this test was determined by the growth around the stab area and by cloudiness on top of the media which was positive for motility.
The MR Test (mixed acid fermentation test): The Methyl Red liquid media was inoculated using the inoculating loop and incubated for 24 hours for a color change. Red meant that the test was positive for mixed acid fermentation, and yellow meant that it was negative.
The VP Test (butanediol fermentation test): The Voges-Proskauer test tested for the presence of butanediol. The liquid media was simply inoculated using the inoculating loop and incubated for 4 days before 18 drops of Barritt¡¯s A and Barritt¡¯s B were added. A pink/red color change meant that the test was positive for production of butanediol.
The Citrate Test (Simmons Citrate agar): The citrate test was used to detect if organisms could utilize citrate as a sole carbon source. The slant was inoculated by streaking the surface and no reagents were added to this experiment. A color change from green to Prussian blue meant that it was positive for utilization of citrate.
The Urease Test: The urease test tested for the presence of the urea hydrolyzing enzyme urease. The liquid media was simply inoculated using an inoculating loop and incubated for up to 8 days. A color change to cerise meant that it was positive for the presence of urease.
The Gelatin Test: The gelatin tested for organisms that produced the metabolic digestive enzyme, gelatinase. After inoculation of the liquid media, it was incubated in the hot room for up to 8 days. If gelatinase was produced, it hydrolyzed gelatin and produced liquefaction, gelatin would not return to solid state when cooled. If the media returned to solid after cooling in the cold room for 30 minutes, the test was negative for gelatinase production.
The Oxidase: The oxidize test tested for the presence of cytochrome oxidase. The unknown bacteria is inoculated onto a piece of filter paper and droplets of 1% oxidase reagent are added. The test was positive if the culture turned from yellow to purple. If the culture remained the same, it was negative for cytochrome oxidase production.

TSIA (K/A + H2S): After the inoculated bacteria were incubated in the hot room for 6 hours, both the slant and the butt were red (K/K) meaning there was no change. However, after 24 hours of incubation the bacteria resulted in a red slant with a yellow butt (K/A) meaning there was glucose fermentation only. There was also black precipitation meaning there was hydrogen sulfide production.

SIM (+): After incubation for 24 hours, the media color changed to black with a cloudy small brown tinted ring at the top due to hydrogen sulfide production. Several drops (4-5) of Kovak¡¯s reagent were added to the media; there was no color change on top of the media, thus, it was negative for indole production. There was also a white cloud around the area of the stab.

Methyl Red (+): After 24 hours of incubation, 3-4 drops of methyl red was added to the media, there was an instant change from yellow to red.

Voges-Proskauer (-): After 4 days of incubation, 18 drops of Barritt¡¯s A reagent was added, followed by the same amount of Barritt¡¯s B reagent. The tube was shaken and let stood for 30 minutes. After 30 minutes and even to 1 hour, it tested negative since there was no change.

Citrate (+): After 24 hours of incubation, a pronounced color changed from green to Prussian blue was visible towards the upper part of media.

Urease (+): After 2 day of incubation, there was a distinct color change to cerise.

Gelatin (+): After 8 days of incubation, the test tube was put back into a cold room to test for solidification. However, the media remained liquid.

Oxidase (-): After a few drops of 1% oxidase reagent was added to the bacteria on the filter paper, there was no color change.

H2S +
MR +
VP -

The TSIA Test: After 6 hours of incubation, the result turned out negative because it was
A/A (acid slant/acid butt) since it needed more time to ferment. After another 24 hours of incubation, it was positive for hydrogen sulfide production due to black precipitation, and it was K/A (alkaline slant/acid butt) meaning there was glucose fermentation only.
The SIM Test: After 24 hours of incubation, it was positive for hydrogen sulfide production because the media color changed to black. The result for indole production was negative due to the brown color on top of media. From the cloudiness that was visible around the stab, the test was positive for motility.
The Methyl Red Test: After 24 hours of incubation, the result was positive due to the color change to red after 3-4 drops of methyl red were added. This indicated the presence of acid; therefore, the organism was a mixed-acid fermenter.
The Voges-Proskauer Test: After 18 drops of Barritt¡¯s A and Barritt¡¯s B were added to the incubated media, there was no change, meaning it tested negative for butanediol production.
The Citrate Test: A positive result was determined after 24 hours of incubation due to the color change from green to Prussian blue. This means that the bacteria used citrate as a sole carbon source.
The Urease Test: The result clearly indicated a positive outcome for urease production since the media turned completely cerise after 1 day of incubation.
The Gelatin Test: The result was positive because it remained a liquid after placing it in the cold room after 8 days of incubation in the hot room. This meant that the enzyme gelatinase was produced inhibiting the resolidification.
The Oxidase Test: This test was negative for the production of cytochrome oxidase since there was no color change to purple after the 1% oxidase reagent was added.

These were all the experiments performed to indicate what the unknown #12 was. I have determined that unknown #12 is Proteus mirabilis. One of the main characteristics of Proteus mirabilis that set it apart from the other bacteria is the swarming morphology of the bacteria after it was inoculated onto the TSA media. The swarming effects and cloudiness around areas of stabs is due to the peritrichous flagella of the bacteria which enables it to be motile. Proteus mirabilis causes 90% of all Proteus infections. Since Proteus mirabilis produces urease, diseases that relate to this bacteria is due to high amounts of urease production which hydrolyzes urea to ammonia. The high levels of ammonia causes the urine to be immensely alkaline which can lead to formation of crystals of struvite, calcium carbonate, and/or apatites. Stones like these can grow if not treated with antibiotics which can eventually lead to renal failure (Wikipedia 2006).

Benson H.J. 2005. Microbiological Applications. Laboratory Manual in General
Microbiology. 9th Ed. Boston. McGraw-Hill.
"Proteus mirabilis." Wikipedia, The Free Encyclopedia. 30 July 2006, 09:07 UTC. 30 July
2006, 16:05 .

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