Materials and Methods
Gram Stain: the test was done to differentiate Gram-positive from Gram-negative organisms. A drop of the unknown 300 was suspended on a slide and the culture spread to an oven thin film. The culture was air dried and fixed over a gentle flame. Crystal violet stain was added over the culture and allowed to stand for 60 seconds. The stain was poured off and rinsed gently. Iodine solution was added on the smear, allowed to stand for 60 seconds and the stain poured off. The slide was rinsed with running water, and a few drops of decolorizer solution added. Rinsing was done after 5 seconds and counterstained using basic fuchsin solution for 60 seconds. Gram-positive cells retain the violet-iodine complex formed during staining and thus do not decolorize when ethanol is added. The Gram-positive organisms will show purple or blue stains while Gram-negative cells show pink to red color.
Carbohydrate fermentation: A culture tube containing a defined liquid medium with either glucose or lactose as a sole carbohydrate source was inoculated with a pure culture of unknown 300. The broth contained the pH indicator phenol red (PR), which changes from red in alkaline conditions to yellow in acidic conditions. This color change was used to detect acid formation from fermentation of the carbohydrate during bacterial growth. The tube also contained an inverted Durham tube filled with the broth. This tube was used to capture any gas formed during the fermentation process.
Catalase Test: The test was conducted by removing a small amount of the unknown 300 from the agar slant from a broth culture and placed on a glass slide. The organism was mixed with a drop of 3% H2O2, and the appearance of gas bubbles checked as the positive indicator of the test.
Motility Test: A tube of motility medium using the inoculating needle rather than the loop was inoculated. The needle was flame sterilized, and when cool, the cells were transferred onto the very tip. The motility medium was stabbed to about two third of its depth and the needle withdrawn straight out using the same path that was used to go in. The needle was sterilized and incubation of the tube done for 48 hours. The test was positive when there was red cloudiness around the stab pathway. Lack of cloudiness outside the area was an indication of negative results.
Starch Hydrolysis: The test was done by inoculating a single starch-gelatin agar plate together with a small quantity of the unknown 300. Incubation of the plate was done for 48 hours at room temperature and then refrigerated. A few drops of Gram’s iodide were added. Areas on the plate that contained starch formed a dark blue or purple complex while areas around the colonies where the starch had been hydrolyzed appeared as clear zones. A clear zone around the test organism was thus an indication of a positive test.
Ammonia from urea Test: Using urea’s broth that was composed of yeast extract urea as well as pH indicator phenol red, incubation of the tube was done in the broth and incubated for 48 hours at room temperature. If the urease was present, ammonia was released, and there was a rise in pH. A positive urease test was thus indicated by color change from yellow to cerise, and no color change was an indication of a negative test.
Indole from tryptophan: The indole production test was done to determine the production of indole from tryptophan by the microbes. The test was done using tryptone broth where Kovac’s reagent was then added. There was a production of a cherry-red ring that is floating on top of the culture medium when the test is positive or a yellow ring when the test is negative.
H2S production: The test was done by inoculating a tube of Kleigler’s iron agar with some of the test organism using the inoculating needle. The stab was made to two third of the way into the agar and incubated for room temperature for 48 hours. A positive test was shown by a dark precipitate that formed in the tube while the absence of the precipitate was an indication of negative test.
Casein hydrolysis: the test is done to determine whether an organism produces exoenzyme casesase. A milk agar plate was inoculated with the unknown 300 and incubated for 48 hours. Agar clearance around the growth indicates casein hydrolysis (Rossbach, 2012).
The culture showed a pink color. The morphological test showed the bacteria to be rod shaped.
Carbohydrate fermentation: Growing the unknown 300 in lactose broth did not change in color. Gas was captured in the Durham tube during lactose fermentation. Growing the unknown 300 in sucrose broth resulted to a change to yellow color. Gas was captured in the Durham tube during sucrose fermentation.
H2S production: Inoculating the unknown 300 culture in a tube of Kleigler’s iron agar resulted to a black precipitate formation.
Indole from tryptophan: Adding tryptone broth and Kovac’s reagent to the unknown 300 resulted in the production of a cherry-red ring that was floating on top of the culture medium.
Ammonia from urea Test: Using urea’s broth that was composed of yeast extract urea as well as pH indicator phenol red, incubation of the tube in the broth and incubated for 48 hours at room temperature resulted to a change in color from yellow to purple.
Starch Hydrolysis: Inoculating a starch-gelatin agar plate together with a small amount of the unknown 300 and incubating for 48 hours at room temperature and then refrigerated, and staining did not produce a clear zone around the test organism.
Casein hydrolysis: Inoculating a milk agar plate with the unknown 300 and incubated for 48 hours resulted into a clear zone around the growth.
Catalase Test: Removing a small amount of the unknown 300 from the agar slant, placing it on a glass slide and mixing with a drop of 3% H2O2 resulted in bubbling.
Motility Test: Testing for motility did not result into a red cloudiness around the stab pathway. These results were recorded in Table 1 below.
The culture showed a pink color, and this was an indication that the unknown 300 was a Gram negative bacterium. The morphological test showed the bacteria to be rod shaped. The unknown 300 did not show change in color, and there was no gas production, therefore, no lactose fermentation. However, growing the unknown 300 in a sucrose broth produced gas and changed the color to yellow. This indicated that unknown 300 ferments sucrose and gas was produced during fermentation.
Unknown 300 appears to be a H2S producer. Formation of a black precipitate indicated hydrogen sulfide production from deamination of sulfur containing amino acid such as cysteine. The unknown 300 was also able to produce indole from tryptophan by the microbes. This was indicated by the production of a cherry-red ring that was floating on top of the culture medium in the test to check for indole production from tryptophan. The organism also has the capability to produce urease enzyme. Urease enzyme was responsible for the production of ammonia from urea, and the released ammonia resulted in the rise in pH rise causing the color of the medium to change from yellow to purple.
The unknown 300 had the capability to produce catalase enzyme that acted on the 3% H2O2 resulting in the production of oxygen and water. Oxygen gas that was produced was responsible for the bubbling that was witnessed. Lack of red cloudiness around the stab pathway indicated that unknown 300 was not motile.
In conclusion, the unknown 300 appears to be Proteus vulgaris. This is as a result of the perfect match that was obtained between the results obtained from the tests and the ones given in the chart in Figure 1 below.
Figure 1: Results and observation chart
Rossbach, S. (2012). Microbiology Lab Procedures. Retrieved October 6, 2013, from Western Michigan University: http://homepages.wmich.edu/~rossbach/bios312/LabProcedures.html