Table 1. Biochemical Test Results and characteristics observed for Kocuria rhizophila (=Micrococcus luteus), Culture G.

Table 2. Biochemical Test Results and characteristics observed from Shigella flexneri, Culture 5.

Discussion

Culture G:

First, culture G was gram stained to identify if the bacteria had a thick or thin cell wall which narrows down possibilities of bacteria. After gram staining the culture appeared purple, indicating a positive thick cell wall. The culture also appeared to form irregular clusters with bacteria in a coccus shape. The culture therefore had to be one of the following bacteria: Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Corynebacterium pseudodiphtheriticum, Enterococcus faecalis (=Streptococcus faecalis), Kocuria rhizophila (=Micrococcus luteus), Kocuria rosea (=Micrococcus roseus), Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus lactis (Lactococcus lactis), Streptococcus pneumoniae, Streptococcus pyogenes.

Second, to isolate culture G and create working and backup cultures a tryptic soy agar plate was streak plated and incubated. After incubation cultures appeared yellow. Next, a fresh tryptic soy broth culture was made to be used as a working culture and a tryptic soy agar slant was prepared for long term storage by using an isolated colony from the agar plate. The agar plate, slant, and original culture were then stored to be used as backups in case of contamination.

Third, a Voges-Proskauer test was done to identify if culture G processes glucose to produce 2,3-butanediol and to further limit possible bacteria. When acetoin (a precursor of 2,3-butendiol) reacts with Barritt’s reagent it produces a red color. The result produced no red color, this is indicative of a negative result. This limited the possibilities of the bacteria to the following: Bacillus megaterium, Corynebacterium pseudodiphtheriticum, Kocuria rhizophila (=Micrococcus luteus), Kocuria rosea (=Micrococcus roseus), Streptococcus pneumoniae, Streptococcus pyogenes.

Forth, due to the slight yellow hue color of culture G on a tryptic soy agar plate a motility test was then preformed to either confirm or rule out Bacillus megaterium. After stab inoculating a SIM agar tube and incubating bacteria only grew on the very edges of the inoculation site, indicating a nonmotile bacteria. This indicated that the culture G was not Bacillus megaterium. This limited the possibilities of the bacteria to the following: Corynebacterium pseudodiphtheriticum, Kocuria rhizophila (=Micrococcus luteus), Kocuria rosea (=Micrococcus roseus), Streptococcus pneumoniae, Streptococcus pyogenes.

Fifth, a triple sugar iron test was preformed to characterize culture G’s acid production from glucose, lactose, and sucrose. As sugars are fermented to acids the pH indicator in the agar changes colors from red to yellow due to a change in pH. After incubation culture G’s tube did not change at all, to ensure that the test was successfully inoculated it was left in the incubator until visible bacteria colonies appeared on top of the surface of the agar. After 2 days the tube was covered in a film of bacteria. This indicated that the tube was successfully inoculated, and changes should have been observed earlier. The test was then repeated to ensure that the results were accurate, the same results were observed. As the butt of the TSI test remained red this indicated that glucose is not fermented to an acid by culture G. This limited the possibilities of the bacteria to the following: Corynebacterium pseudodiphtheriticum, Kocuria rhizophila (=Micrococcus luteus), Kocuria rosea (=Micrococcus roseus).

**As culture G is coccus it must not be Corynebacterium pseudodiphtheriticum which is a rod. As culture G was yellow on agar it must not be Kocuria rosea (=Micrococcus roseus) which is red on agar. Therefore, culture G must be Kocuria rhizophila (=Micrococcus luteus), which is both coccus and yellow on agar. To confirm culture G’s identity further tests were completed.

Sixth, a citrate test was preformed to identify the use of citrate as a sole carbon source. When citrate is used by the bacteria the test turns blue due to an increased pH caused from the reaction of CO2 produced from the bacteria and excess sodium citrate in the agar. After incubation the test remained green, indicating that culture G could not utilize citrate.

Seventh, an Indole test was preformed to determine if culture G could process tryptophan. When tryptophan is processed by the bacteria the metabolite indole is produced. When indole reacts with Kovacs reagent it turns red, indicated indole within the system. After adding Kovacs reagent no red color appeared. This indicates a negative result.

Eighth, a methyl red test was preformed to determine if culture G was a mixed acid fermenter or a butanediol fermenter. Mixed acid fermenters produce more acid’s resulting in a more acidic broth, butanediol fermenters don’t acidify the broth as much due to the production of byproducts other than acids. The if the pH of the media is very low methyl red will stay red, if it is less acidic then the methyl red will turn translucent. After adding methyl red to the test, the methyl red instantly turned translucent, indicating a negative methyl red test.

Ninth, an oxidase test was preformed to determine if culture G posse’s cytochrome oxidase. This enzyme is used to reduce O2 and allow it to be used as the final electron acceptor. In this test a clump of bacteria is placed onto an oxidase test strip. This strip contains a molecule which when reduced it turns blue, and it can only be reduced by cytochrome oxidase. After adding a clump of bacteria onto this strip the color turned from white to blue, indicating that culture G posse’s cytochrome oxidase and is oxidase positive.