To determine that he stock culture was pure, we performed a streak plate using loops of the stock broth on a fresh dish of nutrient agar and then incubated it for 48 hours at Standard Temperature and Pressure; ICC, 1 ATM (STEP). A Gram stain was then carried out to differentiate the unknown sample from a broad class to a more specific category of bacteria. The Gram stain distinguishes between Gram-positive and Gram-negative bacteria based on the composition of the cell walls. Gram-positive bacteria appear purple and Aggregative bacteria appear pink after staining.
The first Gram stain produced unsatisfactory exults and was then repeated with a clear indication of negativity. Light pink staining was evident on the cells in the field of view (OFF) and a search of the slide revealed uniformity in the sample. From these results we concluded that unknown 16 was Gram-negative in nature, and therefore it could possibly be any of the following bacteria: Alignments fiscals, Escherichia coli, Entertainer arrogates, Protest miracles, Protest vulgarism, or Pseudopodia fluorescent.
We then conducted a negative stain to reveal the shape of the cell and any extracurricular features such as the presence of a capsule. The first negative stain was unsuccessful and was therefore repeated. The second stain yielded Cisco and sociability shaped cells occurring singly and also arranged in irregular clusters. These appeared somewhat sparsely distributed at xx with oil immersion. A simple stain was then performed because previously in lab, the negative stain made it hard to visualize the bacteria.
The simple stain was prepared using a sample of bacteria from the 24 hour old broth culture. The appearance of the microbe was again cuscus and sociability in shape, yet clusters were grouped n greater numbers, and their concentration was higher for the same OFF. From the results obtained by the negative and simple stain, we assumed that the unknown sample might be Alignments fiscals, due to the shape of the cells and their clustered pattern. 3/17/05 After 48 hours of incubation, the results of the streak plate were particularly successful.
This revealed moist, colorless, and opaque streaks of colonies the size of pinpricks in quadrant one, diminishing to one 3 mm round colony in the fourth quadrant that had billowing or irregular edges. All colonies appeared to e the same color, size, and shape. We concluded that the stock broth culture was pure. A Potassium Hydroxyl (KOCH) Gram test was then performed to confirm the results of our Gram stain and to assure that our bacteria was Gram negative. The KOCH test revealed an extremely fine filament which adhered to a toothpick and stretched three to five mm before breaking from tension.
This was first performed with a single loop from the streak plate prepared on 3/15/05 with positive results obtained, and repeated with a more generous smear of five loops. The larger smear of bacteria when mixed with KOCH turned nearly opaque ND produced a more prominent filament that again retained adhesion to the toothpick for three to five mm before breaking. Based on the results of the Gram stain and the KOCH Gram test we concluded that our unknown sample was a Gram-negative. To determine if our unknown sample was Alignments fiscals, we decided to perform an oxides test.
Some gram-negative species of bacteria are oxides positive while others, such as those in the Interchangeable family, are oxides negative, so we prepared a tropic soy agar plate and made a single streak-line inoculation using the 72 hour old stock broth culture. We incubated this plate for 24 hours. 3/1 8/05 After the tropic soy agar plate had incubated, we applied a KEY oxides Test Strip to the surface of the agar plate, covering the colony formed from the streak- line inoculation. The KEY oxides Test Strip did not show color change from pink to dark purple.
This test revealed that the unknown sample was gastrointestinal and therefore unlikely to be Alignments fiscals. This led us to believe that our unknown may be Escherichia coli, Entertainer arrogates, Protest miracles, or Protest vulgarism. See figure 2. 3/22/05 To differentiate between Escherichia coli, Entertainer arrogates, Protest miracles, and Protest vulgarism we decided to test carbohydrate fermentation pathways of the unknown sample. We inoculated three Durham tubes containing glucose, sucrose, and lactose, with a loop of our stock broth culture that was 192 hours old and incubated the tubes for 48 hours.
We then prepared a TOGA shake tube to determine the unknown sample’s oxygen requirements. We inoculated the TOGA shake tube with the stock broth culture and incubated this for 48 hours. /24/05 The results of the TOGA shake tube showed consistent growth of the unknown specimen throughout the tube. We concluded that the bacterial sample was a facultative anaerobe because it grew at the bottom of the shake tube in the absence of oxygen and also toward the top of the tube where oxygen was present.
These results further supported our claim that our unknown was not Alignments fiscals because it is an obligate arroba and also it eliminated the possibility of our unknown being Pseudopodia fluorescent because it too is an obligate arroba. The results of our Durham tube test showed that the unknown sample was able to utilize various carbohydrate fermentation pathways. All three test tubes containing Glucose, Sucrose, and Lactose were positive for the presence of acid and gas production.
A positive result was determined by the color change of the medium from red to yellow and the presence of a gas bubble within the Durham tube. From these results we concluded that our unknown sample could not be Protest miracles or Protest vulgarism due to the fact that neither of these bacteria is able to ferment lactose. We determined that the broth culture specimen was either Escherichia coli or Entertainer arrogates because they both are facultative anaerobes that are able to ferment Glucose, Sucrose, and Lactose.
To differentiate between Escherichia coli and Entertainer arrogates we decided to run the Movie tests. For the indolent production test, we inoculated a SIMI deep tube with a loop of stock broth culture that was 240 hours old and incubated it for 24 hours. For the Methyl Red and Vogues-Prosperous Test, we inoculated a MR..- UP broth media tube with the stock broth culture and again incubated it for 24 ours. For the Citrate Utilization Test, we inoculated a Simmons citrate agar slant using a stab-and-streak method and incubated the tube for 24 hours. /25/05 After completing the indolent production test, no color change from colorless or light yellow to deep red was observed therefore a negative result was obtained. As for the Methyl Red Test, a positive result was recorded because a red color change occurred. The Vogues-Prosperous test revealed a negative reaction because no color change occurred and also the citrate utilization test was negative cause the tube remained green in color and did not turn blue. From the results of the Movie Tests we concluded that unknown specimen 16 was Escherichia coli.
From previous tests conducted in lab, it was known that Entertainer arrogates will produce a negative methyl red test, a positive Vogues- Prosperous test, and a positive citrate utilization test. Just the opposite result is true for Escherichia coli and our Movie tests supported these findings. Overall the final conclusion that our unknown sample was in fact Escherichia coli was determined by examining all of our data from the Gram stain, KOCH test, oxides test, oxygen requirement test, carbohydrate fermentation, and the Movie tests to further support our claim.