Bacteria identification through biochemical tests is a common laboratory procedure for unknown substances. Biochemical tests help in stepwise identification of bacteria, helping in sampling and categorization of unknown samples into known bacteria (MacFaddin, 2000). This laboratory exercise involved identification of two unknown bacteria using a stepwise biochemical tests. The findings from the tests indicates that unknown substance 1 is Salmonella T., and unknown substance 2 is Staphylococccus E. The results of the two unknown substances for the various biochemical tests are as indicated in table 1 below.
The first biochemical test to be conducted was the gram stein. This entailed first applying methanol to the prepared slide. The second procedure was adding the primary stain – crystal violet, which stained all the cells purple. The next procedure was application of the mordant. Iodine solution as used as the mordant in this experiment, which helped in forming the violet-iodine (CV-I) complex (MacFaddin, 2000). Decolorization was the next step and involved using acetone to discolor the cells. Counterstaining using safranin was the next step in the gram stein test and helped in revealing two types of bacteria – one that was gram positive whereas the other was gram negative. This was evident through color changes, where the gram-positive bacteria maintained the purple color whereas the gram-negative cells changed to a pink color. From the findings of the gram stein test, the outcome of the dichotomous key, and considering that unknown substance 1 has a negative gram stain, the most ideal bacteria for unknown substance 1 include E. Coli, E. Aerogenes, K. Pneumoniae, A. Faealis, P. Aeruginosa, Salmonella T., P. Vulgaris, S. Flexneri.
After conducting the gram stein, the next step was conducting the MR test, which helped in dividing the possible bacteria into two groups, those that turned positive, and the ones that turned negative. The MR test first involved adding five drops of Methyl Red to the incubated solution. The test gave a consistent yellow color indicating negative results (MacFaddin, 2000). From the findings of the test, and considering that the unknown substance turns negative for the MR test, it indicated that the possible bacteria for the unknown substance are K. Pneumoniae, A. Faealis, P. Aeruginosa, S. Flexneri.
The unknown substance 1 could be K. Pneumoniae, A. since it turns positive for sucrose test, but it is definitely not K. Pneumoniae because the unknown substance turns negative for Urease test whereas K. Pneumoniae turns positive. As such, according to the dichotomous key, K. Pneumoniae is ruled out as being a possible candidate for the unknown substance. For the second section of the tree, both A. Faealis and S. Flexneri do not qualify to be the unknown substance because the unknown tests negative for nitrate whereas A. Faealis tests positive for nitrate. On the other hand, S. Flexneri is not the unknown substance because it tests negative for VP test whereas the unknown substance tests positive for VP test.
From the dichotomous key and the assessment above, the best course of testing for the identification of the unknown substance is the third. This entailed conducting the sorbitol test for the remaining bacteria. The sorbitol fermentation involved using the phenol red broth medium. After selecting the inoculating loop tool, the loop was flamed to sterilize it (MacFaddin, 2000). The caps were removed from the test tubes and the mouth of the tubes was flamed. The inoculum was then inserted into the sterile tubes using the inoculating tool. The inoculum was transferred into fresh sterile medium before the mouth of the tubes was flamed again. The caps were then replaced on the test tubes. The tubes were then incubated at 35-37o C (MacFaddin, 2000). The substance turned positive for the test, which was manifest by showing a yellow color. Considering the outcome of the sorbitol test for the four bacteria, as well as considering the categorization of the dichotomous key, indicated that the possible bacteria for the unknown substance are E. Coli, K. Pneumoniae, and Salmonella T.
After conducting the Sorbitol test, the next test that could be used to separate the two possible bacteria was the Indole test. This involved using 4 ml of tryptophan broth. The tube was inoculated aseptically before being incubated at 37o C for 28 hours (MacFaddin, 2000). The test then involved adding 0.5 ml Kovac reagent to the broth culture before observing for the presence of a ring. The test showed no change in color with formation of a small ring indicating negative results. The unknown substance turned negative for the Indole test, which indicated that the possible bacteria for the unknown substance were K. Pneumoniae, and Salmonella T.
In order to separate the two bacteria and identify the unknown substance, the ornithine test was conducted. This involved using the ornithine-decarboxylase broth medium and then sterilizing the inoculating loop as well as the mouths of the test tubes by flaming (MacFaddin, 2000). The next step was adding 2-3 drops of Vaselin oil before replacing the caps of the test tube; the tube was then incubated at 37o C for 24 hours. The results indicated a purple color after the incubation, showing positive results. The findings of the test showed that the unknown substance turned positive for the test, indicating that the unknown substance was Salmonella T.
Unknown Bacteria 2
The gram stein test indicated positive and negative bacteria in the culture. Considering that unknown substance 1 turned negative for the gram stein, then it followed that unknown substance 2 turned positive. From the findings of the dichotomous key, and considering that unknown substance 2 had a positive gram stain, the possible bacteria include Bacillus subtilis, Bacillus Cereus, Staphylococccus A, and Staphylococccus E.
In order to separate these bacteria, the mannitol test was used. This entailed using the phenol red mannitol broth medium, which was placed in a sterilized test tube. The inoculating loop and the mouth of the test tube was then sterilized using flaming. After closing the mouth of the test tube, the tubes were incubated at 35-37o C for 24 hours (MacFaddin, 2000). The results of the test indicated a pink color after the incubation, indicating a negative result. Considering that the unknown substance is negative for mannitol test, the possible bacteria for the unknown substance include Bacillus subtilis, and Staphylococccus E.
In order to separate these two remaining bacteria, the VP test is used. The VP test entailed first allowing the medium to equilibrate at room temperature before inoculating the medium. The medium was then aerobically incubated at 37o C for 24 hours (MacFaddin, 2000). The next step was to aliquot 2 ml to a test tube before incubating the remaining broth for 24 hours. The next step was adding 6 drops of 5% alpha-naphthol before mixing well for aeration. The next step was adding 2 drops of 40% potassium hydroxide and then mixing well for further aeration. The tube was then shaken vigorously for at least 30 minutes (MacFaddin, 2000). The outcome of the test indicated a yellowish color or generally lack of change in color indicating a negative result. Considering that the unknown substance tests negative for VP test, it follows that the unknown substance is Staphylococccus E.
In conclusion, it is evident that strategic and stepwise use of biochemical tests is a sure way of separating bacteria as well as identification of unknown bacteria. Understandings the required outcome of a test for positive and negative results, as well as following the outcome of a dichotomous key are essential in deciding the identity of an unknown substance. For this particular exercise, use of several tests revealed that the unknown substance 1 was Salmonella T. and unknown substance 2 was Staphylococccus E. Although a hectic process, this experiment helped in understanding how well biochemical tests need to be conducted for effective results.