Unknown Bacteria Lab Report

A. Introduction:

The purpose of this laboratory is to learn about cultural, morphological, and biochemical characteristics that are used in identifying bacterial isolates. Besides identifying the unknown culture, students also gain an understanding of the process of identification and the techniques and theory behind the process. Experiments such as gram stain, negative stain, endospore and other important tests in identifying unknown bacteria are performed. Various chemical tests were done and the results were carefully determined to identify the unknown bacteria. First session of lab started of by the selection of an unknown bacterium then inoculations of 2 tryptic soy gar (TSA) slants, 1 nutrient broth (TSB), 1 nutrient gelatin deep, 1 motility

agar deep (semi-solid medium), 1 FTM tube, 1 TSA plate. The next lab session we examined the results of our inoculations and began the next set of inoculations which included, 1 glucose tube, 1 lactose tube, 1 mannitol tube, 2 MR-VP broth tubes, 1 Simmon’s citrate tube, 1 nitrate broth tube, 2 O/F glucose (one with oil one without), 1 starch agar plate, 1 skim milk agar plate, 1 spirit blue agar plate, 1 urease slant, and 1 phenylalanine agar. During the next few weeks I began to examine all my test results and compared with the lab manual’s charts to narrow down my options and determine the identity of my unknown bacterium.
B. Materials and Methods:
All materials and methods used in this laboratory are detailed in the BIO 2710 Microbiology Laboratory Manual.
C. Results:
The results from the lab exercises are presented in the attached Descriptive Chart. Also attached is a flow chart, of the main tests/experiments that helped me determine my specie’s identity. The main experiments included the gram stain, which determined whether my bacterium was a positive or negative stain, and then the primary tests were nitrate, motility, glucose, citrate, and FTM. Unknown letter V was Micrococcus luteus, gram-positive cocci, grew optimally at 25 and formed round yellow colonies. Growth of the FTM tube was consistent with the O/F glucose test (obligate aerobe). It was non-motile, the nitrate test was negative as were the glucose and citrate. My results all supported my conclusion; some students also swapped my unknown bacteria to use it for various test, since they suspected we had the same bacterium but some of their test results did not support their suspicion and they wanted to make sure that their bacteria were not just contaminated.
D. Discussion:
The identity of unknown letter V was Micrococcus luteus
I began by choosing unknown letter V and inoculating two tryptic soy agar (TSA) slants and incubating both of the slants at 25 °C, because my professor instructed me to do so, instead of incubating one slant at 37 °C and one at 25 °C.

I also inoculated a tryptic soy broth (TSB), a nutrient gelatin deep, a motility agar deep, a fluid thioglycollate medium (FTM) tube, and a TSA plate with my unknown culture. All of these inoculated media were incubated until the next class period (about 48 hours). Then when I came to class most of my inoculated tubes and my streak plate appeared to have growth. The next step I took was making a gram stain to determine the gram reaction and cellular morphology of my unknown. I used my working slant to do this, after careful examination of the gram stain, I learned that my unknown was a gram-positive bacterium. I then preceded by making a negative stain to see the size of the cells of my unknown bacteria. The cell shape was cocci and the cells occurred in clusters of tetrads. After discovering that my unknown bacteria was gram-positive cocci, I turned to page 207 of the lab manual to narrow down my options, there was only four out of the gram-positive list that were

cocci. After doing the gram stain, I looked at the flow chart (figure 42.1) on page 194 of the laboratory manual, which demonstrated the need to do the spore stain for my specific unknown. I then discovered that my bacterium is non-spore forming and since the cell’s shape was cocci and not rods there was no need to do the acid-fast stain. In the next class period, I removed my inoculations from the incubator and studied them. The TSA plate had significant growth that looked yellow, round, smooth. The TSB tube was turbid with some sediment on the bottom. The agar slant also had growth that was white and spread out. Next I decided to figure out the oxygen requirements. The FTM tube looked like my unknown bacterium was obligate aerobe. To double check this, I used the O/F glucose tests to see if my unknown bacteria can grow by respiratory means if oxygen is present and if it can also switch metabolic gears in anaerobic conditions and grow by fermentation. The results of the O/F glucose test showed that my unknown bacterium is an obligate aerobe. After seeing the results of the O/F glucose tests, I carried out specific fermentation tests: Durham tube sugar fermentations, mixed- acid fermentation (methyl red test), 2,3- butanediol fermentation (Voges-Proskauer test), litmus milk, and the citrate test. I inoculated the Durham tubes of glucose, lactose, and mannitol with my unknown organism and all three sugars did not produce gas. The mixed acid fermentation test showed negative results for my unknown. The test for 2,3 butanediol fermentation had positive results. The litmus milk test should no change. The citrate test indicated that my unknown exhibits no citrate utilization. At this point, I turned to figure 39.13 on page 208 of the laboratory manual and followed the flow chart down with my results from the different tests. This told me that my unknown was most likely Micrococcus Luteus. All the tests that I had done so far all supported that my unknown bacterium was Micrococcus luteus. I decided to do the Nitrate test to be sure that it was Micrococcus luteus and not Micrococcus roseus since they are very similar. The result of the Nitrate test was negative which then confirmed that my unknown bacterium was Micrococcus luteus and not Micrococcus roseus. After obtaining all my results, I referred to the Bergey’s Manual of systematic Bacteriology and turned to tables 12.3-12.4 on page1006-1007. I compared all the test results I obtained and found that the table in the manual also supported my conclusion that my unknown bacteria was Micrococcus luteus.