There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that I have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium.
The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were Each person in the class was given a heterogeneous broth mixture of 2 microbes.
This mixture was streaked onto the following plates: TSA, BAP, MAC, CNA some of which are selective for gram (+) or gram (-) microbes, while others aren’t specific. On the nonspecific TSA plate a large hazy white colonies was formed. On the other hand, there were also small orange colonies fewer in growth. Isolation onto a BAP would allow for a pure culture of just gram (+) which would be used to carry out the rest of the differential test. Isolation onto TSA/ MAC allowed for the growth of gram (-) and would be used for further test. Procedure:
The catalase test was performed only on gram (+) bacteria, as this test would not help in differentiating the gram (-) bacteria because all of the possible unknown gram (-) bacteria were catalase positive. This test is used to detect the presence of catalase, which helps to breakdown toxic hydrogen peroxide produced from the transport of high-energy electrons directly to oxygen. Catalase is tested for by adding hydrogen peroxide to the culture, and looking for the production of gas bubbles. If gas bubbles appear immediately, the culture is catalase positive.
However, if no bubbles are observed, the culture is negative for catalase. Results yielded positive. The citrate test was performed only on the gram (-) bacteria and was used to determine the ability of an organism to use citrate as its sole carbon source. Bacteria that possess citrate-permease are able to do this. The medium used for this test contains bromthymol blue dye, which is green at neutral pH, but blue at a basic pH. Bacteria that is able to survive and utilize the citrate, convert ammonium phosphate to ammonia and ammonium hydroxide, which alkalinize the agar, turning it blue.
Thus, the conversion of the medium to blue is a positive citrate test. No color change is negative. However, I believe that the citrate test had failed and should have been positive. There was no growth of bacteria in tube, it just didn’t seem to match up with what results I already had from acquired test. The urease test was performed on the gram (+) and (-) bacteria, testing for the breakdown of urea. Urea hydrolysis to ammonia by urease-positive organisms will overcome the buffer in the medium and change it from orange to fuchsia. Rapid urease-positive bacteria will turn the broth pink within 24 hours.
Urease-negative bacteria will produce no color change or turn the broth yellow. The results for both organisim yielded yellow which indicated negative reaction. A bacitracin test was performed on the gram (-) bacteria, testing for resistance and sensitivity towards antibiotic. If there is a zone of inhibition around the disc then that means the bacteria is sensitive to bacitracin. If there is not a clear zone around disc the bacteria is resistant to the antibiotic. After streaking the unknown S on to BAP plate 1 bacitracin disc was placed on the plate and incubated for 48 hrs.
After 48 hrs of incubation a zone of inhibition was formed around the bacitracin disc which indicated that the bacterium is sensitive to bacitracin. Nitrate reduction was tested for by inoculating a nitrate broth with the unknown gram (-) culture, and allowing growth to take place. Adding 2 drops of both sulfanilic acid and napththylamine to the medium if the first test to see if nitrite is present. If nitrite is present, the medium turns red, indicating a positive test.
However, if the medium does not change, a second test is performed to see if nitrite was further reduced. In this econd test, zinc powder is added to the broth to catalyze the reduction of any nitrate present to nitrite. If nitrate is present when the zinc is added the reduction of this compound will cause the medium to turn red, from the previously added reagents. Red medium on the second addition indicates nitrate was not reduced and a negative test result. However, if the medium does not change after the addition of the zinc, the unknown is positive for nitrate reduction, as the nitrite has just been further reduced, preventing its detection. The result that yielded was positive on the first step.
Triple Sugar Iron agar slant (TSI) was used to test for the fermentation of glucose and lactose, as well as the production of H2S. Gas production was also monitored, looking for fissures produced by production of gas during fermentation. The conversion of the originally red slant and butt of the agar to yellow indicates that glucose fermentation took place. H2S could have been produced by the reduction of thiosulfate contained in the media. Production of this compound will cause a reaction to occur with ferrous sulfate and will result in the production of a black precipitate.
Thus, the culture is positive for H2S production if a black precipitate is seen and negative if one is not observed. The yielding results after incubation was (A/A) for gram (-) bacteria and (K/K) for gram (+) bacteria, there were no black precipitate present in either tubes. Flow chart Unknown 126 Gram Stain (G+ Coccus) Catalase (positive) Positive Negative Staph. aureus Strep. pyogenes Staph. epidermidis Strep. aglactea Staph. saprophyticus Enterococcus faecalis Micrococcus oseus Strep pneumonia BAP Gamma Other Staph. Saprophyticus Staph. Aureus Micrococcus roseus Staph. Epidermidis TSI (k/k) Negative Positive Micrococcus roseus Staph. Saprphyticus Urease (Negative) / Bacitracin (S) Micrococcus roseus Unknown 126- Micrococcus roseus Conclusion: With multiple differential and biochemical test runned on the unknown 126 I was able to single out the bacteria which lead to Micrococcus roseus.
After running the test and observing the results I believe that M. roseus is the unknown 126. Flow chart Unknown S Gram Stain (G- Rod) Urease (Negative) NegativePositive Escherichia coli Citrobacter freundii Enterobacter cloacae Proteus vulgaris Enterobacter aergenes Protues mirabilis Pseudomonas aeruginosa Serratia marcescens Salmonella enteriditis Klebsiella pneumonia TSI (A/A) (A/A)(K/K) (A/K) Escherichia coli Salmonella enteriditis Klebsiella pneumonia Pseudomonas aeruginosa Enterobacter cloacae
Enterobacter aerogenes H2S (Negative) Escherichia coli Enterobacter cloacae Enterobacter aerogenes Klebsiella pneumonia Nitrate (Positive) Escherichia coli Enterobacter aerogenes Enterobacter cloacae Klebsiella pneumonia Urease (Negative) Negative Positive Esherichia coliKlebsiella pneumonia Enterobacter cloacae MR-VP (test tube missing) Go off test that I have. To figure out what the unknown S is. Unknown S- Enterobacter cloacae.
With all the test that was runned I was able to narrow my options, however I ran into a problem for the gram neg. during the MR-VP test, because I was not able to find my test tube to run that test so I became stuck with two microbes and I guess you could say 50-50 chance but I will have to make a guess best to the results that I have. So all in all unknown S (G-) has the possibility to be Enterobacter cloacae. This microbe is an opportunistic pathogen, gram negative, rod shaped, facultative anaerobe. E. cloacae are responsible for various infections, including lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections (UTIs), endocarditic.