There are numerous reasons for identifying unknown bacteria. Some of these organisms have distinct qualities that set them apart from one another, such as the exposure to certain environments. Through out the semester in the laboratory, we are able to encounter some of the few microorganisms that we as humans have come into contact with. With the knowledge gained from the sessions in the laboratory, we can now integrate what we have learned to the process of finding out the unknowns given.
Materials and Methods
The professor gave out the unknown specimens. It contained one-‐gram positive and one-‐gram negative bacteria from the given list. I was assigned unknown A. The process of identification was achieved by utilizing procedures learnt during the semester. Procedures were followed as stated in the lab manual (1). Since the sample contained two unidentified bacteria, the first step was to isolate each bacterium using streak plate technique. Tryptic Soy Agar (TSA) plate, and differential media such as mannitol salt and Eosin methylene blue (EMB) were used for isolation streak technique.
This step is imperative because the bacteria need to be separated and isolated before they can be identified. Moreover, gram staining was used to understand the basic morphology of these bacteria. As these plates were incubated and grown, the presence of two separate bacteria colonies was visible. The colonies from the mannitol salt were used to incubate a TSB broth to grow the gram-‐positive culture. The purity of this broth was tested using gram-‐staining technique. A circular colony from the TSA plate was used to incubate a TSB broth for gram-‐negative growth. Similarly, examining the morphology of the bacteria-‐using gram staining technique tested the purity of the both.
After the isolation of gram-‐positive and gram-‐negative bacteria from unknown A, specific biochemical tests were performed. The results of the biochemical tests along with deductive reasoning and elimination led to the identification the unknown bacteria. The following tests were performed on the gram-‐positive bacteria: 1. Mannitol salt streaking 2. Gram stating of the pure isolate 3. Oxidase test 4. Catalase test 5. Coagulase test The following tests were performed on the gram-‐negative bacteria: 1. Gram staining of the pore isolate 2. Blood agar plate streaking 3. SIM (Sulfide, Motility, Indole) test 4. Catalase test Results
Table 1. Biochemical Tests for the Gram-‐Positive Unknown TESTS PURPOSE REAGENTS/MEDIA OBSERVATION RESULTS Gram stain To determine the gram reaction and morphology �Crystal violet, Iodine, Alcohol, Safranin
Purple cocci, connected Gram positive purple cocci
Mannitol salt Selective growth media for Staphylococci and Micrococcaceae
Mannitol salt plate and gram positive isolate broth After incubation, media turned yellow
Bacteria is mannitol salt positive
Oxidase test To determine if bacterium produces cytochrome c oxidase
No color change Bacterium is oxidase negative Catalase test To identify if bacterium produces catalase
Hydrogen peroxide Bubbling is seen Bacterium is catalase positive Coagulase test Used to identify if bacterium produces coagulase (enzyme that clots blood plasma)
Citrated rabbit plasma Clouding and solidification of plasma is seen Bacterium is coagulase positive
Table 2. Biochemical Tests for the Gram-‐Negative Unknown TESTS PURPOSE REAGENTS/MEDIA OBSERVATION RESULTS Gram stain To determine the gram reaction and morphology
Crystal violet, Iodine, Alcohol, Safranin
Small pink rods Gram negative pink rods Blood agar plateSelective growth media
Blood agar plate After incubation, media displayed beta hemolysis, metallic sheen, and blue-‐green pigment growth (fig 1)
Non fermenting gram negative rods Oxidase test To determine if bacterium produces cytochrome c oxidase
Oxidase reagent (tetramethyl-‐p-‐phenyldiamine)
Color change to dark blue Bacterium is oxidase positive
Catalase test To identify if bacterium produces catalase Hydrogen peroxide Bubbling is seen Bacterium is catalase positive
SIM (Sulfide, Motility, Indole) test 1. To determine the ability of an organism to liberate hydrogen sulfide (H2S) from
SIM medium and indole reagent The medium showed no color change, motility, or color change when indole test was done.
-‐/-‐/-‐ The bacterium is sulfide, motility, and indole negative.
sulphurbearing amino acids producing a visible, black colour reaction. 2. To determine the ability of an organism to split indole from the tryptophan molecule. 3. To determine if the organism is motile or non-‐motile. Discussion and Conclusions:
The biochemical tests performed on the gram-‐positive bacterium worked systematically to narrow down the possible species, and eventually eliminate every organism on the list except the correct one. The gram stain, that showed the gram positive cocci and the transformation of the mannitol media to yellow color left me with two choices that fit this profile-‐ Micrococcus luteus and Staphylococcus aureus. Biochemical tests that differentiate these two were performed. These included, oxidase, catalase, and coagulase test. The results of
these tests (oxidase negative, catalase positive, coagulase positive), confirmed that the gram-‐positive microorganism in unknown A is Staphylococcus aureus. After the Gram+ bacterium was identified as such, further tests were performed to narrow down which particular Gram- species this was. The first test done was blood agar plate. The results show that after incubation the blood agar media displayed beta hemolysis, metallic sheen, and blue-‐green pigmentation.
Colonies of Pseudonomas aeruginosa often display similar result on this medium (2). To confirm the inference, oxidase, SIM, and catalase tests were performed on the gram-‐negative isolate. The results showed that the bacterium was oxidase positive, SIM negative (-‐/-‐/-‐), and catalase positive. Similar results are characteristic of P. aeruginosa. Hence, the gram-‐negative microorganism in unknown A is Pseudonomas aeruginosa. Appendix:
Fig.1. Photograph of the blood agar medium (after incubation) streaked with the gram-‐positive isolate of unknown A. References (1) Harley, J. P. (2014). Laboratory Exercises in Microbiology . University of Kentucky : McGraw Hill
(2) Buxton, R. (2010). Blood Agar Plates and Hemolysis: Non-‐Fermenting Gram-‐ Negative Rods (including Pseudomonas aeruginosa) .
Retrieved from http://www.microbelibrary.org/library/laboratory-‐test/2862-‐blood-‐agar-‐plates-‐and-‐hemolysis-‐non-‐fermenting-‐gram-‐negative-‐rods-‐including-‐pseudomonas-‐aeruginosa (3) Summary of Biochemical Tests. (n.d.). Retrieved from http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm (4) Sigma Aldrich , J. S. (n.d.). Pseudomonas Media and Tests. Retrieved from http://www.sigmaaldrich.com/technical-‐documents/articles/analytix/pseudomonas-‐media.html