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. Tropic Soy Agar (TTS) plate, and differential media such as imitation salt and Eosin methyl blue (EMBED) were used for isolation streak technique. This step is imperative because the bacteria deed 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 imitation salt were used to incubate a TTS broth to grow the gram-positive culture. The purity of this broth was tested using gram-staining technique.
A circular colony from the TTS plate was used to incubate a TTS broth for gram–negative growth. Similarly, examining the morphology of the bacteria”using gram staining technique tested the purity f 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.
Imitation salt streaking 2. Gram stating of the pure isolate 3. Oxides test 4.
Catalane test 5. Coagulate test The following tests were performed on the gram”negative bacteria: 1 . Gram staining of the pore isolate 2. Blood agar plate streaking 3. SIMI (Sulfide, Motility, Indolent) test 4. Catalane 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, Seafaring Purple Cisco, connected Gram positive purple Cisco Imitation salt Selective growth media for Staphylococci and Microwaveable Imitation salt plate and gram positive isolate broth After incubation, media turned yellow Bacteria is imitation salt positive Oxides test To determine if bacterium produces stockroom c oxides No color change Bacterium is oxides negative Catalane test To identify if actinium produces catalane Hydrogen peroxide Bubbling is seen Bacterium is catalane positive Coagulate test Used to identify if bacterium produces coagulate (enzyme that clots blood plasma) Citrated rabbit plasma Clouding and solidification of plasma is seen Bacterium is coagulate positive Table 2.
Biochemical Tests for the Gram–Negative Unknown TESTS PURPOSE REAGENTS/MEDIA OBSERVATION RESULTS Gram stain TO determine the gram Small pink rods Gram negative pink rods Blood agar availabilities growth media Blood agar plate After incubation, media displayed beta hemolytic, metallic sheen, and blue–green pigment growth (fig 1) Non fermenting gram negative rods Oxides test To determine if bacterium produces stockroom c oxides Oxides reagent (tetrameters”p–phenylalanine) Color change to dark blue Bacterium is oxides positive Catalane test To identify if bacterium produces catalane Hydrogen peroxide Bubbling is seen Bacterium is catalane positive SIMI (Sulfide, Motility, Indolent) test 1.To determine the ability of an organism to liberate hydrogen sulfide (HAS) from SIMI medium and indolent reagent The medium showed no color change, motility, or color change when indolent test was done. ”I”I– The bacterium is sulfide, motility, and indolent negative. Lubricating amino acids producing a visible, black color reaction. 2. To determine the ability of an organism to split indolent from the thyrotrophic 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 Cisco and the transformation of the imitation media to yellow color left me with two choices that fit this profile” Microcosmic lutes and Staphylococcus erasures. Biochemical tests that differentiate these two were performed. These included, oxides, catalane, and coagulate test. The results of these tests (oxides negative, catalane positive, coagulate positive), confirmed that the gram”positive microorganism in unknown A is Staphylococcus erasures. 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 hemolytic, metallic sheen, and blue”green pigmentation. Colonies of Pseudonyms organisms often display similar result on this medium (2).
To confirm the inference, oxides, SIMI, and catalane tests were performed on the gram–negative isolate. The results showed that the bacterium was oxides positive, SIMI negative (”1–/”), and catalane positive. Similar results are characteristic of P. Organisms. Hence, the gram”negative microorganism in unknown A is Pseudonyms organisms. Appendix: Fig.
L . Photograph of the blood agar medium (after incubation) streaked with the gram–positive isolate of unknown A.