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Identifying unknown bacteria is a crucial task in microbiology. This laboratory report outlines the procedures and results of identifying two unknown bacteria, one gram-positive and one gram-negative, referred to as unknown A. The isolation of the bacteria, Gram staining, and a series of biochemical tests were performed to narrow down the possibilities. The results revealed that the gram-positive bacterium was Staphylococcus aureus, while the gram-negative bacterium was Pseudomonas aeruginosa.
Identifying unknown bacteria is essential for various reasons, as different microorganisms possess distinct qualities and characteristics that set them apart.
Throughout the semester in the laboratory, we encountered various microorganisms and gained knowledge about their properties. This knowledge is now applied to the process of identifying unknown bacteria, specifically, unknown A.
The identification process began with the professor providing the unknown specimens, including one gram-positive and one gram-negative bacterium from a predetermined list. Unknown A was assigned for identification. The procedures followed closely resembled those outlined in the lab manual (1).
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The initial step involved isolating each bacterium using the streak plate technique. Tryptic Soy Agar (TSA) plates and selective media like mannitol salt and Eosin Methylene Blue (EMB) were employed for isolation.
The isolation step is critical as it separates the bacteria, allowing for individual identification. Gram staining was performed to understand the basic morphology of the bacteria. As the plates incubated, distinct colonies of the two bacteria were observed. Colonies from the mannitol salt plate were used to inoculate Tryptic Soy Broth (TSB) for gram-positive culture growth.
The purity of this broth was verified using gram staining. Similarly, a circular colony from the TSA plate was used to inoculate TSB for gram-negative growth, and its purity was also tested through gram staining.
Once the gram-positive and gram-negative bacteria from unknown A were isolated, specific biochemical tests were conducted for identification. The results of these tests, coupled with deductive reasoning and elimination, led to the identification of the unknown bacteria. The following tests were performed on the gram-positive bacterium: 1. Mannitol salt streaking, 2. Gram staining of the pure isolate, 3. Oxidase test, 4. Catalase test, and 5. Coagulase test. Similarly, the following tests were performed on the gram-negative bacterium: 1. Gram staining of the pure isolate, 2. Blood agar plate streaking, 3. SIM (Sulfide, Motility, Indole) test, and 4. Catalase test.
| Test | 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 | Bacterium 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 |
| Test | 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 plate | Selective growth media | Blood agar plate | After incubation, media displayed beta hemolysis, metallic sheen, and blue-green pigment growth | 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 sulfur-bearing amino acids producing a visible, black color 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. |
SIM medium and indole reagent | The medium showed no color change, motility, or color change when the indole test was done. | Sulfide, motility, and indole negative |
The systematic application of biochemical tests on the gram-positive bacterium allowed us to narrow down the potential species. The Gram stain and mannitol salt test initially pointed towards two options: Micrococcus luteus and Staphylococcus aureus. Subsequent tests, including the oxidase, catalase, and coagulase tests, confirmed that the unknown gram-positive bacterium was Staphylococcus aureus.
After identifying the Gram-positive bacterium, additional tests were conducted to specify the exact species. The blood agar plate test results, showing beta hemolysis and other characteristics, resembled those of Pseudomonas aeruginosa. To validate this inference, oxidase, SIM, and catalase tests were performed on the gram-negative isolate. The results indicated that the bacterium was oxidase positive, SIM negative, and catalase positive, aligning with the characteristics of Pseudomonas aeruginosa. Thus, the gram-negative bacterium in unknown A is Pseudomonas aeruginosa.
Identification of Unknown Bacteria: Lab Report. (2016, Aug 15). Retrieved from https://studymoose.com/document/unknown-lab-report-microbiology
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