While taking a college-level microbiology course, your professor may require you to complete a project where you are given an unknown bacteria, and, by running a series of tests, you must identify what the bacteria is. In order to properly identify your unknown bacteria, you need to have all of the proper lab equipment and knowledge of several laboratory procedures. It is also beneficial to have a flow chart of bacteria and their lab test results so you can identify which bacteria yours is.
Set up all tests prior to beginning the experiment. This includes preparing agar plates and tubes, as well as setting out needed indicators such as Kovac's reagent. Label all tubes and plates appropriately so that no plates or tubes are mixed up, leading to inaccurate results.
Perform a gram stain on your bacteria. This will determine the shape and colour of your bacteria, such as gram positive (purple in colour), gram negative (red or pink in colour), and cocci (circular), spirochaete (spiral-shaped) or rod-shaped. To gram stain your bacteria, you'll need a clean slide, water, crystal violet, iodine, decolorizer, safranine, bibulous paper, a compound light microscope and some paper towels. After flaming your loop, use it to place a drop of water on a slide. Flame your loop again, then place a small amount of your unknown bacteria on the water droplet. Pass the slide through a flame a few times to completely dry the bacteria to the slide. Cover the slide with crystal violet, let it sit for a minute, the gently rinse it off. Next, cover the slide in iodine. Let this sit for 30 seconds, then rinse. Pat the slide with bibulous paper to remove excess dye. Next, pour decolorizer on the slide. After five seconds, rinse the decolorizer off. Finally, cover the slide with safranine. After 40 seconds, rinse the slide and pat it dry with a paper towel. Place a drop of oil on the slide where the bacteria is and observe it under your microscope. Make note of bacteria shapes and colours visible.
Perform an oxidase test on your bacteria. For this test, you need filter paper, oxidase reagent, E. coli, Pseudomonas and your unknown bacteria. In this test, E. coli and Pseudomonas are your controls. Your bacteria must be less than 24 hours old for this test. Flame your loop and place a tiny amount of each bacteria on a piece of filter paper. Use a dropper to place a drop of oxidase reagent on each bacteria. After ten seconds, make note of the colour of each bacteria. E. coli will give you a negative result, while Pseudomonas will give you a positive result. Bacteria positive for use of the enzyme Cytochrome oxidase will turn purple in colour. Negative bacteria will not change colour.
Perform a SIM (sulfer indole motility) test. Flame your loop and use it to pick up some bacteria from your plate. Inoculate the SIM tube with your bacteria and place the cover back on the tube. Let this tube sit for 18 to 24 hours at 37 degrees Celsius. Identify if your bacteria is motile, meaning it has flagella. If there appears to be cloudiness and growth away from where you inoculated the bacteria, your bacteria is motile. Next, place two drops of Kovac's reagent in the tube. If the tube has any red or pink colour change, your bacteria is positive for this test and contains tryptophan, meaning it is an indole producer. If the tube turns black, your bacteria is a hydrogen sulphide producer.
Perform a Simmon's citrate test. With two test tubes of Simmon's agar, inoculate them with your bacteria. Let them sit for 18 to 24 hours at 37 degrees Celsius. The agar is initially green, but if your bacteria uses citrate as a sole carbon source, the colour will change from green to blue. Blue is the positive test result. If your tube remains green, your bacteria is negative for using citrate as a sole carbon source.
Perform a MRVP (methyl red-Voges-Proskauer) test. For this test, you'll need two test tubes filled with broth containing peptone, buffers and dextrose (or glucose). Inoculate each tube with your unknown bacteria and let the bacteria sit in a 37 degree room for 18 to 24 hours. When ready, add three drops of methyl red to one tube. This will be your MR tube. If the tube changes from yellow to red, your bacteria is positive and uses a mixed acid pathway during fermentation (lactic, acetic and formic acid are produced). In your second tube, add three drops of VP reagent. If your tube turns red, it is positive for creating Diacetyl, a fermentation product. If the tube is brown, your bacteria is negative.
Eliminate bacteria from your flow chart as you read each test and determine if your bacteria is negative or positive for that test.
- Truman State University: Identifying Your Unknown
- University of Wisconsin - Madison: An Introduction to Bacterial Identification
- University of Pennsylvania Health System: How to do Gram Stains
- Western Michigan University: Biochemical Characteristics Procedures
- Austin Community College: Differential Tests