![]() Incubate the test until the next lab session. Label the test media with your name, test name, date, and whether the organism is your Gram Negative or Gram-positive unknown. Use your stock culture to perform the first test listed on your dichotomous key. YOU MUST PERFORM THE TESTS IN THE ORDER ON YOUR FLOW CHART.Ģ. As soon as you have your dichotomous keys (flow charts) graded and your organisms growing on stock cultures, you can begin to perform biochemical tests. Day 3: Begin testing your unknown organismsġ. Put the CNA and MAC plate back in the class tray to be saved in the refrigerator in case your stock cultures do not grow on the TSA plates. Incubate the TSA plate of your Gram-Positive stock culture upside down in the Candle Jar.Ĩ. You will use this stock culture to inoculate all future biochemical tests performed on this unknown.ħ. Label this TSA plate with your name, your unknown number and the words “GRAM POSITIVE”. Take an ISOLATED colony from the CNA plate and streak for isolation on a TSA plate. 18) Record your observations on the worksheet.Ħ. Evaluate the hemolytic properties to determine if the organism produces beta, alpha or gamma hemolysis. Observe an isolated colony growing on your CNA plate. Incubate the TSA plate of your Gram-Negative stock culture upside down in the class plate tray until the next lab period.ĥ. soy agar is a “non-inhibitory” media which allows the organism to produce enzymes and metabolize the ingredients in the media.Ĥ. You will use this stock culture to inoculate all future biochemical tests performed on this unknown. ![]() Label this TSA with your name, your unknown number and the words “GRAM NEGATIVE”. Take an ISOLATED colony from the MacConkey Agar and streak for isolation on a TSA plate. If you do not have growth, reincubate your plates and subculture the original broth again.Ģ. Observe your MacConkey Agar plate and determine if your organism ferments lactose (or not) and record your results on the laboratory report form that you will submit for grading. These tubes will be saved in the refrigerator in case your unknowns do not grow on the media. Place the broth in the rack on the front table of the lab. Label your mixed unknown broth with your name and lab section (day & time). Incubate your CNA plate upside down in a candle jar since some of the unknown Gram-Positive organisms are microaerophilic.Ħ. Incubate your MAC plate upside down in the class plate tray until the next lab period.ĥ. Stab the CNA plate in the first section to enhance the hemolysis.Ĥ. Use your best aseptic technique and streak for isolation.ģ. Then, use a sterile inoculating loop to continue streaking for isolation. Roll the swab onto the first section ONLY. To ensure enough sample is collected, use a sterile cotton-tip swab to obtain the inoculum. ![]() Inoculate your unknown broth onto the MAC and the CNA media. Record your unknown number on the worksheet.Ģ. Label the MacConkey and CNA plates with your name (not initials), date, unknown number and the name of the media. PROCEDURE Day 1: Inoculate selective/differential mediaġ. Record the identification of the unknown using correct binomial nomenclature. Record the biochemical tests you perform in the order of your dichotomous key. You must complete the tests in the order of the dichotomous key until you reach an identification of your unknowns. The result of the first test will determine the next test to perform. By using biochemical test charts to differentiate the Gram-positive cocci and the Gram-negative bacilli, you will design two dichotomous keys (flow charts) to guide you in determining which tests will help you identify your unknowns. Once the bacteria have been isolated from each other, the next step is to identify both bacteria. You can determine the hemolytic properties of the Gram-positive cocci. CNA is differential because it contains red blood cells. CNA contains the antibiotics colistin and nalidixic acid which suppress the growth of Gram-negative organisms. ![]() The Colistin-Nalidixic Agar is selective for Gram-positive organisms. Gram-negative bacilli that ferment lactose will appear hot pink and those that do not ferment lactose will appear colorless. MacConkey agar is also a differential media because it contains lactose. The crystal violet dye and the bile salts in the media inhibit the growth of Gram-positive organisms. The MacConkey Agar is selective for Gram-negative organisms. First, you must isolate and separate the two organisms from each other by using selective media. One type is Gram-positive cocci and the other is Gram-negative bacilli. There are two different types of bacteria in the broth of the mixed unknown. Biochemical Test Results Google Photo Album
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