Kristina SimonovicSection O2 March 2016Exercise SevenTitle:Biochemical TestsHypothesis:If several biochemical tests are correctly performed on a pure culture of your unknown, then the bacteria will be easily identified. Methods:-Catalase Test:Remove a small amount of your environment unknown from your agar slant, or a loopful of control test organisms from a broth culture and place it on a glass slide. Mix the organisms with adrop of 3% H2O2 and check for the appearance of gas bubbles (a positive test). No bubble is a negative test. Use Staphylococcus epidermidis for the positive control and Streptococcus lactis for the negative control.-Oxidase Test:For the test, you will use a commercially prepared test called a dry slide oxidase test. These tests are squares of filter paper that have been impregnated with p-phenylenediamine, then sandwiched between two pieces of plastic. There are four windows on the dry slide, enough for two groups to each spot an unknown and a positive control. Using a plastic steri-loop, rub the cells from a plate or slant directly onto the filter paper in one of the windows of the dry slide and record the color change within 20 seconds. If the organism is oxidase positive, the reaction area will turn dark purple. If the organism is oxidase negative, there will either be no color change or a change from colorless to gray. -Carbohydrate Fermentation:Inoculate a tube containing one of each of the four sugars from your TSA slant of your EI. Incubate a room temperature. You must score the tubes in 24-48 hrs.-Oxygen Requirements:In a Thioglycollate tube, inoculate your EI and then cap it. Rotate the tube between your hands todisperse the organism. Incubate at room temperature.-Anaerobic Respiration by Nitrate Reduction:Inoculate a tube of nitrate broth with your culture. Incubate the culture at room temperature for 24-48 hrs until growth appears and then refrigerate until next lab.-Motility Test:Inoculate a tube of motility medium using your inoculating needle. Stab the medium to about 2/3deep, then withdraw the needle straight out of the same hole. Incubate for 24-48 hrs.-Simmon’s Citrate:Stab the agar about 2/3 deep with your EI and then streak the slant in a zigzag fashion. Incubate for 24-48 hrs at room temperature.-Urea Hydrolysis:Inoculate a tube of urea broth with your EI and incubate at room temperature for 24-48 hrs.-Kligler’s Iron Agar:Inoculate a tube of Kligler’s iron agar with your EI using the needle. Incubate at room temperature for 24 hours.-Gelatinase Test:Stab one tube of gelatin media with your EI. Incubate at room temperature for one week. -Starch Hydrolysis:Inoculate a single starch-gelatin agar plate with your EI. Use B. megaterium for the positive control and E. coli for the negative control. Incubate at room temperature for 24-48 hrs and then refrigerate. -Casein Hydrolysis:Use one plate for all organisms. Use B. megaterium for the positive control and E. coli for the negative control. Incubate at room temperature for 24-48 hrs and then refrigerate. -Lipid Hydrolysis:Innoculate a plate of spirit blue agar. Use Serratia spp. for the positive control and S. epidermidisfor the negative control. Incubate at room temperature for 24-48 hrs and then refrigerate. -Facultative Anaerobes:Inoculate a TSA plate with your unknown and place it into the anaerobe jar. The oxygen will be removed chemically. Incubate at room temperature until next lab.-Presence of Biofilm:Heavily inoculate a tube of enriched 1% glucose with your EI. Incubate at room temperature until next lab.Safety Concerns:1. Be sure to use the aseptic technique when performing biochemical tests on your unknown2. Be certain to only test pure cultures of your unknown to avoid uninterpretable results3. Carefully follow the instructions of each biochemical test to ensure accurate results4. Use Bergy’s manual to help you identify your unknownRelevance:Identifying microorganisms is a fundamental part of microbiology and makes people aware of the types of bacterium found