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Unformatted text preview: WEEK 3: September 13 - 19 Culture Transfers, Bacterial Isolation, Gram Staining and Oxygen Needs Culture Transfer Procedure Introduction Your lab instructor will demonstrate the transfer of organisms from a broth culture to two types of media: broth and agar slant. Both the aerobic surface and the oxygen-deficient butt of the slant will be inoculated. The emphasis will be on the importance of sterile technique resulting in pure cultures, i.e., not contaminated with unwanted organisms of other species. Materials (per 2 students): broth culture Serratia marcescens 2 tubes trypticase soy broth (TSB) 2 trypticase soy agar (TSA) slants Procedure for aseptic technique with culture tubes Every student needs to make two transfers, one to each of the media above. All tubes need to be labeled with your drawer code, the microorganism inoculated and the date. A "dry" run with an uncontaminated loop is a good idea to allow you to begin to get a feel for the technique. The technique outlined below describes the proper steps in working aseptically with a bacterial broth in a culture tube. Today you will remove a sample of the broth and use it to inoculate a tube of broth, and later both the surface and butt of an agar slant. In your drawer, there are both inoculating needles and loops. You will use the needles for stab inoculations only and the loops for everything else. Make certain that your loop is correctly formed (a completely closed circle), allowing it to hold a drop of broth, without a jagged edge which can tear the surface of the agar during streaking. 1. Considering the incinerator and the bacteria, tie up any loose hair and sleeves. Avoid a lot of unnecessary moving, as this causes air currents, increasing the risk of contamination of your culture and the environment. If the activity uses screw-capped tubes, you may first wish to loosen any caps that may be tightly screwed down. ( NOTE: Caps should always be left slightly loose after inoculating! ) 2. The inoculating loop should be heated to bright red by holding the loop straight down the heated ceramic tube of the incinerator. This will destroy all living organisms on the loop. NEVER prop a loop or needle in the MCRO 251 Lab Manual 18 incinerator; the handle will quickly heat up, potentially burning your hand. In addition, since the handles are aluminum, they will simply melt. Allow the loop to cool for 10-15 seconds....
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This note was uploaded on 02/11/2012 for the course MCRO 251 taught by Professor Lorrainecramer during the Fall '09 term at UNC.
- Fall '09