Decontamination and Disposal If an autoclave is not available, all solutions and components (loops and pipets) that have come in contact with bacteria can be placed in a fresh 10% bleach solution for at least 20 min for sterilization. A shallow pan of this solution should be placed at every lab station. No matter what you choose, all used loops and pipets should be collected for sterilization. Sterilize petri dishes by covering the agar with 10% bleach solution. Let it stand for 1 hr or more and then pour excess plate liquid down the drain. Once sterilized, the agar plates can be double bagged and treated as normal trash. Safety glasses are recommended when using bleach solutions. Please consult your local environmental health and safety regulations for proper disposal. Incubation This guide is written to reflect the use of a 37°C incubator. The transformation experiment can be conducted without the use of an incubator, however, the number of days required to culture colonies to the optimum size depends on the ambient temperature. Best results are obtained if starter plate colonies are fresh (24–36 hr growth) and measure about 1–1.5 mm in diameter. Refrigeration of cultured plates will significantly lower transformation efficiency. The optimum temperature for growing E. coli is 37°C (98.6°F), and lower temperatures will result in a decreased growth rate. At 28°C (82°F), two days of incubation time are required to obtain optimum colony size. At 21°C (70°F), three days of incubation time are required to obtain optimum colony size. Adjust the advance preparation lead times and laboratory schedule according to your incubation temperature.
Experimental Points – Optimizing Your pGLO Lab Experiment Practicing Techniques Some educators like to do a dry run of the procedures to explain sterile technique, practice using the pipets and loops, and practice streaking and spreading bacteria on the agar’s surface. You will have to decide what is best for your students based upon their laboratory experience and familiarity with these techniques. Paying close attention to the following experimental points will result in successful transformations. Preparing E. coli Starter (Agar) Plates Best results are obtained when using a flask instead of a beaker to prepare the agar. Ensure that the agar is completely dissolved as uneven mixing can result in agar that will not solidify. Follow the directions in "Advance Preparation Step 1" closely to minimize introducing contaminants. After the starter plates are streaked with E. coli and incubated at 37°C, they should be used within 24–36 hr. Delay beyond 36 hr will compromise transformation. Transferring Bacterial Colonies from Agar Plates to Microtubes The process of scraping a single colony off the starter plate leads to the temptation to get more cells than needed. A single colony that is approximately 1 mm in diameter contains millions of bacterial cells. To increase transformation efficiency, students should select 2–4 "fat" colonies that are 1–1.5 mm in diameter. Selecting more than 4 colonies may decrease transformation efficiency. Select individual colonies rather than a swab of bacteria from the dense portion of the plate; the bacteria must be actively growing for transformation to be successful.
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