The plate was then turned a quarter turn and a new section of the plate was streaked, making sure to trace back into the previous section several times. The loop was again sterilized and cooled and the plate was given a quarter turn. Again, the new quarter of the plate was heavily streaked, tracing back into the second quadrant several times. The loop was sterilized and allowed to cool, and the plate was turned a final time. The final streak only traced back into quadrant 3 two or three times before, with a fishtail-like motion, the bacteria was drawn towards the center of the plate. The inoculation loop was sterilized and the top was placed on the plate to avoid contamination. This method of streaking is effective because it decreases cell density until single cells are being distributed on the plate, allowing for pure colonies to grow. .
In the initial quadrant streak, two plates of ENA and two plates of NA were streaked with Unknown 9, one ENA and one NA were incubated at 37∘ C and the remaining two were placed in the drawer to grow at 25∘C. Throughout the experiment, fresh quadrant streaks were made to ensure fresh bacteria for testing. Unknown 9's bacteria grew best on NA plates at 25∘C (Leboffe & Pierce, 2010). Gram staining was the method used to determine if Unknown 9 had been successfully split into two isolated bacteria. First, a drop of DI water was placed on a slide. An inoculation loop was sterilized, cooled, and used to pick up an isolated colony from a streak plate growing Unknown 9. The loop was gently swirled in the drop of water, allowing some of the bacteria on the loop to become suspended in the droplet. Good indication of bacterial concentration is a droplet that is slightly opaque in appearance. The loop was then sterilized and set aside. With a paperclip grasping one end of the slide, the bacteria were heat fixed by gently sweeping the slide over the Bunsen burner several times until all of the water evaporated.