Gram Negative Unknown
Upon being given a bacterial culture containing two bacteria, I was assigned the task of isolating the individual bacteria. The two bacteria of the mixture were one gram positive and one gram negative. The experiment has two parts and this is the part of isolating the gram-negative bacteria. The technique used was called streaking for isolation in which the bacteria were streaked thinly on nutrient agar in different directions in a pattern that left individual colonies on the final growth streak. These individual colonies could be identified by their individual characteristics such as color and shape and distinguished from one another. This was not easy with this culture because both bacteria grew in colonies that were small, white, and shaped similar. To separate these bacteria I had to first gram stain the bacterial mixture to see if I had both types of bacteria and so I used the following steps to perform the gram stain.
1. The bacteria are smeared on a slide and allowed to dry.
2. The bacteria is heat fixed to the slide
3. Crystal violet pigment is added to the smear for 1 minute
4. Wash the pigment off with distilled water
5. Apply Gram’s Iodine for 1 minute
6. Wash iodine off with distilled water
7. Decolorize the smear with 95% ethyl alcohol drop by drop until it runs off clear
8. Wash off alcohol with distilled water
9. Counterstain with Safranin for 45 seconds
10. Wash off Safranin with distilled water
11. Blot dry with Bibulous paper
Following this test, I found that I had a lot more gram negative bacteria than the gram positive and so I could easily take one colony of my streaked culture and have a high chance of growing a pure culture from one of the isolated colonies. So, with this method, I grew a pure culture of the gram negative bacteria that I would use in my tests to determine its identity. I ran several tests on the bacteria staring with the Gram stain that proved that the bacteria were gram negative....