Staining Bacteria (Preparing an Unknown Sample & a Smear)

The class took out our streaking plates and observed the individual colony of bacteria that had grown on each one. We compared them to the Master Bacteria sample and some people had to redo them if individual colonies did not grow. Luckily, my sample grew properly so i did not have to re-streak my plate.
We took microscope glass slides (each person did two) and placed a drop of distilled water on them. We then sterilized loops and took a swab of bacteria from our petri dishes and mixed them into the distilled water. After we swirled them onto the glass microscope slides we let them air dry. Once dry we ran them through the flame from a Bunsen Burner to heat-fix the slide (sterilize). We than stained the slides with Crystal Violet and Safranin. The Crystal Violet slide had to sit for 20 to 30 seconds and the Safranin sat for at least 1 minute. Afterwards we rinsed the excess stain off with distilled water and blotted the slide dry with bibulous paper.

We then were given an unknown sample of Bacteria by our professor: Dr. P. We were instructed to identify the unknown sample of bacteria. Our bacteria was labeled 'L.' Before any tests can be done on the bacteria, we first had to create a pure culture of 'L' by using the aseptic technique. First we sterilized the inoculating loop and the top of the test tube that contained 'L' through a flame from a Bunsen Burner. The inoculating loop was then used to carefully obtain a sample of 'L' and spread it along a slant  agar test tube. The loop and test original test tube top were then sanitized once again through the flame and we placed the bacteria in an incubator at 25 degrees Celsius.

The Master Bacteria Sample (left) With The Streak Plate (right)

About to Heat-Fix The Slide

Checking My Slide To See If I Stained It Correctly

The point of keeping the Master Bacteria Sample was so that if the Streaking Petri Dish did not grow individual colonies we could use the Master Sample to redo the Streak Dish. We learned that isolating bacteria on an agar plate surface creates bacterial colonies in a pure culture from a mixture of bacteria. We also  learned how to properly stain bacteria which is important because staining bacteria makes it easier to be seen more clearly through a microscope. We also learned the proper size, shape, and arrangement of bacteria in a fixed smear. My slide displayed circular bacteria that was a slight green tinge in the crystal violet stain.

Until Thursday!,

Annie B