Staining Day!!

Lab Day 3

“I was Born Ready” -PiCassie

Today's lab began by Mary Rose spraining her ankle playing ultimate frisbee. Woohoo. After being brought into the lab in a wheelchair, Joanna and Cassie joined to begin today's procedures.

Part 1 of today's lab: Gram Stain

When we started preparing to do the gram stain, everyone realized that we, the class, had left our samples in the incubator instead of the refrigerator. According to Dr. P, “One night in there is more than enough.” Oops! To our relief however, no harm had been done to our samples.

Using ascetic technique, we made a fixed smear of our bacteria. We then applied Crystal Violet dye on the air-dried sample for 20 seconds. Using bibulous paper, we pat dry the slide so no excess water would interfere with our observations under the microscope. After this, we applied the Gram Stain and let is sit for 60 seconds. Whoever had the idea to smell this (Mary Rose) made a mistake. It smelled like nasty latex. 

Lab was interrupted by a surprise visit from Dr. Pathakamuri's family which brightened everyone's mood. Dr. P jokingly asked his youngest son to help us look into the microscope but he quickly refused. To remove the Gram Stain, we added 95% Ethanol until the color on the slide stopped running. After this, we applied Gram Safranin, which is a reddish color, also for 60 seconds. We then observed the slide under the microscope and, with the help of Dr. Pathakamuri, we concluded that our unknown bacteria was gram negative and rod shaped. With that knowledge, we knew that our bacteria had a thinner cell wall and thinner peptioglycan layer and that of a gram negative cell wall.

Part 2 of today's lab: Capsule stain

The next experiment we preformed on our unknown bacteria was a Capsule Stain. First, we placed a small amount of Nigrosin dye on a slide (slide #1) with a sample of our unknown bacteria. We then mixed the two together and, using the edge of another slide (slide #2), smeared the dyed bacteria mixture across the surface of slide #1. After the smear dried, we covered it with the crystal-violet dye and let it sit for a minute over the sink. That's when Pathakamuri accidentally knocked a slide over that proceeded to shatter on the ground. Everyone laughed when he turned to Professor Lumbar and said, “Professor Lumbar, someone left a depression slide out and it wasn't one of my students. I don't know whose fault that is!”

Now we know how to handle the situation if we ever break a slide!

We washed off the excess dye with DI H2O but we did not do this for a long period of time due to the possibility of disrupting the placement of the original smear. We then blotted the slide with the bibulous paper and headed over to the microscope. There, we were able to see that our unknown bacteria has no capsule.

Part 3 of today's lab: Acid-fast Stain

Before beginning our final experiment of the day, the acid-fast stain, Dr. Pathakamuri put music on the surround speakers in the lab. This turned out to be one of our favorite memories from lab thus far. We listened to Enya, the Lion King, Dr. Pathakamuri's choir Cd, and various other types of music. That's when we realized that Pete and Cassie have incredible opera voices! (insert video)
We prepared another fixed smear and placed it over a beaker of boiling water. We applied Carbolfuchsin, a green dye, onto a piece of bibulous covering the slide and let it sit for 5 minutes. We were careful to not let the dye dry out by continually saturating the paper with excess Carbolfuchsin.
When 5 minutes was up, we removed the slide from the beaker with thongs and discarded the piece of paper in hazardous waste bin. We let the slide cool and then rinsed it with water to remove excess stain. We decolorized the slide with acid-alcohol while holding the slide at a 45 degree angle. We did this until the color stopped running then immediately rinsed the slide to remove the decolorizing agent.

We covered the smear with Methylene blue and let that dye sit for 2 minutes. We rinsed the stain off with water to remove excess dye and observed it under the microscope. We repeated this same procedure for an unknown bacteria given to us by Dr. Pathakamuri.

The results:

  Our unknown bacteria was negative for being an acid-fast bacteria and Dr. Pathakamuri's unknown was positive for being an acid-fast bacteria.

Part 4 of today's lab: Endospore Staining

This staining for our unknown sample showed that our bacteria had no endospores present. 

End of the Lab Day Results!!!

Joanna Dawyot, Cassie Livingston, Mary Rose Capara 

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