Antibacterial? & A Differential (Gram) Stain of the Pure Air Sample

Antibacterial?
& A Differential (Gram) Stain of the Pure Air Sample

Class: Tuesday, September 10, 2013

Before we started today's lab of determining whether the pure air sample bacteria is gram negative or positive, we retrieved the plate of sample that we were testing for antibacterial properties. As can be recalled, we took three different colored bacteria from our air sample--orange, yellow, and pure culture air sample. We made one zig-zag line with each bacteria on the plate and stored it in the incubator to allow the bacteria to grow. Our objective was to confirm whether or not there is antibacterial properties in any of the bacteria we placed on the plate.

The Pure air sample is Antibacterial!!

Unfortunately, we forgot to mark which bacteria was the yellow and which was the orange. However, we do know that the middle streak is the pure air sample. Also, because we are such great nursing students we kept our cool and made a new plate. This time making sure we marked the colors on the plate!

New and improved plate

After putting this into the incubator to grow, we started our lab assignment for today. In our lab we prepare a differential (gram) stain in order to determine whether our bacteria is gram negative or positive. We start by fixing our bacteria to the microscope using the same correct method we previously learned in our last lab when we prepared a simple stain.

Fixing the bacteria to the slide

After fixing the bacteria to the slide, we had to "flood" the smear with crystal violet. The crystal violet has to sit for 20 seconds before we can rinse the slide with water to wash away any excess coloring. The crystal violet will be used to identify gram positive.

Crystal violet

"Flooding" the smear

Rinsing the excess crystal violet

Next, we have to smear the slide with Gram's iodine. After letting it sit for one minute we have to rinse the excess iodine solution with water. While we're holding the slide at an angle we decolorize the slide with 95% ethanol. We keep adding drops until the crystal violet color stops running off the slide. Immediately following with water to rinse off the decolorizing agent.

(left-right) Gram's Iodine, Ethanol, Safranin

Smear with Gram's iodine

Rinse excess iodine 

Decolorize with 95% ethanol

Rinse off the decolorizing agent

"Flooding the smear with safranin

We then placed the slide back down and "flooded" the smear with safranin for one minute. After the minute had ended, we rinsed off the safranin with water and blot the slide dry with bibulous paper.

Rinsing off excess safranin

                                                                           

Bibulous paper!

Blotting the smear dry with bibulous paper

Bacteria: Dark pink line at the point

What we see on the slide through the microscope is the bacteria mixed in with endospores. Some of the endospores show up with the violet stain, and the rest show up with safranin. The bacteria are the dark pink lines (safranin). One can be seen at the end of the point. The endospores are unimportant right now because they can't determine whether it's gram positive or negative.  We can however, determine whether the bacteria is gram negative or positive. The bacteria took up safranin so therefore we can conclude that the bacteria is gram negative. This means that the bacteria of our air sample has a thin layer of peptidoglycan in the cell wall.