Week 3

Week 3

Preparing a Gram Stain. 

9/12/13

This week the whole lab would be working with another isolated colony of their bacterial specimen and observe the colony by means of differential staining as opposed to simple staining. This week, we would focus on preparing and observing a gram stain. The preparation of this stain begins similar to the simple staining we worked on in week 2. We would again find an isolated colony of bacteria from our specimen plate:

We would then place a drop of distilled H2O (water) on a glass slide and proceed to sterilize an inoculating loop.

We used the sterile inoculating loop to capture one of the isolated, bacterial colonies and would transfer it to the drop of water on the slide. Using the inoculating loop, we thinned out the material sample and water across the whole slide.

We then proceeded to follow the directions like good students, and after the slide had air-dried, we "fixed" the smear by passing the glass through a lighted bunsen burner three times.

We then placed the fixed smear on a rack over a lab sink, and covered the smear with a crystal violet stain for 20 seconds

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After the stain had set on the slide for 20 seconds, we rinsed the slide with distilled water to remove the excess stain. We covered the slide with Gram's iodine for 1 minute and then rinsed the slide again. Next, we held the slide at a 45 degree angle and a 95% ethanol solution (a decolorizing agent) over the slide until the run off was no longer pigmented. Once we achieved this, we rinsed the slide again and then covered it with a safranin dye for 1 minute.

 We then rinsed the slide and blotted it out with a bibulous paper. 

Once the slide was dry, we observed it under a microscope.

The purpose of this experiment was to determine if our bacterial samples were either gram-positive or gram-negative bacteria. Gram-positive bacteria have a thicker peptidogylcan layer than gram-negative bacteria, thus it (in essence) traps the violet and iodine solutions more effectively than the thinner layer found in gram negative bacteria. The 95% ethanol solution dehydrates the the peptidoglycan layer, thinning it out. The thinner layer in gram negative bacteria cannot retain the violet dye, whereas the gram positive bacteria is able to retain the stain. The safranin is added as a counter stain to then stain the now-stainless gram negative bacteria, thus providing us with a differential stain. Here we observed that our bacteria is gram positive, noted by violet stain, as well as spherical shaped. However, we also observed that there was some pinkish dyed bacteria. This could have occurred through heating up the slide too much when fixing the bacterial smear. We would repeat the procedure with a new stain to see if we could correct this error. 

The following lab period we would proceed to finish our second attempt of gram-staining our bacterial smear. We followed the same procedure and, after observation, came up with a more accurate sample. We thereby determined that our bacterial sample was, in fact, gram-positive.