Day 5 - Acid Fast, Capsule, and Endospore Stains

   To start off our day, we scared one of our teammates with a bug we found outside. :P After that entertainment was over, we started in on the staining. The goal for the day is to finish up the gram staining from the day before and to complete capsule, acid fast, and endospore stains. This will aid us in fully figuring out what type of bacteria we have as our sample.

  The first staining that we did was the redo of the Gram staining of the bacteria from Grace's throat. We went through the procedure from the day before. This means that we  placed the slide over the smear rack, and covered the sample with the crystal violet for twenty seconds. After rinsing this off, we covered the sample in Gram's idonine for 1 minute, rinsed this off and put drop by drop of 95% ethanol until no color came off with it. We rinsed this and then added safranin, which we left for one minute. Lastly, we rinsed this off and blotted with bibulous paper. We will wait until tomorrow to view this stain along with all of the others. 

   To begin the first of the new stains, we began a capsule stain, as seen below. This procedure begins with a small drop of nigrosin placed on a slide towards one of the ends, and a small amount of bacteria inoculated into that drop (following aseptic technique below).

After re-sterilizing the inoculating loop in the Bunsen burner, another slide is used to spread the bacteria-nigrosin drop over the initial slide at a 30-45 degree angle; we then allowed the slide to air dry, followed promptly by staining with safranin on top and then gently rinsing the whole slide. The point of this stain is to leave some lighter areas to be able to see bacterial capsules or slime layers.

Our final product :D

    With the finishing of this stain, the next followed and after a slight dilemma of spilled gram's safronin and a rather wasteful use of ethyl alcohol, we moved on to the endospore stain. This procedure consists of holding a fixed bacterial slide over a beaker of boiling water while placing a piece of bibulous paper on top of the slide, and saturating it with malachite green for 6 minutes. After this, the paper is removed, put in a biohazard bag, and the slide is rinsed for 30 seconds, covered with safranin for 90 seconds, rinsed again, and blotted with more bibulous paper.

Rinsing the slide, showing the fight with gram's safranin on my fingers and the sink as well.

  The acid-fast staining process was very similar to the endospore stain, because it also used a fixed bacterial slide over a beaker, to stain the bacteria. This time carbolfuchsin was used instead of malachite green, and stained for 3-5 minutes, while saturating bibulous paper over the slide just as in the endospore stain. The paper was removed after the aloted time and put in a biohazard bag, the slide was rinsed, decolorized with acid-alcohol until color stopped running off, rinsed quickly again, and covered with methylene blue for 2 minutes. The bacterial stain was rinsed again, and blotted with bibulous paper, ready to be examined tomorrow under the microscope.

The lineup of model slides ready to be critiqued in lab tomorrow. Hopefully all celullar structures are now enhanced.

* App of the day: The reason a stagnant bucket of water is slimy, is becuase bacteria is growing on the sides, and protect their territory and growth with a slime layer over their cellular membrane.