October 6th 2011
Today I did two tests simultaneously. I did the Acid-Fast stain to see whether or not my Gram-positive unknown was really Gram-positive or if it was actually an acid-fast bacterium. I also prepared an endospore stain to see whether the bacteria created spores or not. Both procedures required the use of a beaker filled with boiling water and slides with paper on it. The acid-fast stain required Ziehl-Neelsen carbolfuchsin to stain for 3-5 minutes so it was on for 4 minutes; meanwhile the endospore stain required malachite green to stain for 5-6 minutes so it was left on for 5.5 minutes. Each were removed by a forceps and allowed to cool before having the excess stain rinsed off. The acid-fast stain was decolorized by acid-alcohol until the color stopped draining, and then it was rinsed again before methylene blue was used to cover it for 2 minutes. Meanwhile, the endospore stain was covered in safranin for about 60-90 seconds. Both slides had their excess stains rinsed off before being blotted by bibulous paper and examined under a microscope with immersion oil added. The entire acid-fast slide was blue, indicating that the stain was gram-positive and not acid-fast. The endospore stain was completely red with no green to be seen which indicated that the bacteria did not produce spores.
Acid Fast Stain
Endospore Stain
