Background Info
The sole purpose of this experiment is to illustrate the differentiation of bacteria in acid fast and non-acid fast staining groups. Most bacterial organisms are stainable by either simple or Gram staining procedures. The characteristics displayed between mycobacteria and other microorganisms are very different because of the presence of a thick, waxy wall that makes the stain penetration very difficult. Once the stain is penetrated, it is not able to be readily removed even with the vigorous acid-alcohol agent, unlike the 95% ethyl alcohol that is used in gram staining. This a very important property because this is what distinguishes these organisms as acid fast, versus non- acid fast, which are easily decolorized by acid alcohol.
The acid fast staining uses three different reagents which includes the Primary Stain, the Decolorizing Agent, and the Counterstain. The Primary stain being used is Carbol Fushsin which is a dark red stain 5% phenol that is soluble in the lipoidal materials. It establishes most of the cell wall, is retained, and penetrates the bacteria. Acid-Alcohol is used as the Decolorizing Agent, because acid-fast cells will be resistant to decolorization due to the fact that the primary stain is soluble in the cellular waxes than in the decolorizing agent. The final reagent used in this process is Methylene Blue, which is used to stain previously decolorized cells, and serves as the Counterstain.
Methods/ Procedures
In completing this experiment, we used a Bunsen burner, hot plate, 250 ml beaker, inoculating loop, glass slides, bibulous paper, lens paper, staining tray, and a microscope. We also used 72-96 hour soy broth culture of M. Smegmatis and 18-24 hour culture of Staph Aureus and for our Reagents, Carbol Fuschin, acid-alcohol, and methylene blue. In our smear preparation, we took three glass slides and using aseptic technique, we prepared a bacterial smear of each organism plus a third smear which was a mixture of the two onto the third slide. We then allowed the smears to dry, and once they we dry we heat fixed them by quickly running then over the flame of the Bunsen burner.
After the smears were completely dry and heat fixed, we flooded the smears with carbol fuchsin and placed the slide over a beaker on a warm hot plate, and allowed it to steam for 5 minutes. Once the slides were completely cooled, we washed them with tap water. Next, we decolorized them by adding the acid alcohol, drop by drop onto the slides until the alcohol was almost clear with a red tinge, then washed it with tap water again. Then we counterstained with methylene for two minutes and washed with tap water. Once that part was done, we blotted the slides with bibulous paper and examined it under oil immersion.
Results