Lab 2D - Acid-fast [Final].pdf

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Microbiology and Immun (Sci)
MIMM 212
Samantha Gruenheid

A CID-FAST:DIFFERENTIAL STAINING FOR DETECTING M YCOBACTERIUM Yannan Liu - 260469551 TA: Benjamin Ralph Cubicle: C-10 Due: September, 25, 2012 MIMM 212 2 Yannan Liu – 260469551 Abstract The acid-fast staining technique was examined in this experiment on Mycobacterium smegmatis and Staphlyococcus aureus to detect acid-fast bacteria. Ziehl’s carbol fuchsin was used as the primary dye, and stained red, while methylene blue was used after decolourization by acid alcohol, and stained blue. Results indicated that the M. smegmatis stained red and the S. aureus stained blue and any deviations from this ideal result may have been caused by heavy decolourization or other errors. It was ultimately concluded that the M. smegmatis was the acid- fast bacteria due to its colouring after the experiment. Introduction Acid-fast staining is a technique used to treat certain bacteria that cannot be stained with water based chemicals. Bacteria with thick waxy cell walls, such as those of the genus Mycobacterium, are called acid-fast because their walls have a much higher concentration of lipids and are thus hydrophobic (or “water hating”) and repel the water based stains, permitting only staining via the acid-fast method. Acid-fast staining employs the use of a primary stain which is soluble in the bacteria’s cell wall, followed by a decolourization and a counterstain with another chemical. Heat fixing is used in the experiment to both fix the bacteria onto the slides as well as to weaken the cell wall for easier stain access. Through such procedures, acid-fast bacteria will retain the colouration of the primary stain (as with its thick cell wall) and non-acid- fast bacteria will be more prone to decolourization and retain the counterstain. In this experiment, the primary stain used was Ziehl’s carbol fuchsin, which is of a deep red colour comprised of basic fuchsin dissolved in phenol, acid alcohol for decolourization and a MIMM 212 3 Yannan Liu – 260469551 counterstain of methylene blue, which is a shade of dark blue. According to this, we believe that Mycobacterium smegmatis will stain red and Staphylococcus aureus will stain blue. Methods In order to apply the acid-fast stain via the Ziehl-Neelsen method, bacteria are first prepared on slides by heat fixing. Bacteria are extracted from either broth or solid media using a sterilized inoculating loop and placed on microscope slides. For broth mixtures, several drops are needed to ensure there is enough bacteria on the slide. For solid media, however, water must be added and mixed with the solid bacteria to make a similar liquid mixture. (For the bacteria in the Mycobacterium genus, serum is used instead of water.) This mixture is then spread out on the slide as to avoid dense concentrations of bacteria, which would be hard to examine under the microscope. The slide is then passed above a flame to denature the proteins and thus adheres the bacteria to the slide. After heat fixing, Ziehl’s Carbol Fuchsin is added to the slides on a staining rack. The slides are then heated from below with a flame until the mixture is steaming, but not boiling. As the slides dry, more carbol fuchsin is added. This is done for three to five minutes. The slides will then be washed with water, treated with acid alcohol for 10 to 30 seconds then washed again with water. Methylene blue is then applied for 30 to 45 seconds before washing the slide again and blotting it dry. The resulting slides are then observed under a microscope using oil immersion. Results When viewing the slides, we found varying results with both slides. From simply looki
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