WHAT HAVE I LEARN?
This week is we will be doing gram staining and acid- fast staining. Both of them are related to the cell wall composition. The time control of gram staining and acid- fast stain is very important as a little bit change will influence the results. Gram stain is under differential staining which require need of four chemical reagents. Primary stain used in gram staining are Crystal's violet, is to impart colours to all cells. Next is the mordant, Gram's iodine which used to intensify colour of primary stain. To establish a colour contrast, decolorizing agent is used which may or may not remove the primary stain. Decolorizing agent used is 95% ethyl alcohol. The last one is counterstain, safranin has a contrasting colour to that of primary stain. Gram- positive cells have thick peptidoglycan which appear blue in gram staining while gram- negative cells have thin peptidoglycan which appear pink in gram staining.
The genus Mycobacterium are visualised more clearly by acid- fast method. They have thick, waxy ( lipoidal) wall that makes penetration by stains extremely difficult. Primary stain used carbol fuchsin that is soluble in lipoidal materials can penetrate Mycobacterium and is retained. Application of heat in Ziehl- Neelsen method can drives carbol fuchsin throught lipoidal wall and into cytoplasm. Acid- fast cell able resistant to decolorization od acid- alcohol since primary stain is more soluble in cellular waxes. Counterstain used methylene blue to stain previously decolorized cells. Acid- fast cells retain red of primary stain while non- acid- fast cells stained and become blue in colour.
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