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Acid Fast Stain Lab
Terms in this set (21)
What is an acid fast stain?
a differential stain that separates bacteria on the basis of the lipid content of their cell walls
Bacteria categorized as acid-fast (members of the genus Mycobacterium and some other Actinobacteria) have ________________ walls with ________ peptidoglycan containing large quantities of a wax- like ____________.
What is mycolic acid?
a wax-like lipid, in acid-fast cell walls
Bacteria categorized as acid-fast are _______________ to staining with normal procedures, and require severe treatment, e.g., heat and potent dyes, in order to be stained.
What is the acid-fast stain used primarily to diagnose/study?
The acid-fast stain is used primarily in the diagnosis and study of diseases caused by acid-fast bacteria (Tuberculosis and Leprosy)
What are the 3 basic steps in acid-fast staining?
1. Bacteria cells are heat-fixed and stained with a potent primary staining reagent called carbol fuchsin while being subjected to steam heat. An alternative procedure uses a different formulation of carbol fuchsin ("Kinyoun's Acid-Fast Reagent") and does not require the application of steam heat.
2. The stained cells are subjected to decolorizing with an acid-alcohol solution; 95% ethyl alcohol containing 2.5% concentrated nitric or hydrochloric acid (HNO3 or HCl). This step will remove the primary stain from cells that are not acid-fast, rendering them colorless.
3. Non-acid-fast cells are counterstained with a basic dye of a contrasting color (e.g., methylene blue).
When a mixed culture containing both acid-fast and non-acid-fast cells is subjected to this procedure, the acid-fast cells will be stained ____________ and the non-acid-fast cells will be stained ____________.
The presence of purple- colored cells usually indicates the procedure has been __________________________________; however, some cells are only partially acid-fast and others may contain acid-fast structures.
The endospores formed by bacteria in the genus Bacillus will often stain _________ when subjected to the acid-fast stain.
The acid-fast stain is a direct stain and a differential stain, what does this mean?
A differential stain is one that will cause different types of cells to look different even though they are all treated the same way.
Acid-fast cells differ from other cell types because they have high levels of _________________.
The wax-like mycolic acid in acid-fast cell walls causes them to __________________________, or to stain poorly with traditional methods.
Once stained, these (bacteria) cells are difficult to decolorize, even when treated with acid solutions. They tend be ___________________ in _________, so are "acid-fast".
Because acid-fast cells resist staining, we will treat them _____________________ and apply very _____________________ reagent over an extended period of time.
1. steam heat
2. potent stain
What is carbol fuchsin?
is made with basic fuchsin and phenol, or carbolic acid
What are the steps involved in an acid fast stain that are similar to those applied in a Gram stain?
1. Application of the primary stain, carbon fuchsin, will color all the cells present a deep red or fuchsia color.
2. Application of a decolorizing solution, will remove color from cells that are not acid-fast.
3. Application of a counterstain reagent, methylene blue, will color the cells that are not acid-fast, blue
In what ways is the acid-fast stain similar to the Gram stain?
Both the Gram stain and the acid-fast stain are basic stains and both are differential stains that cause differences in cell wall composition to be visible as color differences.
What is mycolic acid and how does it influence staining?
Mycolic acid is the wax-like lipid present in the walls of acid-fast cells. This material makes cells resistant to staining, but also makes them resistant to being decolorized once they have been successfully stained.
Are Gram-negative bacteria likely to be acid-fast?
All acid-fast bacteria are Gram-positive, so no, Gram-negative bacteria are not likely to be acid-fast.
What would you expect to observe if you decolorized an acid-fast stain preparation with acetone- alchol instead of acid-alcohol?
Acetone-alcohol is the decolorizing reagent used in the Gram stain. If this material is used to decolorize cells stained with carbol fuchsin, the red color will not come out of any cells, and non-acid-fast cells will appear to be acid-fast, i.e., will stain red. It is important to use the correct decolorizing reagents when preparing different types of differential stains.
What color do the acid-fast cells appear when stained with the acid-fast stain preparation?
Acid-fast cells stain red, while other cells appear blue (when methylene blue is used as the counterstain).
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