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The acid-fast stain is a microbiological technique used to differentiate bacteria based on the lipid content of their cell walls. the steps for performing the acid-fast stain using both steam heat and Kinyoun's Acid-Fast Reagent. The process involves heat-fixing and staining cells with carbol fuchsin, decolorizing with acid-alcohol, and counterstaining with methylene blue. The document also includes questions related to the acid-fast stain, such as its similarities to the Gram stain, the role of mycolic acid, and the likelihood of Gram-negative bacteria being acid-fast.
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Introduction The acid-fast stain is 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 Gram-positive walls with thick peptidoglycan containing large quantities of a wax- like lipid called mycolic acid. These cells are resistant to staining with normal procedures, and require severe treatment, e.g., heat and potent dyes, in order to be stained. Once stained, their walls do not readily decolorize, and so can be differentiated from non-acid-fast cells. The acid-fast stain is used primarily in the diagnosis and study of diseases caused by acid-fast bacteria (Tuberculosis and Leprosy), however it can also be used on a variety of non-pathogenic organisms. The basic steps in this technique are as follows:
Name ________________________________ Lab Section _______________ WORKSHEET Exercise 6C Staining of Microorganisms: Acid-Fast Stain Goals: __________________________________________________________________________
Materials & Methods: Age of Unknown: ____________ Reagents used: ________________________________________
Data & Results: A) Acid-Fast Positive & Negative Controls B) Morphological unknown Specimen: Total Magnification: Length: units x μm/unit = μm Width: units x μm/unit = μm Notes: Specimen: Total Magnification: Length: units x μm/unit = μm Width: units x μm/unit = μm Notes:
Acid-Fast Results Summary: Specimen Data Result NOTE : If your controls do not appear as expected, SOMETHING IS WRONG. Consult your instructor. Conclusions: Based on your results, what can you conclude about the cell walls of your Morph. Unknown? _____
Does the cell morphology of your Morphological Unknown match what you saw for Exercise 6A (indirect stain) and 6B (Gram stain)? ___________ Additional Comments: ______________________________________________________________