Acid Fast Stain Exercise 20 PDF

UJ Vl u 0::: UJ >< UJ ACID FAST STAIN LEARNING OBJECTIVES------------ 1. Define mycolicacid,including its composition and how it influences survival,growth, and pathogenicity of certain microorganisms. 2. Identify the stains/reagents use...

UJ Vl u 0::: UJ >< UJ ACID FAST STAIN LEARNING OBJECTIVES------------ 1. Define mycolicacid,including its composition and how it influences survival,growth, and pathogenicity of certain microorganisms. 2. Identify the stains/reagents used in each step of the acid-fast stain and describe how each stain/reagent affects acid-fast positive and acid-fast negative bacteria. 3. Use a bright light microscope to view a bacterial culture stained using the acid-fast stain. 4. Differentiate between acid-fast positive and acid-fast negative bacteria using bright light microscopy. INTRODUCTION-------------- The cell walls of certain gram-positive bacteria known as acid-fast bacteria can contain up to 60% of long chain a-alkyl, -hydroxy fatty acids, known as mycolic acids. These waxy lipids protect the cell from certain antibiotics and can also protect the bacteria from phagocytic destruction by macrophages. On the other hand, nutrients cannot cross the cell wall layer as quickly as they do in bacteria that do not contain mycolic acid. Therefore, acid-fast bacteria exhibit extremely slow growth both in vivo and in vitro. Mycolic acids resist staining by most regular stains, including the Gram stain. Because some acid-fast bacteria in the genera Mycobacterium, Nocardia, and Corynebacterium are patho- gens, clinical identification of these organisms in patient samples is important. In such cases, the acid-fast stain is used and can yield important information for clinical diagnosis. The acid-fast stain is another type of differential stain (see Exercise 6), and it allowsinvesti- gators to quickly identify acid-fast positive organisms-those bacteria that contain mycolic acid within their cell walls. In the acid-fast staining procedure, the primary stain, Kinyoun's carbolfuchsin (Kinyoun's acid fast stain), has a high affinity for the lipids in the cell wall of acid-fast positive organisms and stains these organisms red. Decolorization with acid alcohol does not remove this stain, so the addition of a counterstain does not affect the color of the cells. Non-acid-fast organisms, however, are decolorized by the acid alcohol and are able to be stained with Loeffler's alkaline methylene blue during the counterstain step. Accordingly, acid-fast positive organisms will appear red, and non-acid-fast organisms will appear blue following this staining procedure. THEMICROBESAROUNDUS 137  EXERCISE20 ACID FAST STAIN Students will vie a demo slide of an acid-fast stain preparation. The acid fast stain will not be performed with the environmental isolates. As mentioned previously, organisms that at contain m colic acid within their cell walls tend to grow at a much slower rate fast organisms. thann_on acid- The organisms isolated on the environmental sampling plates grew into colonies· colonies within 24 hours; therefore, it is highly unlikely that these plates contain acid-fast positive bacteria. I TODAY'S PROCEDURE----------- t ,. Your graduate teaching assistant has set up a microscope focused on t ecid-faS stain preparation of bacteria. It is each student'sresponsibility to view thisslide. 2. Record all results. - n TU Ml(ROBES AROUND US