Acid-Fast Stain Questions and Answers

Acid-Fast Stain Questions and Answers bacteria used in acid-fast stain Mycobacterium nonchromogenicum & Staphylococcus epidermidis acid-fast stain is a differential stain Previous Play Next Rewind 10 seconds Move forward 10 seconds Unmute 0:00 / 0:15 Full screen Brainpower Read More acid-fast cell wall contains large amount of ____ and ____ linked to the peptidoglycan layer glycolipids; mycolic acids waxy structure of acid-fast cell walls makes the cell wall hydrophobic acid-fast cell wall exclusive to the genera Mycobacterium & Nocardia Acid-fast stain can be used as an important test for pathogenic species of Mycobacterium such as M. tuberculosis M. leprae M. avium-intracellulare complex (MAC) has a higher affinity for lipids than for acid-alcohol carbol fuchsin acid fast bacteria stain red/pink non-acid-fast bacteria stain blue primary stain in acid-fast staining carbol fuchsin Carbol fuchsin contains 5% phenol decolorizing agent in acid-fast staining acid alcohol counterstain in acid-fast staining methylene blue Acid-fast staining method used in lab Ziehls-Neelsen Method Acid-fast bacteria used Mycobacterium nonchromogenicum non-acid fast bacteria used S. epidermidis Acid-fast staining technique 1) prepare slide with mixed culture, air dry 2) heat fix 3) carbol fuchsin, 60 sec 4) heat slide for 2-3 mins with carbol fuchsin 5) cool slide 6) rinse with water 7) acid alcohol - decolorizer 8) methylene blue, 60 sec 9) rinse with water 10) blot with bibulous paper 11) examine under oil immersion Acid-fast bacteria differ from other bacteria because: - cell wall contains glycolipids and mycolic acids - hydrophobic cell wall (make them resistant to disinfectants & desiccation, drying out) Name and explain the role of the different reagents used in an acid-fast stain. Primary stain: Decolorizer: Counterstain: Primary stain: carbol fuchsin - basic red dye that stains both acid-fast and non-acid-fast bacteria. Decolorizer: acid alcohol - washing out primary stain from non-acid-fast bacteria, leaving them colorless while the acid-fast bacteria remains red/hot pink Counterstain: methylene blue - used to stain the decolorized non-acid-fast bacteria so blue. What would happen to both acid-fast and non-acid-fast cells if no heat was used during the staining procedure (after heat fixing the slide)? The acid-fast stain would not be colored, as the heat is necessary to "force" the primary stain, carbol fuchsin, into the cell wall. The non-acid fast stain would still decolorize after the primary stain, and would stain blue after using the methylene blue counterstain. How would you interpret your results if you performed the acid-fast stain on M. smegmatis and saw that the cells were stained blue? M. smegmatis doesn't have an acid-fast cell wall. Often the tuberculosis organism is arranged in "cords". Do you think that the structure of these "cords" plays a role in disease initiation and resistance to the body's primary defenses? Explain your answer. Yes, these "cords" play a role in disease initiation and resistance to the body's primary defense. They are a virulence factor of tuberculosis, and these cords make the Mycobacterium tuberculosis highly resistant to antibiotics and the body is unable to fight it. Compare and contrast the acid-fast stain with the gram stain. (Include theory, reagents, and procedure.) Both the gram stain and the acid-fast stain are differential and staining is based on cell wall components. Gram staining is based on the amount of peptidoglycan present in the cell wall. Acid fast staining is based on the presence of glycolipids and mycolic acids. The gram stain stain helps distinguish between gram positive cell walls (thick layer of peptidoglycan) and gram negative cell walls (thin layer of peptidoglycan with outer membrane composed of lipids). Gram positive cells will appear purple, and the gram negative cells should appear pink/red when viewing them under the microscope. The acid-fast stain specifically helps test for pathogenic species of Mycobacterium, which are hydrophobic due to having a waxy cell wall structure containing large amounts of glycolipids and mycolic acids connected to the peptidoglycan layer. Gram stain: primary stain - crystal violet (60 seconds) mordant - gram's iodine (60 seconds) decolorizing agent - acetone-alcohol mixture (15 seconds) counterstain - safranin (60 seconds) view under oil immersion No heating the slide with the dye on it in gram staining Acid-fast stain: primary stain - carbol fuchsin (60 seconds) heat slide w/ carbol fuchsin stain for 2-3 minutes on warmer w/o boiling or drying it out UNLIKE GRAM STAIN, ACID-FAST STAINING USES NO MORDANT decolorizing agent - acid alcohol (30 seconds) counterstain - methylene blue (1 minute) view under oil immersion