8-Acid-Fast Stain_needs Qs (2)

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Montgomery College *

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210

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Biology

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May 4, 2024

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docx

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3

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8 - Acid-Fast (Ziehl-Neelsen) Stain Objective/ purpose/ learning goals: After performing this lab exercise, students will be able to a. explain principles of acid-fast stain b. perform acid-fast staining procedure c. differentiate bacteria into acid-fast and non-acid-fast groups based on their appearance under microscope Key terms Carbol fuchsin, acid-fast stain, non-acid-fast stain, mycolic acid Introduction / theory/overview: The Acid-Fast stain is a differential staining technique that was first developed by Paul Ehrlich in 1882 and later modified by Franz Ziehl and Friedrich Neelsen in 1883. Therefore the acid-fast stain is also called the Ziehl-Neelsen stain . The acid-fast stain differentiates bacteria into acid- fast and non-acid-fast groups. This method is used for members of the genus Mycobacterium and Nocardia. Acid-fast microorganisms are resistant to simple or Gram staining methods. Principle of Acid-Fast Stain: As the dye carbol fuchsin is applied to a bacterial smear, it solubilizes the lipid material present in the bacterial cell wall. With application of heat, carbol fuchsin further penetrates through the lipid-wall and enters into the cytoplasm. At this point all cells appear red. When these red cells are decolorized with acid-alcohol decolorizing agent (3% HCL in 95% alcohol), the acid-fast cells are resistant to decolonization because of the presence of large amounts of mycolic acid (a specific kind of lipid) in their cell wall, which prevents the penetration of decolorizing solution. The non-acid-fast bacteria lack mycolic acid in their cell walls such that they are easily penetrated by the decolorizing agent and thus decolorized. This results in colorless cells. The smear is then counterstained with methylene blue. Only decolorized cells will absorb the counterstain, taking up its color and appear blue. Acid-fast cells do not absorb methylene blue and retain the red color. Video Links for Principle and Procedure of Acid-Fast Staining: https://www.bing.com/videos/search? q=principle+of+acid+fast+stain+video&docid=608050395440549737&mid=354ADBE14A8139 83005F354ADBE14A813983005F&view=detail&FORM=VIRE
https://www.bing.com/videos/search? q=principle+of+acid+fast+stain+video&docid=608009060688789857&mid=06E0DA9793E0A B04151006E0DA9793E0AB041510&view=detail&FORM=VIRE https://www.bing.com/videos/search? q=principle+of+acid+fast+stain+video&docid=608046706067705648&mid=9A6A60A0D590A 770CDF99A6A60A0D590A770CDF9&view=detail&FORM=VIRE Materials, equipment and organisms (per student/ per group): a. Live Organisms: Mycobacterium pheli, E.coli b. Two slides per student for smears c. Standard materials used for making, drying and heat-fixing smears, d. Staining hot plate, Carbol fuchsin dye, acid alcohol decolorizing solution, and filter paper. Procedure: a. Obtain a slide and make two smears, one for Mycobacterium pheli and one for E. coli on the slide. Take time to break the clumps of Mycobacterium when making smears before drying your smear on designated hotplate. Heat-fix the smears. Each student will make at least two slides, each with smears for both organisms side by side. b. Find the fume hood where acid-fast staining materials are located c. Put heat fixed smears onto staining hot plate in the fume hood. d. Place filter paper(s) on the heat fixed smear and use the dropper to completely saturate the filter paper with carbol fuchsin dye. e. Allow dye to stain your smear for at least 5 minutes. f. Wipe off the bottom of the slide with a wet paper towel and rinse off the dye with tap water. g. Decolorize carefully with acid alcohol (3-5 drops) for a minute and rinse your slide with water. h. Counterstain with methylene blue for a minute. i. Rinse off the counter stain, blot dry ( and place coverslip if required by your instructor). j. View your slide at 100X under oil Safety and disposal:
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