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4.1: Introduction to Acid fast and Endospore Staining

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  • Learning Objectives

    • Learn about microorganisms that have acid-fast cell walls.
    • Perform the acid-fast staining procedure and view cells under oil immersion.
    • Learn about some of the microorganisms that are endospore formers.
    • Perform the endospore staining procedure and view cells under oil immersion.

    Acid-Fast Staining

    Most bacterial species are either Gram positive or Gram negative, however some organisms have different cell wall properties that make them difficult to stain with this method. For example, some species of bacteria have a waxy lipid (mycolic acid) in their cell walls. These organisms generally do not Gram stain very well (those that do would usually appear gram positive) and are more clearly visible with the acid-fast staining technique.

    Acid-fast staining was developed by Robert Koch in 1882 and later modified by other scientists. Koch used the method to observe the “tubercle bacillus”—what we now call Mycobacterium tuberculosis, in sputum samples. While acid-fast and gram staining are both differential stains, the acid-fast stain is much more specific. Many bacteria are either gram positive or gram negative, but very few are acid-fast. Two acid-fast genera that are important as human pathogens are Mycobacterium and Nocardia: Pathogenic species include M. tuberculosis, M leprae, M. bovis, M. avium, and N. asteroides. The protozoan parasite Cryptosporidium can also be stained using this procedure.

    There are 2 different methods of acid-fast staining—both involve techniques that make the cell wall more permeable to the primary stain. The Ziehl-Neelson method uses steam heat to allow stain to penetrate, whereas the Kinyoun (cold method) uses a wetting agent mixed in with the primary stain. In this lab we will be using the Kinyoun method.

    Endospore staining

    Endospores are the most resistant forms of life. They can resist desiccation (drying), boiling and radiation—in addition, disinfectants and antibiotics cannot penetrate an intact spore coat. For this reason they are difficult to eliminate from the environment with standard methods of disinfection, and they are difficult to treat in the case of an infection.

    Endospores are a survival mechanism for the bacterial species that produce them. When conditions are favorable, vegetative bacterial cells will continue to grow and divide; however when nutrients are depleted, cells will begin to form endospores. Endospores are not metabolically active, but contain all the materials needed by cells to survive. When conditions for growth are again favorable, the spore will germinate and form a cell that is identical to the cell that produced it. Endospores are produced by certain types of Gram positive bacilli, like Clostridium and Bacillus, as well as other species. Endospore-forming pathogens include C. tetani, C. botulinum, C. difficile, and B. anthracis.

    In today’s lab we will use the Schaeffer-Fulton method (without heat) to view endospores. Since we are not using heat, it is important to leave the stain on for a long time to allow it to penetrate the spore coat.

    Key Terms

    mycolic acid, endospore, carbol fuchsin, methylene blue, malachite green, Ziehl-Neelson method, Kinyoun method, Schaeffer-Fulton method

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