lab_09_differential_and_special_staining - Miramar College...

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Differential & Special Staining Page 1 of 6 Miramar College Biology 205 Microbiology Lab Exercise 9: Differential & Special Staining Background Special Staining: The Negative Stain All cellular stains can be categorized as either basic/positive stains or acidic/negative stains . Because of the charge associated with these stains, they will adhere to or be repelled by the cell because of the negative resting potential of most all cells (-70mV). Positive stains adhere to and color the cell, while negative stains are repelled by the cell. Because the cells repel the negative stain, it is the background which is colored and cells appear transparent (Figure 1). While positive staining is most often used in the microbiology lab, negative staining can prove invaluable for determining the size and shape of a bacterial cell because negative staining does not employ heat fixing and so does not shrink or distort the cell. In addition, some cells ( e.g . spirochetes) do not stain readily and so cannot be seen using conventional positive staining techniques. Figure 1: Negative stain of Staphylococcus aureus using the negative stain India ink. Cells appear clear against a dark background. Differential Staining: The Spore Stain Certain bacterial species, most commonly Gram positive bacilli, such as those of the genera Bacillus and Clostridium , undergo a complex developmental cycle when faced with environmental adversity that produces a resting endospore . The process of sporulation allows bacteria to survive in harsh environmental conditions where low nutrients, high temperatures, UV radiation, acids and/or toxic chemicals might otherwise prove fatal. If conditions improve, the spore may germinate to form a new vegetative cell and growth will resume. Endospores are very dehydrated structures that are not metabolically active. They possess a protein coat, called an exosporium that forms a barrier around the spore. Because endospores are not easily destroyed by heat or chemicals, they define the conditions necessary to establish sterility. To destroy endospores by heating, they must be exposed for 15–20 minutes to pressurized steam (as found in an autoclave) which generates temperatures of 121°C. The resistant properties of spores also means they are not easily penetrated by stains. They are not readily stained during the Gram stain procedure and appear as clear ovals in a stained cell and causing the tell-tale Gram positive irregular staining result you learned about in Lab Exercise 7 . However, if heat is applied during staining with malachite green , stain penetrates the endospore and becomes trapped as the preparation cools (Figure 2). In this instance, heat is acting as a mordant to facilitate the uptake of the stain. Vegetative cells are counterstained using safranin . Figure 2:
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This note was uploaded on 12/23/2009 for the course BIO 205 taught by Professor Murphy during the Fall '09 term at Miramar College.

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lab_09_differential_and_special_staining - Miramar College...

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