CAPSULE STAINING INTRODUCTION Capsule staining is done by using acidic strain and basic strain to detect the presence

of capsule. Capsules are:• • • • • • • • • Slimy covering which is also called as glycocalyx polymeric substance Usually composed of polysaccharide, polypeptide or both The ability of an organism to produce a capsule is an inherited property of the organism It is often produced only under specific growth conditions Helps bacteria to survive in nature Also helps many pathogenic and normal flora bacteria to initially resist phagocytosis by the host's phagocytic cells In soil and water, capsules help prevent bacteria from being engulfed by protozoans Capsules also help many bacteria to adhere to surfaces and thus resist flushing so not easily stained, this reason negative stain is used It also enables many bacteria to form biofilms o A biofilm consists layers of bacterial populations adhering to host cells and embedded in a common capsular mass. PRINCIPLE • • • • Capsules protect bacteria from the phagocytic action of leukocytes and allow pathogens to invade the body Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces so an acidic stain is used A stain which stains the background against which the uncolored capsule can be seen. Procedure is called the Gin's Method where india ink is used to color the background and crystal violet to stain the bacterial cell "body" or extracellular

= Negative (no clear "halos" around cells when viewed under oil immersion .• • this structure helps the bacterial cell to attach to surfaces and avoid being phagocyted Common example would be Klebsiella pneumoniae METHODS Use a loop to mix a drop of water. a drop of india ink and a small amount of Klebsiella pneumoniae together at the end of a slide Use another slide to spread the smear like a blood smear. observe. Wash with water. RESULTS + = Positive (clear "halos" around cells when viewed under oil immersion . blot. Allow the smear to air dry. dry. 1 minute. Flood the smear with crystal violet.

Terminal endospores are located at the end of the vegetative cell. such as Bacillus . Central endospores are located within the middle of the vegetative cell. By forming spores. or terminal. Spores are metabolically inactive and dehydrated. They can be central. . Subterminal endospores are located between the middle and the end of the cell. bacteria can survive in hostile conditions. These endospore characteristics are consistent within the spore-forming species and can be used to identify the organism. and radiation. Endospores can also be larger or smaller in diameter than the vegetative cell. Endospores can form within different areas of the vegetative cell.ENDOSPORE STAINING INTRODUCTION The endospore stain is a differential stain used to visualize bacterial endospores. subterminal. chemicals. The normallygrowing cell that forms the endospore is called a vegetative cell. When spores are exposed to favorable conditions. Those that are larger in diameter will produce an area of "swelling" in the vegetative cell. they can germinate into a vegetative cell within 90 minutes. Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions. They can remain viable for thousands of years. Endospores are formed by a few genera of bacteria. Spores are resistant to heat. dessication.

Thus endospores are stained green. PRINCIPLE Endospores do not stain easily but. Since malachite green is watersoluble and does not adhere well to the cell. is driven into the cells with heat. malachite green. the smear is covered with paper toweling that has been cut the same size as the microscope slide the paper is soaked with the malachite green staining solution. Vegatative cells are then decolorized with water and 0. air dry (or use a slide warmer). once stained.Because of their tough protein coats made of keratin. while vegetative cells are stained red. This keratin coat resists staining so in order to stain a spore the primary stain. allowing them to readily take up the counterstain. malchite green. Gently heat on the hot plate (just until the stain steams) for 5 to 6 minutes after the malachite green solution begins to steam. The primary stain in the endospore stain procedure.5% safranin is used to counterstain. . Spores have a durable outer coating that is composed of the protein keratin. the malachite green rinses easily from the vegetative cells. This property is the basis of the Schaeffer-Fulton or Wirtz-Conklin method of staining endospores. must be heated to drive the stain into the spores. METHOD With a wax pencil. the names of the respective bacteria placed on the edge of four clean glass slide Aseptically one species of bacterium is transferred with an inoculating loop to each of the respective slides. and heat-fix. and since the vegetative cells have been disrupted by heat.they strongly resist decolorization. Replace the malachite green solution as it evaporates so that the paper remains saturated during heating. the slide to be stained is placed on a hot plate or boiling water bath equipped with a staining loop or rack. spores are highly resistant to normal staining procedures.

A. The spores. and rinse the slide with water for 30 seconds. Vegetative Cell B. RESULTS The stain Shows a bacterium capable of endospore production. Endospore . Blot dry with bibulous paper and examine under oil immersion. vegetative cells stain red. both endospores and free spores. allow the slide to cool. Counter stain with safranin 60 -90seconds Rinse the slide with water for 30 seconds.Remove the paper using forceps. A coverslip is not necessary. stain green.

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