Endospore Staining The name "endospore" is suggestive of a spore or seed-like form (endo- means within). Resting structures formed by some bacteria for survival during adverse environmental conditions (nutrient limitation or extreme environments). Spores are resistant to heat and chemicals because of a tough outer covering made of the protein keratin. The keratin also resists staining, so extreme measures must be taken to stain the spore.

Metabolically inactive and dehydrated. Stable for years. Endospores can remain dormant indefinitely (not reproductive), but germinate quickly when the appropriate trigger is applied. Metabolically inactive and dehydrated. Stable for years. Endospores differ significantly from the vegetative , or normally functioning cells. Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions and germinate into a vegetative cell within 1.5 hour. Sporulation (sporogenesis) Bacteria (vegetative cell) Endospore Germination

Spore formed by Gram-positive bacteria.

Endospore Location The position of the endospore differs among bacterial species and is useful in identification. When describing spore-forming bacteria, the location of the endospore is usually stated as: central (B. cereus, B. anthracis) Terminal (C. tetani) subterminal (B. subtilis)

Endospore Shape Round (spherical) Oval (elliptical) Cylindrical (elongated)

Endospore Size Endospores can also be larger or smaller in diameter than the vegetative cell. Those that are larger in diameter will produce an area of "swelling" in the vegetative cell.

Endospore Staining (Schaeffer-fulton Method) Make smears of each organism on separate slides. Allow the slides to air dry, and then heat fix. Apply a few drops of malachite green to the bacteria. Place the slides directly on a pre-warmed hot plate(enhance malachite green penetration) set on low for 3-5 minutes. Do not allow the stain to evaporate . Apply additional stain, if necessary. Remove the slides from the hot plate and allow them to cool. Gently rinse the slides with water. Apply enough safranin to the slides to cover the bacteria. Allow it to set for 1-2 minutes.

Gently rinse the slides with water. Blot (don't wipe) the slides with bibulous paper. Allow the slides to air dry. Examine the slides under oil immersion. The bacteria should appear pink; the spores should appear green.

Note: In spore staining, water is sufficient to be used as decolorizer because: Malachite green dye(copper salt) is water-soluble and does not adhere well to the cell wall. Vegetative cells have been disrupted by heat, because of these reasons, the malachite green rinses easily from the vegetative cells. 

Capsular Staining Capsules are a gelatinous outer layer structure secreted by bacterial cell that lay outside of an organism's cell wall and thus are in direct contact with the environment. Capsule is synthesized in the cytoplasm and secreted to the outside of the cell where it surrounds the bacterium. Many, perhaps most, bacteria produce capsules under the right conditions.

Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. Most capsules are composed of polysaccharides, but some are composed of polypeptides. Mucoid colonies showing viscous or sticky growth typical of an organism producing large quantities of a carbohydrate capsule .

Capsules Functions Protect the cell from desiccation (drying). Protect the cell from phagocytes (being engulfed by white blood cells). Provide a food reserve when certain organic compounds are in excess. A virulence determinant of pathogenic microbes. They serve as binding or adhesion agents for sticking cells together and/or to a surface such as a rock in flowing stream or a tooth.

Theory Behind Capsule Stain Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces Because most capsule materials are water soluble, simple stains will not adhere to them. Older cultures are more likely to exhibit capsule production.  When performing a capsule stain on your unknown, be sure the culture you take your sample from is at least five days old. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed with a typical compound light microscope.

Capsular Stain Methods Gin’s (India ink) Method In this stain we use acidic and basic dyes: Acidic dye as India Ink and Nigrosen use to stain the background of the slide but basic dye as methylene blue and crystal violet use to stain the cell . The India ink gives a semi opaque background against which the clear capsules can be easily visualized.

Gin’s Method Procedure Use an inoculating needle to suspend the organism in a drop of India Ink at one end of the slide. Place the short end of a clean microscope slide into the suspension and spread the mixture across the slide to form a thin layer. Allow to air dry. Do not heat fix. Cover the smear with methylene blue for 2-3 minutes. Rinse gently with water and allow to air dry. Examine with oil immersion. Diagram the appearance of the organism.

Interpretation Capsules appear as clear zones (halos/transparent) around the refractile organism. Examples: Bacteria with capsules: Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas .

Anthony’s Method Anthony’s procedure employs two reagents. The primary stain is crystal violet, which gives the bacterial cell and its capsular material a dark purple color. Unlike the cell, the capsule is nonionic and the primary stain cannot adhere. A 20% copper sulfate solution(CuSO4) serves a dual role as both the decolorizing agent and counter stain. It removes excess primary stain as well as color from the capsule only and capsule appear faint/light blue. In this procedure, smears should not be heat-fixed since shrinkage is likely to occur and create a clear zone around the bacterium, which can be mistaken for a capsule.

Anthony’s Method Procedure Aseptically transfer a loopful of culture with an inoculating loop to the slide. Allow the slide to air dry. Do not heat-fix! . Place the slide on a staining rack. Flood the slide with crystal violet and let stand for 4 to 7 minutes. Rinse the slide thoroughly with 20% copper sulfate . Blot dry with bibulous paper. Examine under oil immersion (a coverslip is not necessary) Capsules appear as faint halos around dark cells.