CAPSULAR STAINING(ANTHANI METHOD)
Pinciples
Many bacteria have a slimy layer surrounding them,which is usually ret'erred to as a capsule. The capsule's composition, as well as its thickness, varies with individual bacterial species. Polysaccharides, polypeptides, and glycoproteins have all been found in capsules. Often, a pathogenic bacterium with a thick capsule will be more virulent than a strain with little or no capsule since the capsule protects the bacterium against the phagocytic activity of the host's phagocytic cells. The capsule stain is of some importance in clinical microbiology (e.g., in the diagnosis of bacterial pneumonia and the fungus Cryptococcus neoformans). However, one cannot always determine if a capsule is present by simple staining procedures, such as using negative staining andIndia ink. An unstained area around a bacterial cell may be due to the separation of the cell from the surrounding stain upon drying. Two convenient procedures for determining the presence of a capsule are Anthony's capsule staining method and the Graham and Evans procedure. 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. Copper sulfate is the decolorizing agent. It removes excess primary stain as well as color from the capsule. At the same time, the copper sulfate acts as a counterstain by being absorbed into the capsule and turning it a 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.
Procedure:Capsule Staining(Anthony)
1. With a wax pencil, label the left-hand comer of a clean glass slide with the name of the bacterium that will be stained. 2. As shown in figure 15.3, aseptically transfer a loopful of culture with an inoculating loop to the slide. Allow the slide to air dry. Do not heat-fix! 3. Place the slide on a staining rack. Flood the slide with crystal violet and let stand for 4 to 7 minutes. 4. Rinse the slide thoroughly with 20% copper sulfate. 5. Blot dry with bibulous paper. 6. Examine under oil immersion (a coverslip is not necessary) and draw the respective bacteria in the report for exercise 12. Capsules appear as faint blue halos around dark blue to purple cells.
Modified Capsule Stain(Graham and Evans)
1. Thoroughly clean the slide to be used with Bon Ami and alcohol. 2. Mix two loopfuls of culture with a small amount (1 to 2 drops) of India ink at one end of the slide. 3. Spread out the drop using a second slide in the same way one prepares a thin smear. 4. Dry the smear. 5. GENTLY rinse with distilled water. 6. Stain for 1 minute with Gram's crystal violet. 7. Rinse again with water. 8. Stain for 1.5 minutes with safranin stain. 9. Rinse with water and blot dry. 10.If a capsule is present. the pink to red bacteria are surrounded by a clear zone. The background is blue-black