Stain Protocols-BIOL 2420 Capsule Stain 1.Place a single drop of India ink on a clean microscope slide,adjacent to the frosted adge 2.Using a flamed loop and sterile technique,remove some K.pneumoniae (or the organism you want to stain)from your tube or plate and mix it into the drop of India ink.Be sure there are no large clumps of organism,but try to avoid spreading the drop. 3.Place the end of another clean microscope slide at an angle to the end of the slide containing the organism.Spread out the drop out into a film.This is done by contacting the drop of India ink with the clean microscope slide and using the capillary action of the dye/slide to spread the India ink across the smear.Refer to the Negative Stain portion of this handout for a diagram. 4.Allow the film to air dry.DO NOT heat or blot dry!!!!Heat will melt the capsule! 5.Saturate the slide with crystal violet for 1 minute. 6.Rinse the slide gently with water. 7.Allow the slide to air dry.DO NOT heat or blot dry!!!!Heat will melt the capsule! 8.Observe the slide under the microscope,using proper microscope technique The background will be dark. The bacterial cells will be stained purple. The capsule(if present)will appear clear against the dark background. background capsule bacterium 0 TIP:If you are stainin ism he sure ur culture is several days old.Young fresh cult on't ha veloped capsules yet. Smith-2010 Page3of4Stain Protocols – BIOL 2420 Smith – 2010 Page 3 of 4 Capsule Stain 1. Place a single drop of India ink on a clean microscope slide, adjacent to the frosted adge. 2. Using a flamed loop and sterile technique, remove some K. pneumoniae (or the organism you want to stain) from your tube or plate and mix it into the drop of India ink. Be sure there are no large clumps of organism, but try to avoid spreading the drop. 3. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a film. This is done by contacting the drop of India ink with the clean microscope slide and using the capillary action of the dye/ slide to spread the India ink across the smear. Refer to the Negative Stain portion of this handout for a diagram. 4. Allow the film to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule! 5. Saturate the slide with crystal violet for 1 minute. 6. Rinse the slide gently with water. 7. Allow the slide to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule! 8. Observe the slide under the microscope, using proper microscope technique. The background will be dark. The bacterial cells will be stained purple. The capsule (if present) will appear clear against the dark background. background capsule bacterium TIP: If you are staining an unknown organism, be sure your culture is several days old. Young, fresh cultures won’t have developed capsules yet