Clean your microscope with lens cleaner, removing all oil from lenses.You may need to decrease the amount of light in order to make the capsule easier to see. Results: Look for purple cells surrounded by a clear or faint blue halo on transparent background. DO NOT RINSE WITH WATER! Water will remove the capsule from the cell. Tilt the slide and rinse with 20% copper sulfate solution.This is done by contacting the drop of crystal violet with the clean microscope slide and using the capillary action of the dye/ slide to spread the crystal violet across the smear. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism.If you are adding bacteria from a culture plate make sure that there are no large clumps of the organism, but try to avoid spreading the drop. Using a flamed loop and sterile technique, add three loopful of test bacterium (any capsulated bacteria such as Klebsiella pneumoniae, Streptococcus pneumoniae) from broth culture.Place a single drop of crystal violet on a clean microscope slide, adjacent to the frosted edge.Results: Look for purple cells surrounded by a clear halo on a dark background. DO NOT blot the slide! Blotting will remove the bacteria from the slide and/or distort the capsule. Let the slide air dry for a few minutes.Be cautious w ater may remove the capsule from the cell. Saturate the slide with crystal violet for 1 minute and rinse slightly & very gently with water.DO NOT heat or blot dry! Heat will melt the capsule! Allow the film to air dry (will take 5-7 minutes).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. Be sure there are no large clumps of organism, but try to avoid spreading the drop. Using a flamed loop and sterile technique, remove some Klebsiella pneumoniae from culture tube or plate and mix it into the drop of India ink.Place a single drop of India ink on a clean microscope slide, adjacent to the frosted edge.India Ink MethodĬapsule staining by India ink method (at 1000x magnification) Tissue paper Capsule Stain procedure A.Light Microscope with 100x objective lens (oil immersion).Stain solutions: Depending on the method used (crystal violet, India ink, Nigrosin, copper sulfate, Basic carbol fuschin solution, methylene blue solution, etc).Klebsiella pneumoniae growing on a slant of EMB agar or culture of other capsulated bacteria and non-capsulated bacteria Test bacteria: 36-48 hour culture of capsulated bacteria e.g.Thus, the capsule appears as a faint blue halo around a purple cell. As the copper sulfate decolorizes the capsule, it also counterstains the capsule. It decolorizes the capsule by washing out the crystal violet, but will not decolorize the cell. A 20% copper sulfate solution serves a dual role as both the decolorizing agent and counterstain. There is no mordant in the capsule staining procedure. In this type of capsule staining procedure, the primary stain is crystal violet, and all parts of the cell take up the purple crystal violet stain.

The capsule (if present) will appear clear against the dark background (capsule does not take any stain).Įxpert opinion: Tasha Sturm, Microbiologist at Cabrillo College, “I use Nigrosin instead of India ink as Nigrosin gives a more even background and spreads little easier”.The bacterial cells will be stained purple (bacterial cells take crystal violet-basic dyes as they are negatively charged).The background will be dark (color of India ink).This method is used for demonstrating Cryptococcus. The capsule is seen as a clear halo around the microorganism against the black background. In this method, two dyes, crystal violet, and India ink are used. Two commonly used methods are discussed here: A. The results (stain of the cells, background, and capsule) depend on the method used. Various types of methods are available for the demonstration of bacterial capsules.

India ink staining crytpococcois