14
Sep
This unit describes two methods of capsule staining. This unit describes two methods of capsule staining.
Since the surface of most bacterial cells is negatively charged the cell surface repels the stain.
India ink stain bacteria. Unfortunately capsules do not stain well with crystal violet methylene blue or other simple stains. This unit describes two methods of capsule staining. The first is a wet-mount method using india ink.
The capsule is visualized as a refractile zone surrounding a cell. Slime layers and capsules are produced by many bacteria and are best demonstrated by wet preparations because their highly hydrated polymers shrink when fixed and dried. Dulguid Indian ink is the simplest capsule stain.
Many bacteria will produce as a result of metabolism inclusion bodies within the cell. Routine CSF tests include cell count with differential protein glucose Gram stain other special stains including India ink Cryptococcus venereal disease research laboratory VDRL test and cultures including polymerase chain reaction PCR for herpes simplex virus HSV varicella-zoster virus VZV and JC viruses if indicated. India Ink or Nigrosin is an acidic stain.
Since the surface of most bacterial cells is negatively charged the cell surface repels the stain. The glass of the slide will stain but the bacterial. Capsule stain is a type of differential stain which uses acidic and basic dyes to stain background bacterial cells respectively so that presence of capsule.
India ink is difficult to obtain nowadays. However nigrosin is easily acquired. A positive capsule stain requires a mordant that precipitates the capsule.
By counterstaining with dyes like crystal violet or methylene blue bacterial cell wall takes up the dye. Capsules appear colourless with stained cells against dark background. Hardy Diagnostics India Ink is recommended for use in the identification of Cryptococcus neoformans.
Cryptococcus neoformans because of its large polysaccharide capsule can be visualized by the India Ink stain. Organisms that possess a polysaccharide capsule exhibit a halo around the cell against the black background created by the India Ink. Commonly used microbiological stains generally fall into one of two categories - basic stains or acidic stains although there are a few stains such as India Ink which are neutral.
A basic dye is a stain that is positively charged and will therefore react with material that is negatively charged. Bacterial cells have a slight negative charge will therefore attract and bind with basic dyes. India ink is used as a negative staining technique to demonstrate capsule.
This video shows preparation of wet India ink mount for demonstration of Cryptococ. Principle of Negative Staining Negative staining requires the use of an acidic stain such as India ink or nigrosin. The acidic stain with its negatively charged chromogen will not penetrate the cells because of the negative charge on the surface of bacteria.
Therefore the unstained cells are easily discernible against the colored background. Notice how the EPS stains blue Gram positive but the bacteria inside the EPS remain pink in color Gram negative. Zoogloea as EPS Producers Zoogloea is a genus of Gram negative bacteria which include Zoogloea ramigera.
The pink slimy areas show the high polysaccharide coating produced by undesirable bacteria in a wastewater treatment system. You can also perform an India ink staining on a biomass sample to check the levels of polysaccharide coating in the system. It is used to determine if the polysaccharide coating is normal or elevated.
The Bacterial cell body is stained best with the Basic stains like Crystal violet Safranine Methylene blue etc. Whereas the background is stained best with the Acidic stains which include the India ink Nigrosin Eosin Congo red etc. There are numerous methods available for the demonstration of the capsule in bacterial cells.
Used as a negative stain in place of India ink for spirochetes bacteria protozoa and fungi.