In this capsule staining principle and procedure post we have briefly explained about capsule staining principle, objectives, requirements, capsule staining procedure, uses and limitations.
Capsule Staining Principle and Procedure
Capsule staining procedure involves identification of capsular material. The primary function of capsule staining procedure is to differentiate capsular material from bacterial cells. A capsule is a gelatinous outer layer that surrounds and adheres to the cell wall and is secreted by bacterial cells.
The majority of capsules are made of polysaccharides, but some are made of polypeptides. The capsule is distinct from the slime layer produced by most bacterial cells in that it is a thick, detectable, and discrete layer outside the cell wall. To detect capsule production, the capsule staining procedure employs both an acidic and a basic stain.
Capsules shield bacteria from leukocyte phagocytosis, allowing pathogens to enter the body. A pathogen can become a virulent if it loses its ability to form capsules.
Capsule staining procedure very poorly with simple staining reagents, and a capsule staining procedure can be a misnomer depending on the method because the capsule may or may not be stained.
Negative staining methods contrast stained cells with an unstained capsule against a translucent, darker colored background. India ink, nigrosin, or congo red are used to create the background. Nowadays, it is difficult to obtain India ink; however, nigrosin is readily available.
A mordant that precipitates the capsule is required for a positive capsule staining procedure. By staining the bacterial cell wall with dyes such as crystal violet or methylene blue, the dye is taken up by the cell wall. Capsules appear colourless against a dark background, with stained cells.
Capsules are delicate and can be reduced, desiccated, distorted, or destroyed by heating. During smearing, a drop of serum can be added to increase the size of the capsule and make it easier to see with a standard compound light microscope.
Capsule Staining Procedure
1. Apply a little drop of a negative stain to the slide (India Ink, Congo Red, Nigrosin, or Eosin). Using a sterile technique, apply a loop of bacterial culture to the slide and smear it in the dye.
2. Drag the ink-cell combination into a thin film along the first slide with the other slide and let aside for 5-7 minutes. Allow to dry naturally (do not heat fix).
3. For around 1 minute, flood the smear with capsule staining procedure (this will stain the cells but not the capsules). Drain the crystal violet stain by tilting the slide at a 45-degree angle and allowing the stain to run off until it dries naturally.
4. Examine the smear under a microscope (100X) for encapsulated cells, which are identified by clear zones enclosing the cells.
Capsule staining procedure appearance: Clear zone between refractile cell outline against the dark background of Nigrosin stain.
Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Klebsiella pneumoniae, Escherichia coli.
Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa