The primary purpose of capsular staining is to distinguish capsular material from bacterial cells. A capsule is an outer layer of gelatin that is secreted by bacterial cells and surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides.
The capsule is a thick, clear layer outside the cell wall, unlike the mucus layer produced by most bacterial cells. Acidic and alkaline stain is used for capsule staining to detect capsule formation.
What is the Principle of Capsule Staining?
Capsule staining is a type of differential staining that uses acidic and basic dyes to stain the background and bacterial cells, respectively, to easily visualize the presence of capsules. The capsule is synthesized in the cytoplasm and secreted outside the cell, where it surrounds the bacterium.
Most encapsulated bacteria have capsules made of polysaccharide layers, but some bacteria have capsules made of polypeptides or glycoproteins.
Capsules are associated with the virulence of several microorganisms, including Streptococcus pneumoniae and Neisseria meningitides. This is because the capsule resists phagocytosis and evades the host’s immune system.
Capsule staining can be misleading because capsules are rarely stained with reagents used for simple staining, and depending on the method, capsules may or may not be stained.
Negative staining contrasts with the transparent dark background and stained cells, but the capsules are colorless. The background is made with nigrosin or Congo red ink. India ink is hard to obtain nowadays. However, nigrosine is easily mined.
If the capsule stain is positive, a desiccant is needed to precipitate the capsules. When stained with a dye such as crystal violet or methylene blue, the bacterial cell wall absorbs the dye. Capsules appear colorless with staining of cells on a dark background.
Capsules are fragile and can be shrunk, dried, deformed, or destroyed when heated. A drop of serum may be used during the smear to increase the size of the capsule and facilitate observation with a compound light microscope.
Reagents used for Capsule Staining
Crystal Violet (1%)
- Crystal Violet (85% dye content) = 1 gm
- Distilled Water = 100 ml
- Nigrosine, water-soluble = 10 gm
- Distilled Water = 100 ml
The procedure of Capsule Staining
Place a drop of negative stain (India ink, Congo red, nigrosin, or eosin) on the slide.
Congo red is easier to spot, but depending on the type, it doesn’t show up as well. India ink usually works, but contains small particles that exhibit Brownian motion that must be distinguished from bacteria. Nigrosin may need to be kept very dilute.
Using the aseptic technique, add a loopful of bacterial culture to the slide and fill it with dye.
Using the second slide, spread the ink-cell mixture along the first slide in a thin layer and leave for 5-7 minutes.
Allow to air dry (do not heat fix).
Immerse the smear in crystal violet (which stains the cells but not the capsules) for about 1 minute. Tilt the slide at a 45° angle to remove the crystal violet until air dry.
Examine the smear under a microscope (at 100x magnification) for the presence of encapsulated cells. This is indicated by a light area (clear Zone) around the cell.
Result of Capsule Staining
- Capsule: Clear halos zone against a dark background
- No Capsule: No Clear halos zone
Examples of Capsule Positive and Negative
Bacillus anthracis, Klebsiella pneumoniae, Streptococcus pneumonia Neisseria meningitidis Clostridium spp, Rhizaobium spp, etc.
Neisseria gonorrhoreae, etc.
Mneomonics to remember capsulated bacteria- Some Killers Have Pretty Nice Capsule
- Streptococcus pneumoniae
- Klebsiella pneumoniae
- Haemophilus influenzae
- Pseudomonas aeruginosa
- Neisseria meningitidis
- Cryptococcus neoformans
Quality control of Capsule Staining
- Positive control: Klebsiella pneumoniae (ATCC e13883)
- Negative control: Alacilgenes denitrificans (ATCC 15173)