Capsule staining (Anthony method): is atypical and cells that have a gelatinous capsule (complex polysaccharides, levans, dextrans, cellulose) are often by nature virulent. Capsule staining is difficult because the capsule is water-soluble—easy to washout stain or destroy the capsule. There are 2 reagents.
The primary stain is crystal violet (stains cell and capsule).
The decolorizing agent is copper sulfate (20%) which takes out the primary stain from the capsule but does not remove color bound to the cell wall. Copper sulfate will also bind to the capsular material.
Clinical significance. Helps to identify capsule-forming Gram-negative bacteria such as Haemophilus influenzae and Klebsiella pneumoniae. Capsule-forming Gram-positive bacteria include Bacillus anthracis and Streptococcus pneumoniae.
Summary of procedure.
- On a clean slide, put several drops of crystal violet.
- Inoculate the slide (with crystal violet) with 3 loopfuls of a culture.
- Using another clean slide, smear the first slide such that the inoculated crystal violet spreads thinly across.
- Allow this to stand for 5-7 minutes such that it is dry.
- Do not heatfix.
- Gently flush smear with copper sulfate solution.
- Gently blot with bibulous paper.
Reference
Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.
