Re: Acid-Fast Bacillus

From:"Ms. Evelyn Kaplan"

Hi Mary Ann,
I will answer your question in two parts.
1. The principle of the ZN/Wade-Fite methods: mycobacterium possess a capsule containing a long-chain fatty acid, mycolic acid, and makes them  hydrophobic.  This fatty capsule influences the penetration and resistance to removal of stain by acid and alcohol. It takes a great deal of effort to force a stain through this capsule into the organism. Phenol/heat are used to reduce the surface tension, increasing porosity and forcing dyes to penetrate this capsule. The speed of removal by differentiation with acid/alcohol of the primary dye is proportional to the extent of the fatty coat. The basis of the technique is that the red dye basic fuchsin is forced in to the bacteria and other substances and then is removed from the other structures with acid/alcohol. The avoidance of defatting agents such as alcohol and xylene in methods for M.leprae are an attempt to save its fragile fatty capsule. This is because it is thought that M.leprae is much less acid/alcohol fast and is frequently decolourised by the ZN method.
2. In the south west region of England, the Cellular Pathology Technical Audit Group run an EQA scheme. Several hospitals take part and every two months, sections are sent to all participants along with instructions of which methods to carry out. On one particular run, a Wade-Fite was requested on a known positive control for M.leprae. Only 2 participants got a positive result. On questioning at the next audit group meeting, only two hospitals had actually carried out the Wade-Fite method and hadn't thought it necessary to carry out the "gentle" de-waxing procedure. Everyone else had actually carried out a ZN method, and their tests came up negative. It is not just the gentle dewaxing which preserves the fatty coat in the WF method; it is also the fact that after the oil/xylene step, sections are blotted dry instead of going through the alcohols to water; decolourisation is carried out by using 10% sulphuric acid; after counterstaining, sections are dehydrated by blotting and drying in an oven before coverslipping.
Evelyn Kaplan,
Sultan Qaboos University,
Muscat, Oman

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