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Acid-Alcohol for slide and specimen preparation

Acid-Alcohol is a simple mixture compounded from an acid and an alcohol, it's usual method of use is in bleaching or decolourising samples that contain a mixture of cells, some of which are resistant to acid bleaching and others that are not. These samples have usually been previously stained. The acid is generally hydrochloric and the alcohol can be ethanol or a mixture of ethanol, isopropanol and methanol, but other recipes are used in some cases.

The table below gives some typical recipes, but others may be found for special purposes and any other percentages can be calculated from the information the table contains.

SolnHclEthanolMethanolIsopropanolH2O
0.5%O.5 ml69.8 ml29.7ml
1%1 ml69.3 ml29.7ml
3%3 ml87 ml5 ml5 ml
3%3 ml92 ml5 ml

In order to illustrate the way that it may be used, the Ziehl-Neelsen method of staining acid-fast bacteria is described.

Acid-fast mycobacteria usually appear as slender, rod-shaped bacilli with dimensions in the range 1 to 10 µm long by 0.2 to 0.6 µm wide. They are generally straight, but can appear curved. The high lipid content (about 60%) of the cell wall makes the bacteria resistant to penetration by many dyes and chemicals.

  1. The prepared slide is first stained with hot Carbol-Fuchsin, which is a red dye that contains detergents. All the bacteria that are present become stained red.
  2. The slide is washed with acid-alcohol. The bacteria that retain the red dye are termed 'acid-fast bacteria'.
  3. The slide is then counterstained with Methylene blue dye (Swiss blue). Those bacteria that have been bleached turn blue, the acid-fast bacteria remain red, giving a colour differential that can easily be seen.

A similar procedure, known as the Kinyoun method uses a wetting agent in the primary dye formulation, removing the need for heating.