Principle and Procedure of Acid Fast Staining: Acid-fast staining is another widely used differential staining procedure in bacteriology. This stain was developed by Paul Ehrlich in 1882. Ziehl and Neelsen independently proposed acid-fast stains in 1882-1883, which are commonly used today. Some bacteria resist decolourisation by both acid and alcohol and hence they are referred to as acid-fast organisms. Acid-alcohol (3% HCl 95% ethanol) is a very intensive decolouriser. This staining technique divides bacteria into two groups. (1) Acid-fast and (2) Non-acid-fast. This procedure is extensively used in the diagnosis of M. tuberculosis and M. leprae. Acid fastness property in certain mycobacteria and some species of Nocardia is correlated with their high lipid content (60% w/w). Due to the high lipid content of the cell wall, acid-fast cells have relatively low permeability to dye and hence it is difficult to stain. For the staining of these bacteria, penetration of primary dye is facilitated with the use of 5% aqueous phenol which acts as a chemical intensifier. Heat is also used which acts as a physical intensifier. Once these cells are stained, it is difficult to decolourise with acid and alcohol.
In acid-fast staining, three different reagents or stains are used.
1. Primary stain (carbol fuchsin):
Carbol fuchsin, a phenolic stain that is soluble in the lipoidal material which constitutes the major portion of the mycobacterial cell wall allows penetration and retention of the red stain. Penetration is increased by the application of heat, which drives the carbol fuchsin through the lipoidal wall and into the cytoplasm. All cells appear red after the application of the primary stain.
2. Decolourising agent (acid-alcohol or 20% H2SO4):
The smear is cooled before decolourisation which allows the waxy cell substances to harden. On application of decolourising agent, acid-fast cells show resistance to decolourisation. Primary stain is more soluble in the cellular waxes than decolourising reagents. Hence primary stain is retained by mycobacteria species but this is not the case for non-acid fast bacteria that lack cellular waxes. Primary stain is easily removed during decolourisation and non-acid fast cells are observed as colourless.
3. Counterstain (methylene blue or malachite green):
Non-acid-fast bacteria absorb the counter stain and appear blue or green colour while acid-fast cells retain the red colour of the primary stain.
The spore staining technique is used for the detection of spore-carrying bacteria and types of spores. Spores are not easily stained by primary stain (malachite green). Hence, the smear is heated for staining the spores. Once the spore accepts the malachite green, it cannot be decolourised by tap water. Vegetative cells easily get decolourized by tap water and take counter-stain and appear red (safranin). This method is called Schaeffer and Fulton method, modified by Ashby. This method is a modified method in which direct heating is replaced by steam heating. Dorner method is also used for spore staining in which carbol fuchsin is used as the primary stain. After heating the slide with stain for 5 to 10 minutes, wash it and perform negative staining.
Capsule staining is used to detect the presence of capsules surrounding cells. It can be performed by using positive as well as negative staining techniques. Due to the nonionic nature of the capsule, it has very less affinity for dye. In positive stains, crystal violet is applied which stains bacterial cells. The osmotic difference is created by the application of copper sulphate (20%) solution, which causes diffusion of the stain towards the outer surface of the cell. On removal of the copper sulphate solution and drying the slide, some of the stains have not crossed the capsular surrounding during diffusion. This stain is retained in the capsular layer as light violet colour against the deep violet cell. Capsules are also easily demonstrated by negative staining techniques. When stain solution is applied, it stains the cell and on the application of a nigrosin background is stained leaving the capsule colourless.
Different other staining techniques are also used to study the morphology of bacteria. Flagella staining is used to detect the presence and arrangement of flagella and cytoplasmic inclusion staining is used to identify intracellular deposits of starch, glycogen, polyphosphates, hydroxybutyrate and other substances.
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