Staining mycobacteria with periodic acid-carbol-pararosanilin: principle and practice of the method.

Mycobacteria may be acid-fast, non-acid-fast or even chromophobic in staining under different conditions. The pretreatments with oxidants including periodic acid increase effectually the acid-fastness of acid-fast bacilli. This is caused by additional free carboxyl groups resulting from non-acid-fast wax in the cell walls by demethylation with oxidants. Only by prolonged periodic oxidation the aldehyde groups formed as oxidation products of 1-amino-2-hydroxy groups in the cells can be demonstrated with carbol-fuchsin stain. This reaction is most probably attributable to the formation of Schiff's bases between fuchsin and aldehydes. Since pararosanilin is the active molecule in the diphenamine reaction, periodic acid (10%, 24 hours) followed by carbol-pararosanilin stain is a most sensitive and selective method to demonstrate mycobacteria including chromophobic forms.