The use of fluorescein diacetate and ethidium bromide as a stain for evaluating viability of mycobacteria.
A method for evaluating the viability of mycobacterial cultures using fluorescein diacetate and ethidium bromide is described. Smears from 2-4 week old cultures of mycobacteria were stained with a 0.005% Dubos albumin broth dilution of fluorescein diacetate for thirty minutes at 37 C. The smears were counterstained with a 0.005% aqueous solution of ethidium bromide for ten minutes at room temperature and blotted dry. One drop of glycerol was placed on the smear and covered with a 22 X 40 mm coverslip. Stained smears were observed with fluorescence microscopy at 450 X. Viable organisms hydrolyzed the fluorescein diacetate and appeared yellow-green while dead organisms absorbed the ethidium bromide and appeared red-orange. Aliquots of material from which the slides were made were concurrently placed on Lowenstein-Jensen media and cultured for growth as a confirmation of viability. Investigation of 104 mycobacterial isolates using the described test showed that the viability of mycobacterial cultures can be determined by this method.
D001431:Bacteriological Techniques; D004996:Ethidium; D005452:Fluoresceins; D009161:Mycobacterium / Q000254:growth & development*; D009162:Mycobacterium avium / Q000254:growth & development; D009163:Mycobacterium bovis / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000254:growth & development; D009170:Nontuberculous Mycobacteria / Q000254:growth & development; D013194:Staining and Labeling / Q000379:methods
D005452:Fluoresceins; D004996:Ethidium; C018506:diacetylfluorescein