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.