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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.

Interest in tuberculosis in elephants has been increasing over the past several years in the United States. Several techniques have been used to diagnose mammalian tuberculosis. Currently, the test considered most reliable for diagnosis of TB in elephants is based on the culture of respiratory secretions obtained by trunk washes.

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Abstract. Mycobacterium bovis isolates from cattle, captive elk, and free-ranging mule deer and coyotes were examined by restriction fragment length polymorphism (RFLP) analysis. DNA extracted from each isolate was digested with restriction endonucleases AluI and PvuII. DNA probes used for Southern hybridizations were a 37-base oligonucleotide and a 123-base-pair sequence specific for the insertion sequence IS6110 and a plasmid, pTBN12, which contains a polymorphic GC-rich repetitive sequence present in several species of mycobacteria. Generally, M. bovis isolates originating from a single herd of either cattle or captive elk had identical RFLP patterns, whereas isolates from unrelated sources had distinct patterns. The RFLP patterns for M. bovis isolates from free-ranging mule deer and coyotes were identical to patterns observed for isolates from a captive elk herd that was located in the area where the free-ranging animals were found. These results indicate that the captive elk herd may have been the source of M. bovis that infected the free-ranging animals. Results of this study show that RFLP analysis is a useful tool for differentiation of M. bovis isolates and for molecular epidemiology studies to determine possible sources of infection in outbreaks of tuberculosis in animals.Most cases of tuberculosis in cattle, elk, deer, and many other species of animals are caused by Mycobacterium bovis, a member of a closely related group of organisms referred to as the M. tuberculosis complex. 20 This complex also includes M. tuberculosis, M. microti, and M. africanum. Species within the M. tuberculosis complex usually are differentiated by results of standard biochemical tests and animal pathogenicity studies. 40 Isolates within a species of the M. tuberculosis complex have been differentiated using a variety of methods, including phage typing, 11 restriction endonuclease analysis, 5,6 pulse-field gel electrophoresis, 43 and several polymerase chain reaction assays. 2,18,19,21,23,26,30 Restriction fragment length polymorphism (RFLP) analysis is currently one of the most widely used methods for differentiation of M. tuberculosis complex isolates. 3,8,12,14,15,17,22,25,27,[31][32][33][37][38][39] A recommended standardized method for RFLP analysis of M. tuberculosis isolates includes digestion of DNA with restriction endonuclease PvuII and hybridization with a probe specific for the repetitive sequence IS6110. 38 Mycobacterium tuberculosis isolates usually have 5-20 copies of IS6110 in sites dispersed throughout the genome that vary among strains. This makes IS6110 a useful target sequence to detect genomic differences among isoFrom the USDA-ARS National Animal Disease Center, Zoonotic Diseases Research Unit, Ames, IA (Whipple), the USDA/APHIS/ VS, Billings, MT (Clarke), and the USDA/APHIS/VS National Veterinary Services Laboratories, Diagnostic Bacteriology Laboratory, Ames, IA (Jarnagin, Payeur).Received for publication December 10, 1996. lates. 33 However, most M. bovis isolates have fewer than 6 copies of IS611...