Simplified Fluorescence Microscopy of Tubercle Bacilli

SUMMARY: The fluorescence of tubercle bacilli stained with auramine and rhoda- mine does not require ultra-violet light. It can be caused by blue light up to 406mp. A'copper sdphate ammonia liquid filter, suitably diluted, will transmit this wave- band. A gelatin screen filter, absorbing all light below 510mp. is used in the eyepiece. Normal, high intensity projection Alament lamps, combined with lamp condensers of large aperture, provide suitable light sources. The numerical aperture of the microsoope condenser must be fully used by immersion in glycerol. The fluorescence is vellry bright with the usual biological, and certain binocular, microscopes. The simplicity of the equipment enables fluorescence microscopy of tubercle bacilli to be used at low cost in any laboratory. The phenomenon of fluorescence as applied to stained bacilli may be described as follows. Certain fluorochroms (e.g. auramine) absorb light of a certain wave- length, and convert it into light energy of another wave-length. This effect is observed by illuminating the specimen with suitable light passed through a filter which transmits a wave-band capable of causing fluorescence, and by incorporating into the observing part of the microscope a filter which transmits the dour of the fluorescence, but absorbs the light which has caused it. The use of auramine as an acid-fast stain for the examination of tubercle bacilli in fluorescent light under the microscope was first introduced by Hagemann in 1988. Later workers made several improvements in Hagemann's method, but these were mainly technical. In essence the principle has remained the same, and is to-day recognized as having great advantages over the usual Ziehl-Neelsen technique: the staining process is quick and the result very clear-cut because of the great contrast between bacilli and background, the appearance of the field being almost that of a dark-ground with the bacilli a bright golden-yellow colour. Moreover, because of the distinctive appearance of the bacilli, a much lower magnification than usual can be used: x 800-$00 instead of the usual x 1850. Thus an area from ten to twenty times that of the average oil-immersion field can be covered, and the time of searching the slide correspondingly decreased. The instruments used in the early development of fluorescence microscopy were equipped with a quartz optical system and a carbon arc with ultra-violet filter to produce the necessary fluorescence. Later workers (see Ellinger, 1940) considered that the fluorescence could be obtained with light of 8oo-BWmp. wave-length, and since most of this is transmitted by the usual optical glass, the use of special quartz lenses and condensers became unnecessary. The mercury arc, with a very strong emission band at SSSrnp., is now used as the most practicable light source, together with a liquid copper sulphate ammonia filter to absorb all the light of more than 436mp. Richards, Kline & Leach (1941) described a high-intensity low-voltage