By C. H. Collins, J. M. Grange, M. D. Yates
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The mixture is allowed to stand at room temperature for 12-18 hours. Distilled water, 16 ml, is added and mixed with the digested sputum. The bottle is then centrifuged. The deposit is cultured. Trisodium phosphate-Zephir an method About 2 ml of sputum is added to a bottle containing 3 ml of a mixture of 23 per cent trisodium phosphate solution containing 2 ml per litre of 17 per cent Zephiran* * Winthrop Laboratories, New York. 46 Cultural methods (benzalkonium chloride). After blending for 2 minutes on a Vortex mixer the homogenate is allowed to stand at room temperature for up to 2 hours.
Slopes showing confluent growth or isolated colonies of a buff, yellow or orange colour should be removed for microscopical examination. Close inspection, preferably with a hand lens and under a good bench lamp, is necessary to reveal very small colonies or the effuse growth which is characteristic of some opportunist mycobacteria. In Kirchner medium tubercle bacilli grow as spherical colonies, 1-2 mm in diameter, at the bottom of the tube. The bottle should be shaken gently to observe them. They may be removed with a plastic Pasteur pipette for microscopic examination and subculture on solid medium.
The slides are then washed again with running water and the counterstain poured on. This is left for 1-2 minutes and washed off. The slides are then placed on edge to drain and allowed to dry. Alternatively each slide may be blotted dry with a fresh piece of filter paper or bacteriological blotting paper (which should not be used again) and dried either by passing them once through a bunsen flame or on a drying rack. The Tan Thiam Hok (TTH) method This is a cold staining method, developed for field use when heating slides is difficult.