Preparation of stains for Ziehl-Neelsen staining

preparation of stains for Ziehl-Neelsen staining Fixed smears are then arranged on a staining rack over a sink or bucket or basin, taking care that smears are up and slides do not touch each other. The rack can be made locally of various materials (best 2 glass or metal rods joined by pieces of tubing or the like); it must be straight and placed almost perfectly horizontally so that stains do not run off. Cover the smear with carbolfuchsin, pouring it through a small funnel with no. 1 filter paper held directly over the smear, moving the funnel to the next when the slide is almost completely covered. At the end of the series, place the funnel on the open bottle till drained (later close the bottle). Keep the funnel with its paper hanging in a jar; it can be used further for a week or so before a new filter paper has to be used. Discard the last 20-50 ml of stain of a stock bottle, it contains too many crystals. Heat the carbolfuchsin on the smears till they give off a bit of vapor (best seen from the side). Use a metal stick (i.e.bicycle spoke) with a piece of cotton wool or rag wound around its end, which is lighted after putting a bit of burning spirit on it. Hold this flame under the slides on the rack, but keep moving continuously from one slide to the other (to avoid boiling or breaking the slides). Stop when the first vapor is seen, and observe. If needed continue heating targeting slides where no vapor was seen yet. Add carbolfuchsin if it has become too little on some slides. Then leave the slides covered with the stain for at least 10 minutes. Use a forceps to drain off the stain, and gently rinse with pure water (filtered or deep tube well) till no visible stain comes off anymore. For rinsing, do not use tubing attached to a tap that may become colonized by mycobacteria but a jug that is easy to clean. Let drain off excess water and replace the slides on the rack. Cover them with sulphuric acid 25% for about 3 minutes, and rinse again. Repeat this for smears that still look quite red. Let the water drain off and replace the slides on the rack, then cover with methylene blue 0.1% for maximum one minute. Let the stain drain off and rinse with pure water. Take slides from the rack, drain off water, clean the underside by wiping with toilet paper soaked in alcohol if necessary, and place them on drying rack out of sunlight. Microscopy should be done on well dried smears only