Chemicals needed for AFB microscopy

Chemicals needed for AFB microscopy For carbolfuchsin: basic fuchsin powder; phenol crystals of good quality; alcohol: denatured 95% ethanol or pure methanol; water, distilled or purified (see below) For acid solution: concentrated sulphuric acid, which can be of industrial grade if it is not too dirty; water, distilled or purified (see below) For methylene blue solution: methylene blue powder of good quality; water, distilled or purified. Water for preparation of stains does not have to be distilled, but it should not contain AFB. This can be obtained by regular cleaning (scrubbing, rinsing) of water containers and by using a household candle type filter to prepare water if distilled is not available. The filter should be maintained regularly (scrubbing, boiling, rinsing with purified water or alcohol).

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

Equipment needed

Equipment needed The following equipment is needed for preparation of the stains: • A balance or weighting scale, sensitivity 0.1 gram • Measuring cylinders, one of 100 ml and one of 500 or 1000 ml • Conical flasks (big) or flat-bottomed balloon flasks • A spirit lamp for heating • Containers for the newly prepared stains. These can be well closing plastic bottles of 1 liter • Labels for the bottles • Brushes to clean the bottles before re-use • Big funnel for each solution to fill up the bottles • AFB-positive and negative unstained control smears. The positives are preferably low positive (1+); the negative can be made from the technologist’s own sputum or from white of egg. Do not use suspect sputa for the negative controls. Make as many slides as possible from the same low positive sputum, after letting it stand one day and mixing it carefully (container closed); check what the average number of AFB is by staining a few smears, write this in your notebook. Keep fixed unstained smears protected from dust and sunlight, best in a slide box

Staining procedure

Staining procedure Good stains, especially good carbolfuchsin, are essential to detect high numbers of AFB-positives. Only a good stain will be able to show the AFB, also when these are rare. AFB damaged by treatment are difficult to stain, so all follow-up smears will often be negative when the quality of the carbolfuchsine is substandard. Stain preparation requires some equipment for weighing and measuring, and also pure water without mycobacteria from the environment (they often colonize water tanks and taps). Therefore preparation of stains should be done at one laboratory at district level, with checks for quality before they are distributed to the peripheral laboratories.

Procedure of Smear preparation

Procedure of Smear preparation The best tool for smearing sputum is a short, thin stick made of bamboo, Whit ragged end (obtained by breaking a +/-15 cm long stick in tow). The two parts are easily used to pick up a bit of sputum , also from thick specimens, or to apply just the right quantity on the slide (removing and discarding excess using one half stick), and to made a fine smear using the ragged end as a brush (holding it almost vertical in the slide ) . Coconut sticks are less good and wood sticks are less good at all since they yield a pointed , not good at all since they yield a pointed , not a ragged end not ragged end, Wire-loops can be used but should be thicker than usual ((1-2 mm wire) to handle of the slide, and they need flaming in between two samples .Take the slide, holding it at the sides or over the numbering, and put it a on the newspaper protection Verify if the number corresponds with that on the sputum container, open the container carefully , and using bamboo stick or other tool pick up a little bit from a good part of the specimen (not bigger than then the head of match) Close the container then apply the sputum particle in the middle of the slide, and smear it out as cleanly as possibly over an area of 3 cm 2 cm. using slow circular and spiral movements. if too much sputum was taken remove some using the other part of the stick. The thickness is correct if the newspaper print under it can only just be read . Replace the slide on its container of slide to dry. Drying should complete and may take at least half an hour , if humidity is very high , drying can be assisted by heating the slide over the spirit lamp just a little bit (passing once over it) and repeating this may be one or two times. Excess heat will spoil the AFB when it is still wet.

BJECTIVES OF TREATMENT OF TB

OBJECTIVES OF TREATMENT OF TB Culture individual patient. Prevent death from active TB or its late effects.prevent relapse of TB . Reduce transmition .prevent the development of Drug resistance.

GROWTH ASSESSMENT

GROWTH ASSESSMENT Documented loss or failure to gain weight, especially After being treated in a nutritional rehabilition programe Is agood indicator of chronic disease in children of Which TB may be the cause .