Every techniques have advantages and disadvantages depending on circumstances. For this reason, the procedures to be employed in NTP must be carefully selected taking into account of sensitivity, specificity, simplicity, economy, and maximal security. Once selected they must be well standardized and always carried out under strict quality control and supervision. In other words, they must not be deviated from the standard techniques under any circumstance.
Clinicians or other health personnel, who are changed in diagnosis and treatment of tuberculosis patients, always want fast and accurate results of laboratory examinations. It makes laboratory personnel look for the technique to meet such demand, often forgetting the costs. If you emphasize fast results, you may lose both sensitivity and specificity.
A. Smear microscopy
In all aspects, smear microscopy is a priority procedure over any other techniques in case-finding and in monitoring patients response to chemotherapy in NTP. It permits to screen 40-60% of the cases.
1. Smear preparation
Before preparing smear, utmost care should be taken to exact numbering and recording identification number or particulars on sputum containers, slides, worksheets and logbooks, otherwise serious mistakes will occur. Numbering of slide glasses must remain clearly even after staining. If slide glasses are frosted at one end, it could be marked with a pencil. If unfrosted slides are used, the identification number should be engraved on the end of the slide with the diamond-pointed stylus. Materials necessary for smear preparation are as follows: (1) a non-porous surface plate made of galvanized metal or aluminum. It can be substituted with paper towel; (2) engraved slides for smears; (3) special box for transporting with specimens to be examined; (4) diamond-pointed stylus; (5) a nichrome wire loop or wooden applicators; (6) alcohol sand flask; (7) Bunsen-burner of spirit lamp: (8) dryer on which to place the finished smears; and (9) forceps.
Smear preparation can be made either by the direct method (DS) or by the concentration method (CS). Preparation of concentrated smear using a centrifuge is complex and laborious and requires much more materials, equipment, and precaution than the direct smears so that a few laboratories can afford it for routine use in diagnosis.
Therefore, a simple and cheap DS microscopy is most widely used.
The quality of sputum must be inspected macroscopically and recorded on work sheet prior to smear preparation. DS must be prepared by transferring opaque mucopurulent particles on the slide with nichrome wire loop or wooden applicator stick and then spreading evenly in an appropriate size of 1-2 x 2-3cms. Smear should be air-dried without exposing to direct sunlight and then fixed by passing slowly over the flame for a few seconds and must be careful not to scorch it. It is recommended not to attempt processing more than 10 or 12 specimens at one time.
For the preparation of CS, sputum homogenate must be diluted more than six times with a sterile distilled water and centrifuged at over 3000xg for 15 minutes. The sediment can be smeared on a slide. However, CS is no better than DS in case-yield unless it is performed properly. As seen in Table 7, centrifugal force affect case-yield rate to a great extent.
2. Staining and microscopy
For staining, the following materials are required: (1) slide-rack for staining; (2) funnel with filter paper; (3) forceps; (4) plastic flask with Ziehl's carbol fuchsin; (5) alarm clock; (6) plastic flask with spirit; (7) cotton holder made of metal; (8) waste receptacle for used filter papers; (9) plastic flask with 25% sulfuric acid; (10) plastic flask with 0.3% methylene blue; (11) basin.
Ziehl-Nelseen stained DS microscopy (DZN) is simple and cheap and provides fast results and the cases found are epidemiologically very important. This technique is of adequate sensitivity and specificity and economical particularly in a small health center (HC) laboratory examining no more than 30 specimens a day.
