A cid-fast m icroscopy (AFM ).

Sputum samples and skin-tissue fluid specimens were prepared, fixed, and stained by the Ziehl-Neelsen (ZN) method for acid-fast bacilli (APB) using strong carbol fuchsin, acid-alcohol as a decolourant, and methylene blue as the counterstain. To demonstrate the leprosy bacilli, the ZN staining method was modified by the use of a weaker decolourizer than used for tubercle bacilli, and 5% H2SO4 instead of

2 0 % H2SO4 in alcohol was used but 2 0 % H2SO4 in alcohol was used to

demonstrate the tubercle bacilli.

In the acid-fast staining procedure, the red dye carbol fuchsin was applied to a fixed smear, and the slide was gently heated for several minutes (heating enhances penetration and retention of the dye). Then

the slide was cooled and washed with water. The smear was next treated with acid-alcohol, a decolourizer, which removes the red stain from tissues and bacteria that are not acid-fast (The acid-fast microorganisms retain the red colour since the carbol fuchsin is more soluble in the cell wall waxes than in the acid-alcohol. In non-acid-fast bacteria, whose cell wall lack the waxy components, the carbol fuchsin is rapidly removed during decolourization, leaving the cell colourless). The smear was then stained with a methylene blue counterstain (Non-acid- fast cells appear blue after application of the counterstain) (Laidlaw,

1989).

After staining, more than 20 fields of each stained smear were examined carefully under the light microscope using an oil immersion (XIGO) lens.

4.4. Culture

For the tuberculosis study of leprosy patients, all collected sputum samples were treated with the protocol of A-acetyl-L-cysteine (NALC)- 2% NaOH, inoculated onto Lowenstein-Jensen (LJ) media, and incubated at 37°C. The slopes were examined for growth every week

4.5. Procedures of DNA extraction from clinical specimens for PCR assays

Three different and simple DNA extraction methods for preparation of template DNA from samples were used as follows:

(1) DNA extraction by using guanidine thiocyanate and diatom s. This method was based on the lysing and nuclease- inactivating properties of guanidine thiocyanate (GuSCN) together with the nucleic acid-binding properties of diatoms. Diatoms are the fossilized cell walls of unicellular algae and consist almost entirely of silica allowing more disruption in diatom-nucleic acid networks by vortexing (Boom et ah, 1990; 1991).

For skin-tissue fluid swab specimens, each was placed in a 10 ml conical tube containing 1 ml phosphate buffered saline (PBS) at pH 7.4 plus a 2X concentration of Fungi-Bact (Innis et al., 1990), and dispersed on a vortex-mixer. Next, the specimen was allowed to stand at room temperature overnight, after which it was again vortex-mixed.

In the case of sputum samples, each was liquefied as follows:

An equal volume of a 4% (wt/vol) solution of NaOH containing 1 g of A-acetyl-L-cysteine per 100 ml was added to a sputum sample, and the tube was periodically vortex-mixed for 20 min. The sample was made up to 20 ml phosphate buffer (67 mM Na2HP0 ^, 67 mM KH2PO4 at pH

6 .8 ) and centrifuged at 3,000 X g for 20 min. The supernatant was

decanted, and the resulting pellet was suspended in 1 ml of the same

phosphate buffer (Victor etaL, 1992).

For subsequent DNA extraction, 50 \il of either type of treated specimen was prepared by the procedure described below.

Diatom suspension (40 pi) was mixed with 900 pi of lysis buffer L6

(GuSCN 120 g; 0.1 M Tris-HCl at pH 6.4, 100 ml; 0.2 M EDTA at pH 8.0, 22 ml; and Triton X-100, 2.6 g) in a 1.5 ml Eppendorf microcentrifuge tube, and briefly vortex-mixed. The clinical specimen (50 pi) was added to the above, vortex-mixed for 5 sec, and allowed to stand at room temperature for 10 min. Next, it was vortex-mixed again, and centrifuged at 12,000 X g for 15 sec. The supernatant was discarded, and the nucleic acid (NA)-pellet was washed twice with washing buffer L2 (GuSCN 120 g; and 0.1 M Tris-HCl at pH 6.4, 100 ml), twice with 70% ethanol and once with acetone. The acetone was removed and the NA-pellet was dried at 56°C for 10 min. Next, 100 pi of TE buffer (Tris-HCl at pH 8.0, 10 mM; and EDTA at pH 8.0, 1 mM) was added to the NA-pellet, vortex-mixed, and incubated for 10 min at 56°C. It was mixed again, centrifuged at 12,0(X) X g for 2 min, and 5 pi of the supernatant was used directly for PCR assay.

(2) DNA extraction by using Chelex^ 100. This procedure was based on the utilization of Chelex^ 100, a chelating ion exchange

resin. The alkalinity of Chelex^ suspensions and the exposure to 100°C temperatures result in disruption of the cell membranes and dénaturation of the DNA, and can obtain a DNA solution suitable for PCR (Stein and Raoult, 1992).

After treating the skin-tissue fluid swab specimens, as mentioned above (in method 1), for subsequent DNA extraction by this method, 50 pi of the treated specimen was added to 200 pi of a suspension containing 20% Chelex^ 100 in a 0.1% Lauryl sulphate, 1% Nonidet P40, and 1% Tween 20 aqueous solution in a 1.5 ml Eppendorf microcentrifuge tube, and briefly vortex-mixed on a vortex-mixer. The mixture was then boiled for 10 min, centrifuged at 12,000 X g for 10 min, and 5 pi of the supernatant was used directly for PCR examination.

(3) DNA extraction by boiling. This method was based on heating the sample in a boiling water bath to break down the bacterial cell wall, and release the DNA (Kocagoz et al., 1993).

After treating the skin-tissue fluid swab specimens, as described above (in method 1), 50 pi of treated specimen was added to 100 pi of TE buffer (10 mM Tris at pH 8.0, and 1 mM EDTA at pH 8.0) in a 1.5 ml Eppendorf microcentrifuge tube, and vortex-mixed. Next, it was placed in a boiling water bath for 10 min, and then 5 pi of the suspension was used directly for PCR examination.