For proper staining uniform smears must be made. The material. General principle of collection of specimens: (a) Proper selection of the material. (b) Avoidance of all extraneous contamination. (c) Maintain strict asepsis while collecting specimens. Gram stain constitutes the most important single differential stain in bacteriological work. Wipe the urethra with sterile saline. Take the pus discharge with the help of a platinum loop. N. gonorrhea as gram-negative Diplococci intracellularly in leucocyte and extracellularly. Zieh­-Neelsen Staining Method for M. Leprae: 1. Thoroughly clear the selected portion of the skin with a small pledged of spirit cotton. 2. With a sterile scalp0el (15 Bard Parker scalpel blade) held in the right hand, make an incision on the pinched skin so that the edge of the knife goes into the dermis of skin. 3. With the scalpel turned at right angle, scrape the bottom and side of the slit with the point of the blade, to obtain sufficient material for making the smear. 4. Transfer the material from the point of the scalpel to a clear microscopic glass slide. Technique for Staining the Smear: 1. Pour over the smear a requisite quantity of carbolfuchsin. 2. After requisite period wash the smear preferably in running water. 3. Decolourize with acid alcohol for 5 minutes. 4. Counter stain with methylene blue for half a minute. 6. Examine under a microscope—oil immersion objective. The MI is undertaken on the same stained slit skin smears used to measures the BI. Only bacteria showing uniform and bright staining down their length are called regularly stained. The MI as the portion of percentage of regularly stained bacteria of the total scored.