4. Treat with Lugol's iodine solution until the film is black or dark brown. To do this treat with iodine solution for a few seconds, wash in water, and examine the film over a piece of white filter paper. Note the colour. Repeat this process until the film ceases to darken with the fresh application of iodine solution. Lugol's solution is prepared by dissolving Iodine 1 gramme Iodide of potassium 3 grammes In distilled water 300 c.c. 5. Wash in water. 6. Wash with alcohol until no more colour is discharged and the alcohol runs away clear and colourless. The following mixture may be substituted for absolute alcohol as a decolouriser Acetone 10 c.c. Absolute alcohol 100 c.c. 7. Wash in water. 8. Counterstain very lightly with aqueous solution of Neutral Red. Other counterstains may be used such as dilute eosin, dilute fuchsin, or vesuvin. NOTE.--This section may be omitted when dealing with films prepared from pure cultivations. 9. Wash in water. 10. Dry and mount. ~Gram-Claudius Method.~-- 1. Prepare a cover-slip film and fix in the usual way. 2. Stain in methyl violet, 1 per cent. aqueous solution for three to five minutes. 3. Treat with two lots picric acid, saturated aqueous solution. 4. Wash in water and dry. 5. Decolourise with clove oil. 6. Wash off clove oil with xylol. 7. Mount in xylol balsam. ~Gram-Weigert Method.~-- 1-5. Proceed as for the corresponding sections of Gram's method (_quod vide_). 6. Dry in the air. 7. Wash in aniline oil, 1 part, xylol, 2 parts, until no more colour is discharged. 8. Wash in xylol. 9. Mount in xylol balsam. ~Modified Gram-Weigert Method.~--(To demonstrate trichophyta in hair.) 1. Soak the hairs in ether for ten minutes to remove the fat. 2. Stain thirty minutes in a tar-like solution of aniline gentian violet (prepared by adding 15 drops of the alcoholic solution of gentian violet to 3 drops of aniline water). 3. Dry the hairs between pieces of blotting paper. 4. Treat with perfectly fresh iodine solution. 5. Again dry between blotting paper. 6. Treat with aniline oil to remove excess of stain. (If necessary, add a drop or two of nitric acid to the oil.) 7. Again treat with aniline oil. 8. Treat with aniline oil and xylol, equal parts. 9. Clear with xylol. 10. Mount in xylol balsam. To obtain the best differentiation the preparation should be repeatedly examined microscopically (with a 1/6-inch objective) between steps 5 and 9, as the actual time involved varies with different specimens. ~Ziehl-Neelsen's Method.~--(To demonstrate tubercle and other acid-fast bacilli.) 1. Smear a thin, even film of the specimen on the cover-slip by means of the platinum loop. (In the case of sputum, if it is a very watery specimen, allow the film to dry, then spread a second and even a third layer over the first.) 2. Fix by passing three times through the flame.