1. Mordant with a mixture of 10 c.c. 20 per cent. tannin solution, 5 c.c. cold saturated ferrous sulphate, 1 c.c. fuchsin or wool-black solution. This solution is put on the preparation, which is then heated for one minute until steam is given off. 2. Wash under the tap, then in alcohol. 3. Stain with anilin-water-fuchsin solution, prepared by dissolving the dye in anilin-water and adding 1 per cent. of a 1 per cent. solution of caustic soda until the liquid begins to go cloudy. 4. Wash off the stain with distilled water. B. coli does not liquefy the gelatin. When grown in nutrient solutions containing sugars, it produces much acid, and at the same time gases are given off, consisting of CO_{2} and hydrogen. If the growth in this solution be allowed to continue a secondary fermentation ensues, and the culture eventually becomes alkaline. Indol is produced by B. coli, and may be demonstrated by adding to 10 c.c. of the culture, 1 c.c. of a 1/50 per cent. solution of pure potassium nitrite; then adding a few drops of concentrated sulphuric acid, when, if indol be present, a red coloration (nitroso-indol) is produced. This bacterium reduces nitrates to nitrites. Cultivated in a 1 per cent. solution of peptone, to which 1/10000 per cent. of potassium nitrate has been added, after four hours at 37° C., the presence of nitrite may be shown; after the growth has continued for seventeen hours, the nitrite is further reduced to ammonia. Among other products of B. coli, Harden found lactic, formic, acetic, and succinic acids, ethyl-alcohol, CO_{2} and hydrogen. In Germany, W. Lembke and H. Becker have specially investigated the bacterial flora of the dog’s intestines. Lembke, in 1896[79] cultivated the bacteria from the fæces of the dog, fed in various ways--bread, meat, and fat diet--and found B. coli constantly present, although the form of the individuals, as well as the colonies, and the intensity of the indol and gas formation, showed great variations. [79] “Beitrag zur Bakterienflora des Darmes,” Archiv f. Hygiene, Bd. 26, p. 293. The other species of bacteria present varied with the kind of food; this has a great influence on the flora of the intestines, which was found to be very different when the dogs were fed on bread to what it was when they were fed on meat. Lembke describes two other species of bacteria closely resembling B. coli, one of which he calls B. coli anindolicum, which, as the name implies, gives no indol reaction; the other, B. coli anaerogenes, is non-motile, possesses no flagellæ, and differs from B. coli by the absence of gas production in the fermentation of sugars.