The styles of incubators for lower temperatures, 20° and below, are not so numerous nor so satisfactory. The author has constructed a device which answers every purpose for a small class. The diagram, Fig. 143, explains it. [Illustration: FIG. 143.--Diagram of fittings for a cold incubator. _1._ small tank for constant head, about 1 foot in each dimension. _a_, inflow; _b_, overflow; _c_, lead pipe. _2_, refrigerator. _a′_, ice; _b′_, flat coil under ice; _c′_, outflow to incubator. _3_, incubator. _a″_, cold water inflow; _b″_, overflow; thermometer and burner omitted. The diagram explains the construction. The water cooled to about 14° with artificial ice by flowing through the lead coil under the ice, flows into the incubator which may be heated and regulated in the usual way.] The thermal death-point is determined by exposing the organisms in thin tubes of broth at varying temperatures for ten-minute periods and then plating out to determine growth. The effect of heat may also be determined by exposing at a given temperature, _e.g._, 60°, for varying lengths of time and plating out. _B._ Oxygen relations--whether the organism is aërobic, anaërobic, or facultative is determined by inoculation in gelatin or agar puncture or stab cultures and noting whether the most abundant growth is at the top, the bottom or all along the line of inoculation. _C._ Reaction of the medium--acid, alkaline or neutral as influencing the rate and amount of growth. _D._ The kind of medium on which the organism grows best. _E._ The effect of injurious chemicals, as various disinfectants, on the growth. _F._ Osmotic pressure conditions, though modifying decidedly the growth of bacteria, are not usually studied as aids in their recognition, nor are the effects of various forms of energy, such as light, electricity, _x_-rays, etc. Among the “Physiological Activities” discussed in Chapters IX-XII those which, in addition to the staining reactions described, are of most use in the identification of non-pathogenic bacteria are the first ten listed below. For pathogenic bacteria the entire thirteen are needed. 1. Liquefaction of gelatin. 2. Digestion of blood serum. 3. Coagulation and digestion of milk. 4. Acid or gaseous fermentation in milk, or both. 5. Acid or gaseous fermentation of various carbohydrates in carbohydrate broth, or both. 6. Production of indol in “indol solution.” 7. Production of pigments on various media. 8. Reduction of nitrates to nitrites, ammonia, or free nitrogen. 9. Production of enzymes as illustrated in the above activities. 10. Appearance of growth on different culture media. 11. Production of free toxins as determined by injection of animals with broth cultures filtered free from bacteria. 12. Causation of disease as ascertained by the injection of animals with the bacteria themselves, and recovery of the organism from the animals.