Sat. alcoholic solution of methylene blue 1.0 part Glacial acetic acid 2.5 parts Distilled water 50.0 parts _Bismarck Brown_: Bismarck brown (dry dye) 2 parts Distilled Water 1000 parts By the use of the hanging drop slide and the methods of staining just described all the various morphological features of the bacterial cell may be ascertained. It is necessary when _cell groupings_ as characteristic of definite modes of division are to be determined to make slides from a liquid culture, as broth. Place a drop of the material, preferably from the bottom of the tube in most instances, from the top in case a pellicle or scum is formed on the surface, on the slide and allow this to dry _without spreading it out_, fix, wash gently with water, then stain lightly with Löffler’s blue. Such slides also show characteristic _cell forms_ as well. Slides should be made from solid media to show variations in form and size and involution forms. These latter are especially apt to occur on potato media. CHAPTER XX. STUDY OF THE PHYSIOLOGY OF BACTERIA. Of the environmental conditions influencing the growth of bacteria the following are the chief ones ordinarily determined: _A._ Temperature.--The optimum temperature for growth is usually about the temperature of the natural environment and ordinarily one determines merely whether the organism grows at body temperature (37°) and at room temperature (20°) or not. For exact work the maximum, minimum and optimum temperature must be ascertained by growing in “incubators” with varying temperatures. A bacteriological incubator is an apparatus for growing bacteria at a constant temperature. This may be any temperature within the limits for bacterial growth. If temperatures above that of an ordinary room are desired, some source of artificial heat is needed. Electricity, gas or oil may be used. A necessary adjunct is some device for maintaining the temperature constant, a “thermoregulator” or “thermostat.” For lower temperatures a cooling arrangement must be installed. For the great part of bacteriological work only two temperatures are used, 20° so-called “room temperature” (this applies to European “rooms” not to American) and 37° or body temperature. Incubators for 37° of almost any size and style desired may be secured from supply houses and need not be further described. Figs. 141 and 142 illustrate some of the types. For use with large classes “incubator rooms” are to be preferred. The author has one such room for 37° work with 200 compartments for student use which did not cost over $60 to install. [Illustration: FIG. 141.--Small laboratory incubator, gas heated.] [Illustration: FIG. 142.--Electric incubator.]