224 LABORATORY EXERCISES IN BACTERIOLOGY. 



dried in the air. Before it is quite dry, the film is flooded with carbol-fuchsin solution, 

 which is heated nearly to boiling and evaporated ; the slide with the evaporated stain 

 being passed several times through the flame to fix the film. More of the stain is 

 now added and boiled for five minutes, fresh solution being furnished as evaporation 

 takes place. At the end of this time the excess of stain is poured off and the film 

 well washed in hot water until the color is discharged from the organisms (but not from 

 the spores). This usually requires several minutes; and it is well from time to time 

 to drop a cover on the wet film and examine the preparation with the one-seventh 

 inch lens to determine when to refrain from further decolorization. This accom- 

 plished, the film is stained for a few seconds with Loemer's blue, washed in cold water, 

 dried, and examined. The spores will be found red, the bacteria blue. 



In addition to the possibility of determining the presence of spores by actual 

 microscopic observation, it may be inferred that they are present (and further search 

 in microscopic preparations should then be made) if after exposing a culture for ten 

 minutes to 80 C. inoculations made from it upon fresh medium are followed by growth. 

 In this the adult forms of bacteria are destroyed by the heat exposure and only spores 

 are likely to persist in their vital condition, further growth depending upon their 

 germination. 



It is believed that a third mode of reproduction occurs, at least in the higher 

 forms, as the mycobacteria, from segmentation of the rods into small coccoid bodies, 

 analogous to the gonidia of the hyphomycetes, and spoken of as such in this connec- 

 tion. They are small, not so refractive as spores; and, like spores, they are believed 

 to be very resistant to adverse conditions of life. Under favorable conditions they 

 develop by simple growth into the adult types. 



Exercise 54. From an agar culture of Bacillus subtilis, grown in the 

 incubator for several days, one can usually obtain numerous spores or 

 sporulating individuals; often nearly the whole preparation seems made 

 up of these to the exclusion of the ordinary rods. Prepare and stain a film 

 from such source according to the above instruction. What is the position 

 of the spores in the bacilli ? 



Exercise 55. In a few drops of sterile bouillon place a number of spores 

 obtained from culture used in previous exercise, and keep in incubator. 

 Every ten minutes for an hour or more prepare films, staining them quickly 

 with Loeflfler's solution, warm. The spores will be unstained, the germinat- 

 ing organisms blue. Is germination equatorial, polar, or bipolar? 



CHEMICAL ACTIVITIES OF BACTERIA. 



In the growth of bacteria, both in the living host and upon laboratory media, it 

 is well known that important metabolic changes proceed in the bacterial cells, as well 

 as chemical alterations in the medium on which they develop. Little is known of 

 these processes, but in connection with them a number of phenomena, as production 

 of pigment, light, heat, various types of fermentation and putrefaction, and other 

 manifestations, take place along with the growth, multiplication, and death of the 

 bacterial cell. The demonstration of such changes is in a large measure favored or 

 prevented by the character of the medium upon which the bacteria are grown; and 



