258 LABORATORY EXERCISES IN BACTERIOLOGY. 



Bacillus coli, the other bacteria of the fecal matter having probably entirely 

 failed of development. Plant from such colonies to other sterile media 

 for comparison with the known characteristics of Bacillus coli. 



Exercise 77. From a known mixture of typhoid and colon bacilli plant 

 in the same manner upon the same medium. Note at the close of twenty- 

 four hours the appearance of the colonies of the colon bacillus. After a 

 second day the colonies of Bacillus typhosus appear like tiny, clear, color- 

 less droplets. Separate to fresh media (agar, gelatine, and potato) and 

 study for each organism the macroscopic cultural features, and the flagel- 

 lation, staining by Gram's method, liquefaction of gelatine, gas formation in 

 sugar-containing bouillon, acid production, milk coagulation, indol pro- 

 duction, and the agglutination phenomenon on addition of serum from a 

 suitable typhoid subject. 



3. Selection by Culture Temperature. From reflection upon the differences 

 of optimum temperature for growth of the different groups it may be readily appre- 

 ciated that this feature may be utilized in efforts to separate from complex mixtures 

 the psychrophilic, mesophilic, or thermophilic bacteria (vide exercise 35). It need 

 scarcely be added that, having provided the condition of temperature requisite for 

 growth of some bacterium which it is desired to separate from such a mixture, the 

 first colonies appearing in the inoculated medium are, other things being equal, those 

 of the sought-for organism. These are at once to be transferred to fresh medium 

 for inquiry as to perfect isolation and confirmation of type, lest if allowed a longer 

 period of development other germs may follow if the temperature to which the culture 

 has been subjected be within the vital range of such forms. 



4. Selection by Culture Atmosphere. Similarly, the possibility of growth in 

 the ordinary air or in anaerobic surroundings may be utilized in the separation of the 

 obligate forms of the two varieties of bacteria, it being a matter of no great difficulty 

 to isolate and obtain in pure culture one or other variety where only the obligate 

 forms are present in a mixture. The facultative forms may, however, grow along 

 with the aerobes in ordinary air or along with the obligate anaerobes in atmospheres from 

 which oxygen has been displaced by hydrogen or nitrogen. In such cases it may be that 

 a difference in the rate of development will serve to render possible the distinction of 

 obligates from the facultative varieties, or a proper selection of temperature or medium 

 of growth in connection with atmosphere may lead to the same results. The capillary 

 tubes of Salomonsen, or Koch's suggestion that on the surface of a plated medium 

 in ordinary air a small sterile plate of glass or mica be applied to render the under- 

 lying medium relatively anaerobic, may be utilized in this relation ; but]} commonly 

 the anaerobic jar with special interior atmosphere or the special anaerobic tubes de- 

 scribed in a previous lesson are employed in such work. 



Exercise 78. Make an infusion of ordinary garden earth in sterile water 

 and from this make smear inoculations on agar surfaces. Grow in an at- 

 mosphere of hydrogen at body temperature. Observe after twenty -four 

 hours the appearance of small, whitish, round colonies with delicate veil- 

 like margins. Transfer by stab inoculation to gelatine and agar in at- 



