234 LABORATORY EXERCISES IN BACTERIOLOGY. 



temperature; or at least the conditions of pressure and temperature noted 

 in the records. Moreover, daily records should be kept, and the amount 

 accepted as final only four or five days after gas production has ceased. 

 Repeat with Micrococcus pyogenes, Bacillus subtilis, Bacillus typhosus, 

 Microspira comma, and Mycobacterium diphtheria. 



5. Acid Production. It is asserted that free acids are produced only in carbo- 

 hydrate-holding media, and are producing in sugar-holding media either from the 

 sugar directly or from the alcohol after alcoholic fermentation. Of these acids, lactic 

 acid is the most prominent, but there may also be traces of acetic acid, butyric acid, 

 and a few others. The actual analysis of the media for such acids is too complicated 

 for discussion in this place, and the mere demonstration of an acid change in the reaction 

 of the medium must be sufficient. 



Exercise 64. To neutral bouillon is added a drop or two of alcoholic 

 solution of rosolic acid, so as to produce a distinct pink color. Inoculate 

 the tube with Bacillus coli and place in the incubator. Note that for a 

 time the color is diminished, indicating the production of an acid reaction ; 

 after a few days, however, owing to exhaustion of sugar, this ceases, and 

 from the production of -ammoniacal compounds the reaction changes to 

 alkaline, causing the color to reappear and grow more distinct. 



Repeat with cultures of Micrococcus pyogenes, Bacillus subtilis, Bacillus 

 typhosus, Mycobacterium diphtheria, and Microspira comma. 



Exercise 65. To sterilized milk a little sterile litmus solution is added 

 so as to cause the medium to become slightly blue. To such preparation 

 is added a loopful of Bacillus coli, and it is allowed to remain in the incu- 

 bator for a time. At first the blue color changes to red, the latter later 

 disappearing and a deeper blue assumed from the alkaline change. The 

 milk-sugar at first was changed to lactic acid ; when the sugar was used up, 

 the proteids were progressively changed into alkaline compounds. 



6. Alkaline Production. This, as already referred to, is believed to be due 

 to the splitting of nitrogen compounds in the medium employed by certain bacteria 

 in a manner of the same type if not identical to metabolism; and the products as 

 far as known are ammonia, amine, and ammonium bases. It is the same process 

 as is concerned in the so-called urea-fermentation, by which the urea of fresh urine 

 is converted into ammonium carbonate, from which other ammonium bases may 

 thereafter develop, as ammonium urate or phosphate. To exhibit this alkaline -pro- 

 duction, which is common to a large number of bacteria grown on sugar-free proteids, 

 the following will serve: 



Exercise 66. Bacillus coli is inoculated into a tube of peptone solution 

 and grown at body temperature or lower (20 to 37 C.), for from ten to 

 fourteen days. The medium should have been exactly neutral to phenol- 

 phthalein, or if this has not been used, a fresh tube of the same reaction as 



