MEDIA MAKING AND APPARATUS 271 



pet cock first a little at a time, until pressure inside and out is 

 equal. This process should not take more than one to one and 

 one-half hours. 



The general principal of the above was devised in the Minn. 

 State Board of Health Laboratories, but is applied there to chloro- 

 formed serum, and requires about four times as long. As given, 

 the method was devised in the Boston Board of Health Bacteriolog- 

 ical Laboratory, and is for fresh serum only. 



Test for Indol. — Grow the bacteria which it is desired to test 

 for 10 days in muscle-sugar-free broth (" indol broth "). Add 10 

 drops of sulphuric acid and 1 c.c. of an aqueous solution of sodium 

 nitrite (1 in 10,000) ; a pink color developing at once or within half 

 an hour, especially on warming gently to 50 C, indicates presence 

 of indol. Certain bacteria produce nitrites. If such produce indol 

 also (as in the case of the cholera spirillum (microspira comma), 

 sulphuric acid, added as described, produces the color without 

 further addition of nitrite. This is called the cholera red reaction. 



Test for Formation of Nitrites from Nitrates. — Grow the 

 bacteria for 5 days in " nitrate broth." Add to the culture 2 c.c. 

 of freshly made mixture of the following solutions: (a) naphyla- 

 mine, 1% aqueous solution; (b) sulphanilic acid, 3% solution in 

 dilute acetic acid. A pink color shows presence of nitrite. Nitrite 

 is absorbed from the air, and in laboratory air much nitrite is 

 usually present; hence care should be taken at every stage to avoid 

 air-exposure so far as possible, and uninoculated tubes of the ni- 

 trate medium, exposed to the same conditions as those inoculated, 

 should be tested at the same time as a control. 



Litmus Solution. — Take 200 to 500 grams of litmus cubes 

 (chalk saturated in crude litmus and dried). Pulverize in mortar; 

 wash all blue out of chalk with water on a filter paper. Evaporate 

 water to dryness. Pulverize residue and wash on filter with al- 

 cohol (95%) to extract the red portion. When the alcohol pass- 

 ing through the litmus comes away colorless (8 or 9 litres of al- 



