vi4^-14 MANUAL OF METHODS FOR PURE CULTURE STUDY 



To 1 volume of the culture fluid add 9 volumes of water and bring the reaction to pH 

 8 by the addition of N/20 NaOH or HCl. Add 8 volumes of formaldehyde solution 

 (approximately 40% formaldehyde). Immediately titrate the mixture to pH 8 with 

 N/20 NaOH. Determine the amount of N/20 NaOH required to bring 8 volumes of 

 the formaldehyde solution to pH 8 and subtract this from the titration of the mixture. 

 The result is the formol titration expressed as ml. of N/20 NaOH per 100 ml. of the 

 culture fluid. 



Deductions as to the amino-N content of the fluid should take into 

 account the ammonia inchided in the determination. 



The Van Slyke (1913) Amino-N Method: This procedure depends 

 upon the production of gaseous nitrogen when nitrous acid acts on an 

 aHphatic amine. Special apparatus is required. The recently intro- 

 duced (Van Slyke, 1929) manometric method is more generally useful 

 than the older volumetric. For details of the procedure consult the 

 original references (also, Peters and Van Slyke, 1932, 385). 



Ammonia. The quantitative determination of ammonia must be 

 carried out by a procedure which will not decompose potential am- 

 monia-producing compounds. From this standpoint, the Folin (1902) 

 aeration method as modified by Van Slyke and Cullen (1916) is prob- 

 ably safer than the usual distillation from a solution treated with 

 MgO. The procedure involves a cautious aeration of the alkalinized 

 solution with ammonia-free air into standard acid which is subse- 

 quently titrated. 



HoS and Volatile Mercaptans. Aeration of the acidified culture fluid 

 with HoS-free air and absorption of the volatile sulfides in a solution of 

 zinc or lead acetate would be the first step. Oxidation of the sulfide 

 with NaoOo would produce sulfate which is precipitable as BaS04; 

 oxidation of the mercaptans woidd produce sulfonic acid which is not 

 precipitable as lJaS04. Intensive oxidation in the presence of nitrate 

 and chlorate would convert all of the sulfur to sulfate. These are the 

 general principles upon which a method of analysis can be based. 



Action on Inorganic Nitrogenous Compounds 

 There are many bacteria that are capable of utilizing inorganic 

 sources of nitrogen, such as nitrates or ammonium salts. Some utilize 

 such nitrogen sources in preference to organic forms, others in addi- 

 tion to the latter. When action on such compounds occurs it is of value 

 to make more of a study of it than is given in Leaflet V under "Action 

 on Nitrates". 



Action on Ammonium Salts. There are a few bacteria that utilize 

 ammonium salts when furnished with no other source of nitrogen. In 

 such cases it is frequently of value to determine what percentage of 

 the ammonia furnished is used by the organisms. For such purposes 

 the ammonia can best be determined by distillation with magnesium 

 oxide and collection of the ammonia in standard acid solution, in 

 which it can be determined by titration. 



Action on Nitrates. The most common action of bacteria on nitrates 

 is one of reduction to nitrite, to ammonia, or to free nitrogen, or pos- 



