FURTHER BIOCHEMICAL METHODS VI4.-I3 



A constant temperature water-bath is essential to bring the re- 

 actants quickly to the desired temperature and to maintain that 

 temperature throughout the experiment. 



The buffer must be carefully selected as to type of buffer and con- 

 centration. One must have sufficient buffer to maintain the desired 

 pH throughout the duration of the experiment. 



The standard (90% reduction) may be prepared by substituting 

 distilled water for the substrate and by adding 0.1 the regular amount 

 of methylene blue and leaving it open to the air. 



The reactants may consist of 1 ml. each of buffer, substrate (N/10), 

 methylene blue solution (1/5,000) and the bacterial suspension. The 

 buffer, substrate and methylene blue are mixed together. The sus- 

 pension is placed in the side arm or in the hollow stopper. The system 

 is made anaerobic and placed in the water-bath at a predetermined 

 temperature, usually 30°, 37° or 40°C. When the temperature has 

 reached that of the water-bath, the suspension is mixed with the 

 other substances and the time recorded. The time required by the 

 substrate (Ho-donator) to reduce the methylene blue, until the color 

 matches that of the standard, is compared to the endogenous reduc- 

 tion time; the latter is the time required by the suspension to reduce 

 the methylene blue in the absence of the substrate. 



A dehydrogenase is considered present when the reduction time 

 in the presence of substrate is less than the endogenous time. 



Cleavage of Proteins and Their Products 

 The liquefaction of insoluble nitrogenous organic material such as 

 gelatin, coagulated casein or blood serum is one criterion of the cleav- 

 age of these substances. As the process continues, progressive changes 

 occur in the biuret reaction and in the number of "free" amino and 

 carboxyl groups. In addition, there appear certain more or less 

 characteristic end-products, such as ammonia, hydrogen sulfide, 

 mercaptans, and tyrosine (depending on the constitution of the nitrog- 

 enous substrate) which are often readily perceptible. 



The Biuret Reaciion. Proteins form colored complexes with cup- 

 ric ions in alkaline solution. This is one of a general type of reactions 

 by ammonia or substituted ammonias. The color of the complex is 

 violet with the more complex polypeptides and proteins, and pinkish 

 lavender with peptones. 



The test is carried out by making the culture solution alkaline (about molar) with 

 iVaOH and then adding 0.1% CUSO4 dropwise imtil the minimum amovint has been 

 added to produce the pink to violet color. Ammonium salts interfere and, if present, 

 should be removed before testing. 



Amino Nitrogen. The commonly employed measures of amino com- 

 pounds are the Sorensen formol titration and the well-known Van 

 Slyke procedure. 



The Formol Titration: This method depends on the increase in 

 acidity brought about when neutralized formaldehyde is added to a 

 solution containing ammonia, primary amines, amino acids or poly- 

 peptides. A practical procedure is given by Brown (1923). 



