FURTHER BIOCHEMICAL METHODS vi^.-S 



determinations with several strains believed to be of the same species 

 or at least very closely related one to another. 



Each fermentation is a problem of its own, and the choice of analyt- 

 ical methods must vary with the group of bacteria under investiga- 

 tion. To give specific directions here for even the most common con- 

 tingencies would consume an inordinate amount of space. Except in 

 one instance (action on nitrates) which seems nowhere to have been 

 treated adequately, only the main features will be considered here. Of 

 the various compilations of methods, the three following may be parti- 

 cularly useful for purposes of reference: A.O.A.C., Official and Tenta- 

 tive Methods of Analysis, 5th Ed. 1940; Abderhalden, E., Handbuch 

 der biologischen Arbeitsmethoden (Urban, Berlin); and Peters and 

 Van Slyke (1931, 1932). 



Preparation of Bacterial Juices 



Cell-free juices prepared from bacteria are receiving increasing use 

 in physiological studies and are serving in the elucidation of problems 

 dealing with mechanism of bacterial action on substrates. Juices are 

 obtained usually by one of the following methods: (a) Extraction of 

 juice, (b) press juice, (c) filtrates, (d) grinding, or a combination of 

 methods. The Booth-Green (1938) mill has been used to good ad- 

 vantage; in the United States, the powdered glass-grinding-extraction 

 technic has given good results. The Booth-Green mill is unobtainable 

 at present; it has been used especially by the English workers. In 

 general the technic of grinding with powdered glass, followed by ex- 

 traction, has certain advantages both in cost of equipment and 

 breadth of application. Bacteria are grown in liquid culture, centri- 

 fuged in a Sharpies super-centrifuge at 30,000 r.p.m. and the resulting 

 paste mixed with a quantity of powdered glass (generally two parts 

 paste: 1 part glass) with a particle size of about 2 /i. The powdered 

 glass is prepared by grinding clean pyrex in a ball mill with steel balls 

 for one hour. A mask should be worn. The bacteria-glass mixture is 

 forced through a grinding apparatus comprising two glass cones, one 

 turning within the other. The bacteria are cut by the fine glass 

 particles. See: Wiggert, et al (1940); Werkman and Wood (1940). 

 The mixture is extracted with water or a buffer solution, and after 

 that is centrifuged to throw down the glass. The extract then may be 

 centrifuged in a Beams air-driven centrifuge until clear. A differential 

 separation of enzymes may be accomplished by the Beams centri- 

 fugation. The supernatant liquid may be dialyzed through collodion or 

 cellophane membranes to remove coenzymes and inorganic ions. 

 Juices are desirable when separate enzyme systems are under in- 

 vestigation; also when the cell wall is impermeable to a substrate. 



