PROBLEMS OF METHOD 205 



culture may be transferred to a respiromctcr flask (36, 78, 214, 313), 

 but most workers have preferred to use when possible the more homo- 

 geneous mycelium obtained from shaken or aerated cultures (65, 70, 

 102, 163). The mycelium from such cultures is often in the form of 

 small pellets; if these are too large, above 0.4 mm. for Myrothecium 

 verrucaria (70), the rate of diffusion of oxygen into the interior of the 

 pellet may be limiting. Either surface or shake culture mycelium 

 may be homogenized briefly, but there is a considerable risk that 

 mechanical injury may reduce respiratory capacity (70, 80). 



The preparation of active cell-free extracts from fungi is an even 

 more vexing problem. We may list here, in outline, some of the 

 methods which have been successful: 



1. Hand grinding with abrasives (7, 44, 50, 65, 71, 140, 182, 301). 



2. Acetone drying (14, 129, 212, 288). 



3. Physical drying, with or without a preliminary freezing (89, 155, 

 204, 218, 331). 



4. Mechanical disruption in a ball mill (240) or in a motor-driven 

 homogenizer (46, 274). 



5. Freezing, followed by grinding with abrasives or by homogeniza- 

 tion (190, 201, 258, 274). 



6. Sonic oscillation (210, 264). 



7. Disruption in the Hughes press (142) or in a small-orifice press 

 (55, 200, 228). 



8. Treatment of frozen mycelium with dilute ammonium hydroxide, 

 followed by mild pressure (148). 



Of these, the most generally useful appear to be hand grinding with 

 sand or powdered glass, sonic oscillation, and treatment in the 

 Hughes or similar presses. Hugo (143) reviews the methods available 

 for the preparation of extracts from bacteria. 



Suspending media for extraction may be water, phosphate buffer, 

 or salt mixtures. Protective materials are often essential — both cys- 

 teine (62, 209) and ethylenediaminetetraacetate (110) protect against 

 inactivation of enzymes by heavy metals liberated during the extrac- 

 tion process. 



During the preparation of cell-free extracts the terminal respiratory 

 system is usually disrupted and must be replaced by an added hydro- 

 gen carrier or hydrogen acceptor. Methylene blue, 2,6-dichlorophenol- 

 indophenol, and other dyes have often been used; phenazine deriva- 

 tives (76), ferricyanide (134, 235), and manganese dioxide (133) are of 

 value for the same purpose. 



