PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 175 



all types of high metabolic activity. In practice it is knoA^^i that the 

 metabolism of a given organism reflects many contributing factors: the 

 nature and age of the organism, its nutritional requirements, and reac- 

 tions to physical and chemical conditions, etc. (see Werkman and Wilson, 

 1951). A medium yielding a large crop of cells is not necessarily one 

 which will produce cells of high metabolic activity. The reader is 

 referred to Chap. Ill for some details of media composition, to Snell 

 (1951) for a discussion of bacterial nutrition, and to Gale (1943) and 

 Stanier (1951) for consideration of the many factors affecting the enzy- 

 matic activities of cells. Prior to a biochemical study of a given organ- 

 ism, it is desirable and often necessary to investigate closely the relation- 

 ship between the conditions of growth and of metabolic activity. Culti- 

 vation of the organism under conditions which enhance the quantitative 

 presence of the biochemical reaction of interest (e.g., adaptive enzyme 

 and apoenzyme formation) is a povrerful tool available to the microbial 

 biochemist, and once these conditions are recognized, their control is 

 relatively simple. 



Harvesting of cells from a liquid medium is achieved by centrifugation, 

 the type of machine employed depending primarily on the speed desired 

 and the volume of medium involved. For large volumes the Sharpies 

 type of ultracentrifuge is emploj^ed, while for smaller volumes many types 

 of angle head centrifuges are available. Although refrigeration during 

 centrifugation may be desirable and even obligatory in some cases, in 

 most instances room-temperature centrifugation is adequate. Harvest- 

 ing of cells from a solid medium is done simply by washing the surface of 

 the medium with water or saline, using a spatula or glass rod and collec- 

 tion of the resultant suspension. 



Preparation of Resting Cell Suspensions 



After centrifugation the medium is discarded and the packed cells 

 resuspended in a suitable menstruum and washed once or twice to remove 

 nutrients absorbed to the cell surface. The menstruum employed varies, 

 although distilled water, saline, or buffer is most com.monly used. The 

 washed cell paste is resuspended, and since it is often necessary in bio- 

 chemical work to relate a given metabolic activity to cell number or cell 

 mass, the cell suspension density is usually controlled and measured. 

 Determination of cell number is performed in the usual manner : dilution 

 and plate count or total microscopic count. How^ever, metabolic activity 

 is not necessarily related to viability or total cell number, a measure of 

 cell mass or protoplasm, being more directly related to biochemical activ- 

 ity. If dry weight of cells is related to the turbidity of a cell suspension, 

 a quick and accurate relationship of cell mass is obtained (see the previous 

 section) . 



Si^ch a suspension is referred to as a resting cell suspension because the 



