1/6 MANUAL OF MICROBIOLOGICAL METHODS 



organisms are washed free of nutrients and are suspended in a nutrient- 

 free liquid, thus rendering them nonproliferative. In actual use, the 

 endogenous activity of the cell suspension must be determined as well as 

 the activity upon the addition of a specific substrate. When the endog- 

 enous activity is low, the experimental values can be readily corrected 

 by subtraction of the endogenous rate. However, if the endogenous rate 

 is high, certain complications may arise which may best be avoided by 

 attempts to lower this rate. Bubbling air at room temperature into the 

 suspension for an hour or more may result in lowered endogenous values 

 by the oxidation of nutrients still present within the cells. 



Some of the advantages of the resting cell technic include (1) elimina- 

 tion of growth in nonprolif crating cells, (2) ease of preparation with good 

 reproducibility of data, (3) uniformity of suspensions for quantitative 

 pipetting, (4) excellent spectrum of enzymes stable under these condi- 

 tions, and (5) maintenance of activity at refrigerator storage tempera- 

 ture, the time depending on the lability of the particular enzyme system. 

 The major difficulties are (1) selective permeability of the living cell 

 which prevents entrance of many types of compounds into the cell and 

 (2) the number of reactions which may be competing for the substrate or 

 the products or both. 



Dried Cells 



The use of dried preparations of microbial cells has certain advantages 

 over the resting cell technic. The permeability barrier is markedly 

 reduced and in some cases eliminated; the number of reactions occurring 

 is reduced; the preparations are often stable for months or even years, 

 thus allowing excellent reproducibility of results. Although some 

 enzyme systems appear quite labile to drying, persistent efforts with some 

 modification in technic have resulted in stable preparations of many 

 enzymes. 



There are two methods of drying in general use : vacuum and acetone 

 drying. Vacuum drying consists of drying the cell suspension in vacuo 

 over a suitable desiccant, such as Drierite or phosphorus pentoxide, in a 

 desiccator. A minimum volume of water is used to prepare the cell 

 paste, since thin layers of cell paste present the maximum surface to the 

 desiccant. A good high-vacuum pump and an amount of desiccant 

 capable of absorbing the volume of water present are required. Acetone 

 drying consists of the dropwise addition of a heavy cell paste to about 

 10 vol of ice-cold acetone with constant stirring. The cells precipitate 

 and are removed immediately by vacuum filtration, the acetone being 

 removed by suction. The dried cell preparations and, in most cases, the 

 cell-free enzyme extracts below should be stored at to — 20°C to prevent 

 destruction of enzyme activity. 



