174 MANUAL OF MICROBIOLOGICAL METHODS 



of a digestion mixture (500 ml of H2SO4, sp gr 1.84; 75 g of Na2S04; 2.0 g 

 of CuSeOs; and 500 ml of H2O), and boil gently in a hood or on a Kjeldahl 

 rack for 1 hr longer than is necessary to clarify the solution. Allow the 

 mixture to cool; add a small amount of antifoam agent (caprylic or oleic 

 acid, or silicone) and 5.0 ml of ION NaOH. Immediately connect the 

 flask to a small distillation apparatus fitted with a graduated receiver 

 tube containing 2.0 ml of 0.Q5N H2SO4. The delivery tube should 

 extend well belov/ the surface of the acid in the receiver. Gently boil the 

 sample for 5 min ; during the last minute, raise the delivery tube slightly 

 above the surface of the acid. This procedure allows all the distilled 

 ammonia to drip free of the delivery tube and prevents drawing the 

 receiving solution back into the distilling flask when the flame is removed 

 from the latter. Dilute the receiving solution to contain 10-15 ng per ml 

 of ammonia-nitrogen. In a colorimeter tube, mix 2.0 ml of diluted dis- 

 tillate, 2.0 ml of Nessler's reagent (contains per liter: 4.0 g of Hgl, 4.0 g oi 

 KI, and 1.75 g of gum ghatti), and 3.0 ml of 2N NaOH. Allow to stand 

 for 15 min at room temperature, and read in the colorimeter at 540 m/z. 

 The amount of ammonia-nitrogen is determined from a curve previously 

 constructed using known samples of ammonia-nitrogen. 



It should be remembered that this method cannot be used with aliquots 

 of a culture taken directly from the medium, since the sample will include 

 medium constituents and will yield falsely high results. The nessleriza- 

 tion procedure can also be replaced by direct titration of the distillate 

 when the receiver acid solution is measured quantitatively. 



Discussion and Recommendations 



It is obvious that application of any of the methods described above 

 depends upon the experimental approach and the type of apparatus and 

 materials available. The technic selected also will depend to a certain 

 extent upon the investigator's definition of growth. If growth is viewed 

 as a simple increase in size, then a cell count will not reveal the extent of 

 growth. On the other hand, if growth is recognized as increase in both 

 the amount of protein and the number of cells, then expressions of total 

 N, turbidity, and cell count will each represent growth, although not pre- 

 cisely, and will also be in some measure of disagreement, since each is 

 based upon different physical and chemical attributes of the cells. Thus, 

 where possible, the method selected should be used throughout a com- 

 plete study, since comparisons among experiments will then be valid. 



PREPARATION OF CELLS AND EXTRACTS 

 Growth and Harvest of Metabolically Active Cells 



It is impossible to suggest specific growth media and conditions of 

 incubation suitable for the production of microbial cells demonstrating 



