GENERAL PROCEDURES FOR RADIOASSAY 161 



down the initial reaction by using increased amounts of acid and applying 

 heat very cautiously with frequent stirring. In the case of teeth, bone, 

 or cartilage samples, care must be taken not to reduce the acid solution 

 to the point where the salts start to precipitate; it is very difficult to get 

 the precipitated salts back into solution. In handling samples such as 

 white bone marrow, lymph glands, and adipose tissue, which contain 

 large amounts of fat, there is a tendency for bumping and spattering, so 

 that it is advisable to heat them gently at all times, preferably on a water 

 bath. With this type of sample, a solvent mixture of 10 parts ethyl alco- 

 hol, 25 parts diethyl ether, and 25 parts petroleum ether may be more 

 efficient than isoamyl alcohol. 



Some feces samples, depending on the nature of the diet, may require 

 more than the usual amount of nitric acid for complete digestion. It has 

 been observed that, if these samples are evaporated to dryness, it is very 

 difficult to get the residue into solution again. In some experiments, col- 

 lections are made on filter paper, and in such cases wet-ashing may be 

 carried out as follows: The filter paper is first dampened with water, and 

 4 to 5 ml concentrated sulfuric acid is added. About 10 ml nitric acid is 

 then added, and the beaker is heated gently until the violent reaction has 

 subsided, then heated further until the contents are charred. At that 

 stage 25 to 30 ml nitric acid is added, and the digestion continued as pre- 

 viously described. 



Bahner et al. (14) have suggested a modification of this procedure which 

 is based on the use of acetone or dioxane to bring both the fat and the 

 water into a single phase. The tissue sample is heated with a minimum 

 amount of concentrated nitric acid until all particulate matter is dissolved 

 and most of the excess acid boiled away. The solution is cooled and 

 diluted with acetone or dioxane and a little water to form a clear solution. 

 The optimum mixture contains 60 to 80 per cent dioxane or acetone 

 depending on the relative amounts of fat and inorganic salts. Howarth 

 (15) was able to obtain satisfactory tissue suspensions for radioassay by 

 boiling under reflux with a solution of lithium hydroxide containing 20 per 

 cent alcohol. Tabern (16) has proposed the disintegration of tissue in a 

 Waring Blendor or Potter Elvejhem homogenizer followed by treatment 

 with formamide to give a concentration of at least 40 per cent; this pro- 

 duced a homogeneous colloidal solution suitable for liquid counting. 



Wet-ashing. Conventional wet-ashing procedures have been ade- 

 quately described in the literature (4, 5, 17 to 21). The Kjeldahl proce- 

 dure, using concentrated H2SO4 as the oxidizing agent and various cat- 

 alysts, is widely employed. Various combinations of nitric, sulfuric, 

 hydrochloric, and perchloric acids and hydrogen peroxide have been suc- 

 cessfully used. A typical procedure for plant samples has been described 

 by Piper (4) : One to five grams of the finely ground sample is transferred 



