MICRO-NUTRIENT PROBLEMS 25 



salts. In all cases 65 g. calcium carbonate and a small quantity 

 of a solution of some other salt were added to 5 1. of the solu- 

 tion to be purified. For calcium nitrate and potassium nitrate 

 the solution added along with the calcium carbonate was 

 50 ml. of molar dipotassium phosphate; with dipotassium phos- 

 phate and diammonium phosphate the added solution con- 

 sisted of 25 ml. of molar calcium nitrate, and with magnesium 

 sulphate and ammonium sulphate the added solution was 50 ml. 

 of molar calcium nitrate +50 ml. of molar dipotassium phos- 

 phate. The purification of all the solutions except that of 

 ammonium sulphate was then effected by autoclaving the mix- 

 ture for an hour at 20 lb. pressure, allowing the solution to stand 

 overnight and then filtering. The ammonium sulphate was 

 treated similarly except that the solution was heated for 45 min. 

 in a steamer instead of in an autoclave. The final filtrates of the 

 dipotassium and diammonium phosphate were acidified to 

 pH 5-5 with pure sulphuric or nitric acid. 



For testing the purity of the solutions so prepared Stout and 

 Arnon made use of dithizone (diphenylthiocarbazone). The 

 testing reagent is prepared by dissolving 0-1 g. of purified 

 dithizone in 100 ml. of redistilled chloroform. This reagent gives 

 a red or purple colour in the chloroform layer when it is added 

 to a solution containing zinc, copper, lead, nickel, cobalt, 

 cadmium, thallium, mercury or bismuth. By comparing the 

 colour produced by standard solutions with that produced by 

 the purified nutrient solutions it was found that the latter 

 usually contained less than one part of metal contaminants in 

 10 8 parts of solution, a degree of purity which was deemed 

 sufficient for culture work on micro -nutrients. Although man- 

 ganese does not give the colour reaction with dithizone it was 

 concluded that if the other metal contaminants which do 

 produce the colour with dithizone are removed, the manganese 

 will have been removed also. 



The salts so purified did not include iron. This was provided 

 as a solution containing 0-5 per cent ferrous sulphate +0-5 per 

 cent tartaric acid which was added twice weekly to the extent 

 of 0-5 ml. per litre of culture solution. 



That the method of purification used by Stout and Arnon was 

 justified is clear from the fact that they obtained definite effects 



