472 Prof. A. B. Macallum. Detection and Localisation 



compound. What the conditions are, under which this coloured 

 compound is produced, have not been determined, but this reaction 

 cannot interfere with or confuse the results of the action of the 

 reducing reagent on the phospho-molybdate compound. 



On the molybdate and phospho-molybdate compounds distributed 

 in animal and vegetable tissues, the phenylhydrazin hydrochloride 

 :acts as it does on these in the test-tube. It is not necessary to free 

 the tissue preparations from ammonium molybdate. They may be 

 placed for a minute or two in a dilute solution of nitric acid, after 

 which they are transferred to the reducing solution, which, in less 

 than two minutes, brings out the green colour where the phospho- 

 molybdate compound occurs, bat a faint yellow reaction where 

 ammonium molybdate alone is present. Instead of dilute nitric acid, 

 one may use distilled water, but it is not necessary to do even this, 

 for if the preparations are transferred directly to the reducing fluid 

 with but what may adhere to them of the nitric-molybdate solution, 

 the result is the same. 



When the reducing fluid has been allowed to act for the proper 

 length of time the preparations are washed in distilled water, then 

 dehydrated, cleared in oil of cedar, and mounted in balsam. Pre- 

 parations made in this way four months ago are now quite as satis- 

 factory as they were at first. 



Reference to the other reagents and methods which have been 

 used is also necessary. The nitric-molybdate reagent was made by 

 dissolving one part of pure molybdic acid (Mo0 3 ) in four parts of 

 strong ammonia, and adding thereto, slowly, fifteen parts of nitric 

 acid, sp. gr. 1*2. The proportions indicate weights. The resulting 

 solution had a faint yellow tinge, and, after decantation from the 

 very slight sediment, remained free from a precipitate as long as 

 any of it was unused. 



Fresh tissue material was used as well as that which had been 

 hardened in alcohol. The alcohol material is the best, for the nitric 

 acid, before it converts the phosphorus compounds in fresh tissue 

 elements into orthophosphoric acid, must dissolve a portion at least 

 of the phosphorus-holding proteids, and thus the phosphorus when 

 converted may not be distributed as intra vitam. I have, however, 

 used fresh material, wherever possible, to compare with that hard- 

 ened in alcohol. The latter offers advantages in the fixed form of 

 the elements, and in the preparation of thin sections which readily 

 permit the uniform action of the reagent as well as the extraction of 

 lecithin and inorganic phosphates. 



The time during which the reagent was allowed to act on the pre- 

 parations varied from, ten minutes to twenty- four, and even, in some 

 cases, forty-eight hours. It was found that a temperature of 35 C. 

 favoured considerably the formation of the phospho-molybdate. The 





