358 
ROBERT AARON STEINBERG 
the salt itself or to the impurities present in the salt. Recourse was had 
to further chemical purification. 
The method of purification used depends on the fact that the "stimu- 
lants" in question are salts of cations whose hydroxides (and carbonates 
also) are quite insoluble and but weakly ionized. The barium carbonate 
method (66) of separation of ferric, aluminum, chromic, titanic, and uranyl 
salts from manganese, nickel, cobalt, zinc, and ferrous salts has long been 
known and is a standard method in the chemical laboratory. In using this 
method of separation it is necessary to carry out the hydrolysis in the cold, 
since on warming the salts of the bivalent metals are also hydrolyzed to- an 
appreciable extent and are then precipitated by the barium carbonate. 
It is evident, however, that the use of barium carbonate would be 
objectionable from a physiological point of view, since some barium neces- 
sarily goes into solution. Calcium carbonate, however, with use of higher 
temperatures (20 minutes at 14^ pounds in the autoclave) apparently 
causes the precipitation of these metals (iron, zinc, etc.) as effectually, and 
was therefore used in place of the barium carbonate. 
The details of the method of treating the Pfeffer solution as I have used 
it are as follows : The Pfeffer solution is prepared by weighing out the sugar 
and salts (except iron sulphate) into the same beaker, redistilled water is 
added, and the compounds are permitted to go into solution. The solution 
is now placed in liter pyrex flasks, 15 grams of dry CaCOa (Baker's 
"Analyzed") are added per liter, and the neck of the flask is plugged with 
absorbent cotton. The contents are now thoroughly rotated so as to 
insure mixing and placed in the autoclave for twenty minutes at 14^^ pounds 
pressure. On removal from^ the autoclave the flask is again rotated to cause 
mixing of the contents and set aside in a cupboard for 12 to 24 hours, or 
even for several days. When required for use it is necessary only to remove 
the cotton plug and to decant the supernatant liquid into a beaker. Fifty 
cc. of culture medium are now poured into each flask, the flask is plugged 
with absorbent cotton and sterilized. The substance to be tested, iron, 
zinc, etc., is added the next morning shortly before inoculation. 
The Pfeffer solution so treated is still slightly acid to litmus, gives 
excellent tests for phosphates and sulphates, and without doubt contains 
calcium. The CaCOs residue contains appreciable amounts of phosphates 
and sulphates from the nutrient solution, but a test for sugar (Fehling 
solution) gave negative results. A slight precipitate forms in the treated 
solution on sterilization. The composition of this precipitate as well as 
that of the nutrient medium has as yet not been definitely determined, but 
it is planned, in connection with the testing of various other heavy metals 
and their combinations as Substitutes for iron and zinc, to ascertain analyt- 
ically just how and in what degree the components of the Pfeffer nutrient 
solution undergo modification as the result of the treatment. Reference 
to the earlier experiments given in this paper indicate that the variations 
