78 
Proceedings of Boyal Society of Edinburgh. [sess. 
After having added rennet to a solution containing caseinogen 
and kept the mixture for sometime (30 minutes) at 40° C., the 
non-appearance of a clot is not now-a-days held to prove that 
caseinogen has not been acted on by rennet, but merely that the 
casein has not been precipitated. 
I have corroborated the value of Ringer’s test of the formation of 
this soluble casein, which, for brevity, I shall call giro-casein, based 
on his belief in the two-fold nature of the process of clotting, viz. : — 
(1.) Stage one . — The conversion of caseinogen by rennet into a 
soluble form of casein (gerinnung). 
(2.) Stage two . — The precipitation or solidification of pro-casein 
into a more or less cohesive clot or curd — casein — in presence of or 
by the action of certain substances (abscheidung). This is shown 
by boiling the solution containing the rennet, so as to kill the 
enzyme and prevent its further action. When cold, some preci- 
pitant of pro-casein is added to the tube, usually CaCl 2 , and if after 
heating, a precipitate now falls, it is assumed that the rennet did 
its work independently of the CaCl 2 or other precipitant. One 
cannot demonstrate stage two in ordinary milk, because clotting 
occurs in it without the addition of any precipitant ; it can be well 
shown in diluted milk or in pure solutions of caseinogen ; thus, 
dilute milk (1 in 4) heated at 40° C. with M.R. gave no clot, it 
was then boiled , and on cooling 4 m. of CaCl 2 (10 per cent, solu- 
tion) were added, when a clot appeared. Had the solution not 
been boiled, we should have called it a case of clotting due to the 
adjuvancy of CaCl 2 ; we now know the rennet acted without any 
assistance from the CaCl 2 , which merely precipitated the preformed 
pro-casein. (CaCl 2 precipitates pro-casein with great rapidity, 
and, except in dilute solutions, as a firm curd, whereas it precipi- 
tates caseinogen in milk very much more slowly, at a much higher 
temperature, and often only as a slimy deposit). The importance 
of a free acid in effecting stage two is well brought out by the follow- 
ing experiments. Ringer’s pure caseinogen or milk + N.R., heated 
gave no clot ; boil each solution, and add 1 in acetic acid to each, 
when a clot forms in each. Had we not previously boiled we 
might have imagined the rennet had not been able to act until the 
acid was added, whereas what really occurred was that the rennet 
did its work in the neutral medium, the result of that work 
