240 26 



the latter case, and also the apparent loss in agglutinating power experienced hy 

 the serum is much less. It is evident, too, that the important factor liere is not 

 the strength of the acid to which the immune-serum was exposed; but that it is 

 the amount of acid (in the form of acidified serum) added to the tubes at the 

 "zones" upper and lower limits. 



The experimental results detailed in Series 88 and 89 shew that although the 

 agglutinating serum employed in one set of tubes may be treated with 10 or 

 15 times as much acid as in the serum used in another set, yet the "zone of 

 inhibited agglutination" will come to an end, in either case, at that tube in which 

 the amount of acid added in the form of acidified serum is less than about 

 02 mgrm.; yet in the one case the amount of agglutinating scrum present in this 

 tube is some 22 times as great as it is in the other. 



Hence there can be no (jiiestion here of the conversion of agglutinine into ag<jliit- 

 inoid, as Eisenberg & Volk, and others have supposed to be the case. The experi- 

 ments just detailed prove this, and furtlier proof is given by the fact that /»;/ siiilal>le 

 neutralisation of the acidified serum, both the zone of inhibited agglutination (Uid the 

 apparent increase or reduction in the agglutinative power of the serum disappear, com- 

 pletely or nearly so, this depending upon the amount of acid with which the 

 immune-serum was originally treated. 



Since the results obtained here by ourselves stand in absolute contradiction 

 to those found by Eisenberg & Volk, it is necessary to look more closely into 

 the significance of this neutralisation of the acidified serum and certain other 

 points. 



The method of experiment was as follows. The immune-serum was exposed 

 to the action of the acid for 2 hours at 37°, in a water-bath. The subsequent 

 neutralisation of a portion of it is, however, a matter of no little difficulty; for 

 some compound is formed between the acid and the serum that is but slowly 

 decomposed by the alkali (NaOH solution) towards the end of the neutralisation, 

 or even when the alkali is present in slight excess. The effect of this is that when 

 almost the whole of the calculated quantity of NaOH solution has been added to 

 the acidified serum that is to be neutralised, the mixture shews a markedly alkaline 

 reaction with phenol-phthalein; but less than half an hour later it will be acid again, 

 and this alternation can be repeated several times in succession by the slow addi- 

 tion of the alkaline solution. Hence it is impossible to neutralise an acidified 

 serum with any great degree of chemical precision. A further difficulty arises from 

 the fact that a serum unites ecpially readily with eillier an acid or an alkali {HCl 

 or NaOH). As the destructive effect of an alkali upon the agglutinine is much 

 stronger than that of an acid, a new factor appears which must not be overlooked 

 when an estimate of the destructive efiect of an acid upon an agglutinating immune- 

 serum is being estimated by the results of neutralisation experiments such as those 

 described above: and il must be mentioned finally lliat when even very small 



