230 16 



When the serum was heated to 65°, dilTerences appeared according as it was 

 heated after dilution with normal saline or hy itself alone; and when such heated 

 sera were used to agglutinate an agar-culture suspension, "inhibition-zones" appeared. 

 For example, the experiments in Series 54 shew that an agglutinating serum heated 

 alone to 65° for Va hour, when present in from 2500 to 29 times the unit quantity 

 of the unheated serum needed to produce the definite degree of agglutination present 

 in the test-tube selected as standard, may cause either no agglutination at all, or 

 else a less degree of agglutination than that present in the standard tube; while 

 the agglutination in the tubes containing from 28 to 2 of the agglutinating units is 

 either total or more marked than it is in the standard tube. 



Hut if the serum is diluted with 9 volumes of normal saline before being 

 heated, the results obtained are quite different. The tubes containing from 250 to 

 125 agglutinating units of serum may shew no agglutination, while the tubes con- 

 taining from 125 to 2 agglutinating units may be more strongly agglutinated than 

 is the standard tube containing the single agglutinating unit of the unheated serum. 

 So in the second of these two cases the "zone of inhibited agglutination" is less 

 extensive than it is in the first; in one case it ends at the tube containing 125, in 

 the other, at the lube containing 29 agglutinating units of the heated serum. Exam- 

 ination of the other figures obtained with sera heated to 65° or 70° will be found 

 to shew very similar results. 



Thus, the more concentrated the solution in which the immune-serum is Iwated, 

 the more readily do "zones of inhibited agglutination" appear. Also, insj)ection of 

 the measurements recorded after the lapse of various periods of time will shew that 

 the longer the time that passes before the measurements are made, the less extensive 

 will the "zone of inhibited agglutination" be found to be; see Series 63 and 65. In 

 fact, a "zone" may entirely disappear if the tubes are examined a second time after 

 they have stood for a further 18 hours at the temperature of the room, as was the 

 case in the experiments of Series 59 and 61. 



If the agar-culture suspension of B. coli used for the measurements be now 

 replaced by a bouillon-culture, it is probable that no "zone of inhibited agglutina- 

 tion" will be found to occur, as may be observed in the figures in Series 58, 60, 

 62 and 64; or if it does occur, as in Series 66 and 68, it will only be feebly marked. 

 The employment of sera that have been heated may also lead to the appearance of 

 other curious phenomena in addition to the "zones of inhibited agglutination". Thus 

 if an agglutinating serum diluted with nine volumes of normal saline be warmed 

 to 70° for periods of 30 minutes, 1 hour, and 2 hours (see Series 65) and if the 

 agglulinaling powers of these three diluted specimens be then measured on an agar- 

 culture suspension, it will be found that lliey have not diminished, but have, on the 

 contrary, actually increased, so that Iheir agglutinating powers are now -/i, '-7i and 

 ^li, respectively, of that of the unheated serum in liie same state of dilution. But if 

 these same healed sera be tested with a bouillon-culture (see Series 66), their sliengths 



