THE LYSIS OF RED BLOOD CELLS 227 



bacteria. It may be noted (see, for instance, Markl 1902, Topley 1915) that electrolytes 

 in high concentration have an inhibitory effect on the union of complement with sensitized 

 red ■ cells, and on the resulting haemolysis. 



The effect of temperature on haemolysis differs somewhat from its effect on precipitation 

 and agglutination, because we have an additional reagent to consider — the complement — 

 and this reagent is thermolabile. Union between red cells and hsemolysin occurs readily 

 at O'" C (see, for example, Ueno 1938). At this temperatm-e complement also unites with 

 sensitized red cells, though very much more slowly, but lysis fails to occiir over any ordinary 

 period of observation, unless excess of hsemolysin and complement is present. At room 

 temperature lysis occurs, but so slowly that this temperature is unsuitable for experimental 

 purposes. The optimal temperature for haemolysis is in the neighbourhood of 37° C. At 

 temjjeratures much above this the complement is inactivated, the rapidity of inactivation 

 increasing as the temperature is raised. 



The problem of the titration of a hsemolytic serum differs from that of titrating 

 precipitins or agglutinins in that we areiaced with three dependent variables instead 

 of two. Our essential reagents are red cells, hsemolysin and complement. Of 

 these reagents, it is natural to keep our red cells constant, regarding as our end-point 

 the lysis of a specified quantity of red cells, in a specified time, under specified 

 conditions. It is a common practice to use a 5 per cent, suspension of red cells in 

 saline, and to employ some convenient volume of this suspension, such as 0-5 ml., 

 in our tests. We have two reagents left, hsemolysin and complement. The natural, 

 and usual, plan is to vary the amount of the reagent that we want to measure — 

 the hsemolysin — while keeping the complement constant. But here we meet a 

 difficulty. We can measure our reagents only in terms of their activity, and they 

 are dependent variables ; the more complement we add, up to a point, the less 

 hsemolysin we need, and vice versa. We get out of our difiiculty by making use 

 of the kind of relation that exists between our variables. However much com- 

 plement we add, we need a certain amount of hsemolysin. However much hsemoly- 

 sin we add, we need a certain amount of complement. Moreover, the limit at which 

 further additions of one reagent make no appreciable difference to the required 

 amount of the other is not a high multiple of the minimal dose. So we define our 

 units of measurement as follows : 



The Minimal Hcemolytic Dose [M.H.D.) of hcemolysin is the smallest amount that 

 U'ill cause complete lysis of an arbitrarily selected amount of red cells, in the presence of 

 excess of complement, in 1 hour at 37° C. 



The Minimal Hcemolytic Dose {M.H.D.) of complement is the smallest amount that 

 will cause complete lysis of an arbitrarily selected amount of red cells, in the presence of 

 excess of hcemolysin, in 1 hour at 37° C. 



In an actual titration we proceed as follows : We first titrate our complement, which 

 must be used fresh, adding varying dilutions of our complement-containing serum to 

 mixtures of red cells and a haemolytic serum of known titre. A hsemolytic serum, it may 

 be noted, remains stable over a considerable period of time. In this titration we use 

 excess of hsemolysin say 5 or 6 M.H.D. We note the last tube that shows complete 

 haemolysis, and this gives us the M.H.D. of our complement. We now dilute our comple- 

 ment so that the volume we intend to use, commonly 0-5 ml., contains 3-5 M.H.D., and to 

 a series of tubes we add 0-5 ml. of our red cell suspension, 0-5 ml. of our diluted comple- 

 ment, and 0-5 ml. of the hEemolysin under test, the dilution of the latter increasing from 

 tube to tube. After 1 hour at 37° C. we note the last tube that shows complete haemolysis, 

 and this gives us the M.H.D. of our haemolysin. 



