TOXIN PRODUCTION 615 



used in the production of the toxin. In a fluid medium it develops rather slowly. 

 Thus Neisser and Wechsberg (1901), by testing broth cultures filtered at intervals, 

 found that the toxin, as judged by the haemolytic titre, was first demonstrable on 

 the 4th day of incubation at 37° C, and that it rose to a maximum between the 

 10th and 14th days, after which it gradually diminished. Burnet's (1930) technique 

 of growing the organisms on 0-8 per cent, nutrient agar for 24 hours in air containing 

 10-20 per cent. COg, and extracting the toxin from the agar with saline, is very 

 satisfactory, and is widely used, either in its original or a slightly modified form 

 (Parish and Clark 1932, Dolman 1932). The medium used should have a re- 

 action of between pH 6-0 and 7-0 (Walbum 1922). Though the toxin can traverse a 

 Seitz filter, some of its activity is lost in passage, and it is therefore advisable to 

 separate the organisms from the toxin by centrifugation. A good toxin should 

 haemolyse a 1 per cent, suspension of rabbit cells in a dose of 0-0005-0-002 ml. ; 

 0"001-0-005 ml. should cause necrosis on intradermal inoculation, and 0-25-0-8 ml. 

 per kilo injected intravenously should kill a rabbit within a few minutes (Burnet 

 1929, Gross 1931c, Parish and Clark 1932). Satisfactory toxins can be prepared 

 by growth in a chemically defined medium (Gladstone 1938, Smith and Price 1938a). 

 Though Burnet (1929), Gross (1931c) and Gengou (1932) formed the opinion 

 that the various activities of a toxic filtrate were due to one and the same toxin, 

 subsequent workers have inclined to the opposite view. Without prejudicing the 

 issue, we shall, for the sake of simplicity, describe each of the manifestations 

 separately. 



ix-hcemolysin. — This is active against rabbit, but not against human, red corpuscles, 

 and causes lysis rapidly at 37° C. It has some action on sheep red corpuscles, but this 

 is destroyed by heating at 57° C. for 30 minutes (Flaum 1938). It can be specifically 

 neutralized in multiple proportions by an antiserum. Its resistance to heat is peculiar, 

 since it is inactivated more readily by lower than by higher temperatures (Arrhenius 1907, 

 Landsteiner and von Rauchenbichler 1909, SeifFert 1935-36, Kodama and Nisiyama 

 1938, Rigdon 1938a, Beumer 1939, Fulton 1943). According to Arrhenius (1907), lysin 

 heated to 70° C. loses a great part of its hfemolytic activity, but regains it if it is heated 

 for a further 5 minutes to 100° C. This fundamental observation was confirmed by 

 Landsteiner and von Rauchenbichler (1909), using a temperature of 65° C. for 30 minutes 

 to inactivate the lysin. As the result of experimental observations they were led to 

 beUeve that at the lower temperature the lysin enters into combination with a protein 

 constituent, dei?ived from the broken down organisms, the medium, or added serum, to 

 form an inactive compound that can be destroyed by heating to a higher temperature, 

 thus Uberating the lysin. The failure to realize this unusual reaction to heat has led 

 to much confusion by workers who have assumed that, because the lysin is apparently 

 destroyed by heating for 2 hours at 57° C, 1 hour at 60° C, or 30 minutes at 65° C, it 

 is necessarily destroyed by boihng. According to Burnet (1931), Kodama and Nisiyama 

 (1938), and Fulton (1943), a strong -toxin is not destroyed completely even in 30 minutes 

 at 100° C. Its activity is said to be inhibited by ascorbic acid in a concentration of 30 mgm. 

 per ml. (Mercier 1938), and by azochloramid (Heise and Starin 1940). There is a close 

 paraUeUsm between the a-hsemolysin content of a filtrate and (1) the dermonecrotic and 

 lethal factors (see Panton and Valentine 1932, Levine 1939), (2) the ability of a given 

 strain to reduce methylene blue (McBroom 1937), and (3) the leucocidin content, as esti- 

 mated by the Neisser-Wechsberg method (Wright 1936), though this is still subject to 

 dispute (see Flaum 1938). Morgan and Graydon (1936) have brought evidence to show 

 that in most toxic filtrates the a-lysin contains two antigenically distinct components, 

 referred to as a^ and ag, which have different combining powers for antitoxin ; both are 

 dermonecrotic. Treatment of the toxin with 0-2-0-5 per cent, formol at 37° C. leads 



