568 STREPTOCOCCUS 



a-haemolysin. — -When we turn to those streptococci that produce a-haemolysis 

 on blood agar plates, we have two mechanisms to consider : the reaction that 

 causes the lysis of the red cells, and the reaction that causes the green coloration. 



Cole (1914) described the presence in pneumococci of a labile intracellular hsemolysin 

 which was liberated from the cells on autolysis. It was commonly stated by subsequent 

 workers that the pneumococcus produced no filtrable hsemolysin in fluid media ; and 

 the agent causing a-hsemolysis was supposed to be of some quite different nature. It 

 has, however, been quite clearly demonstrated within recent years (see Neill 1926, Sickles 

 and Coffey 1928, Cowan 1934, Todd 1934) that the pneumococcus, when grown under suit- 

 able conditions, produces a soluble hsemolysin of the oxygen-sensitive, heat-sensitive type, 

 which undergoes reversible oxidation at low temperatures. Whether other streptococci 

 that produce a-hsemolysis would also elaborate a filtrable hsemolysin under suitable con- 

 ditions is at present unknown. 



Until recently the most widely accepted view in regard to the green coloration associated 

 with a-hsemolysis was that it was due to the formation of methsemoglobin or of some closely 

 allied substance (Schnabel 1921, McLeod and Gordon 1922, Rother 1925). It was shown 

 by McLeod and Gordon that the pneumococcus produces hydrogen peroxide and that 

 hydrogen peroxide will discolour heated blood agar, in which the blood catalase has been 

 inactivated. The mechanisms involved in the production of hydrogen peroxide by the 

 pneumococcus, or by pneumococcal extracts, have been studied in considerable detail by 

 Avery, Morgan and Neill (Avery and Morgan 1924, Morgan and Avery 1924, Avery and 

 NeiU 1924a, h, c, Neill and Avery 1924o, h, 1925, Morgan and NeiU 1924, NeiU 1925). 

 The systems involved appear to include the catalysed oxidation-reduction and peroxidase 

 mechanisms discussed in Chapter 3, and the actual course of the reaction seems to be 

 determined in the main by the oxygen pressure to which the reacting system is exposed. 

 It is, however, clear that the formation of methsemoglobin is not itself the cause of the 

 green pigmentation, unless the apparent greenness is due to an optical illusion resulting 

 from a colour contrast ; and, in view of the amount of catalase present in unheated blood, 

 it is difficult to believe that hydrogen peroxide is the active agent in cultures on unheated 

 blood agar plates. 



This problem has been brought nearer solution by the studies of Hart and Anderson 

 (1933) (see also Anderson and Hart 1934a). Working with the pneumococcus, they found 

 that when small quantities of laked blood were added to broth cultures in the presence of 

 an alkaline buffer solution, an olive-green precipitate was formed. This could be separated, 

 washed, and dissolved in dilute alkali to give a green solution. Crystalline haemoglobin, 

 or methsemoglobin, gave the same green pigment when incubated under suitable conditions 

 with pneumococcal cultures. The spectroscopic and chemical analysis of this pigment 

 suggest that it is an iron -containing derivative of haemoglobin. It is rapidly bleached by 

 hydrogen peroxide, but it is not affected by reducing agents. An identical, or very similar, 

 green pigment can be obtained by incubating laked blood, hsemoglobin, or methsemoglobin 

 with various chemical reducing systems, such as ascorbic acid, cysteine-glucose, etc. From 

 the results obtained with autolysed bacterial cells, washed bacteria, bacterial extracts, etc., 

 it would seem that the green pigment results from the activity of a bacterial oxidation- 

 reduction system, one component of which is intracellular. This system is not peculiar 

 to the pneumococcus ; it is shared, not only by those streptococci that produce the green 

 pigment on blood agar plates, but by Str. pyogenes which gives ^-haemolysis, and by the 

 enterococci which usually produce no change on unheated blood media. It is also 

 possessed by unrelated bacteria, such as Staph, aureus and Bact. coli. The production of 

 green pigmentation by some species and not by others would seem to be due, not so much 

 to the presence or absence of the necessary enzyme system as to secondary factors, them- 

 selves determined by the metabolic activities of the bacteria concerned, which sometimes 

 permit this system to function, and sometimes suppress it. We have already noted 

 that some strains of hsemolytic streptococci may produce /?-hsemolysis under anaerobic 



