BIOCHEMICAL REACTIONS 463 



sugars ; others again, such as C. hofmanni, attack none of them. No species 

 produces gas. Within the species C. diphthericB the gravis type ferments starch 

 and glycogen ; the mitis and intermedius types do not. In practice great care 

 is necessary in the preparation of the medium if reliable results are to be obtained. 

 A medium containing serum is almost indispensable, but it has certain drawbacks. 

 The serum, for example, may contain suflB.cient fermentable carbohydrate to give 

 a false reaction ; this can be overcome by suitable buffering. Unheated horse 

 serum may hydrolyse starch due to the presence of a natural diastase (Hendry 1938) ; 

 this may be destroyed by heating. Not all samples of soluble starch are satis 

 factory ; each sample should be tested before use. 



A suitable medium may be prepared by dissolving 0-5 per cent, peptone and 0-1 per cent. 

 Na2HP04 in 1,400 ml. of distilled water, steaming for 15 minutes, filtering, adjusting 

 to pH 7-4, adding 250 ml. of horse serum, steaming for 20 minutes, adding 11 ml. of 

 Andrade's indicator, adjusting to pH 7-6-7-8, tubing in 3 ml. quantities, autoclaving at 

 10 lb. for 10 minutes, and adding to each tube separately sufficient of a sterile solution 

 of the sugar in distilled water to give a final concentration of 0-4 per cent, starch or 

 1 per cent, of other sugars (Robinson 1940). 



A more detailed and extended table of the fermentation reactions of ten 

 named species within this genus, and of eleven unnamed diphtheroids examined 

 by Barratt (see Andre wes et al. 1923) is appended to this chapter, but experience 

 has revealed the existence of several more fermentative types. The fermenta- 

 tion of certain other substrates by different varieties of the type species is con- 

 sidered below. 



We may note here that certain species and types within this group, for instance 

 C. ovis, C. pyogenes and certain unnamed diphtheroids liquefy gelatin, while C. diph- 

 thericB and most diphtheroid organisms do not. 



Many strains of C. diphtliericB, but not all, produce areas of haemolysis on blood- 

 agar plates, and lyse red cells when these are added to broth cultures (Schwoner 

 1904, Costa et al. 1918, Goldie 1933). The red cells of the guinea-pig are the most 

 sensitive, then those of the rabbit, horse, man, pig, mouse and sheep in this order. 

 There is a conflict of evidence in regard to the production of a soluble hsemolysin. 

 The earlier observers stated that culture-filtrates did not cause haemolysis ; but 

 Goldie (1933) reports that cell-free filtrates are haemolytic, and that their activity 

 runs roughly parallel to their toxin content, though the hsemolysin is not neutralized 

 by antitoxin. There is a similar conflict in regard to the heat-resistance of the 

 haemolytic agent, whether it be intracellular or extracellular. It was originally 

 recorded as being inactivated by heating to 58° C. for half an hour, Goldie states 

 that it is not inactivated by boiling. Lysis around colonies on blood agar is of 

 some value in distinguishing between the three types of the diphtheria bacillus. 

 Most mitis strains are haemolytic, intermedius strains are not, and gravis strains 

 vary in their activity. 



Two species of diphtheroids that are pathogenic for animals, G. ovis and C. 

 pyogenes, also exert a haemolytic action on the red blood corpuscles of various 

 species, including the rabbit and horse. The other species and types within this 

 genus that have been examined from this point of view appear to be non-haemolytic. 



Some diphtheroid strains form phosphatase ; the true diphtheria bacillus never 

 does. The production of this enzyme may be tested for on a medium containing 

 phenolphthalein phosphate (see Bray 1944). 



