790 HEMOPHILUS 



H. influenzoB stains with some difficulty, and many of the ordinary bacteriological 

 dyes are unsuitable for this purpose. Dilute carbol-fuchsin applied for 5 to 15 

 minutes usually gives satisfactory results. All species within this genus are frankly 

 Gram-negative and non-acid-fast. 



It may be added that it is impossible to differentiate any given strain of H. per- 

 tussis from H. influenzcB on grounds of morphology alone. If, however, large 

 samples of strains are compared, certain modal differences may be observed. 

 H. pertussis displays a more constant morphology than H. injluenzce, and there is 

 a marked tendency towards the predominance of the short oval form of cell. 

 Longer bacillary or thread forms may occur, but they are relatively uncommon. 



Cultural Characters. Growth Requirements. — Since certain nutritional require- 

 ments provide the criteria by which this genus has been defined, and also the basis 

 on which many of the species included within it are differentiated from one 

 another, it will be convenient to discuss this aspect of their behaviour before dealing 

 with their type of growth, enzymic activities, antigenic structure or pathogenicity. 

 The most characteristic feature of the hamophilic bacilli is their failure to grow 

 in the absence of certain factors which are present in blood. The ability of the 

 influenza bacillus to grow on blood agar, and its inability to grow on agar, with 

 or without the addition of serum, or other native protein, has been noted by all 

 workers fromPfeiffer onwards. Grassberger (1897) described another phenomenon 

 — that of satellitism — which is highly characteristic of H. influenzcB. In cultures 

 on blood agar plates, streaked with sputum or with bronchial secretion, he noted 

 the appearance of relatively large colonies of the influenza bacillus, with a slightly 

 granular central portion. These large colonies (1 mm. or more in diameter) always 

 developed in the immediate vicinity of a colony of staphylococcus. Studying this 

 phenomenon in greater detail, Grassberger streaked agar plates with a suspension of 

 H. influenzoB mixed with a small quantity of blood, and then inoculated the central 

 portion of the plate with a trace of a pure culture of Staph, aureus. After 

 24 hours' incubation such plates showed a well-defined zone of colonies of 

 H. influenzcB, surrounding each colony of staphylococcus. A similar result was 

 obtained with Staph, albus, Staph, citreus, and certain other chromogenic 

 micrococci. These observations have been repeatedly confirmed (see Davis 1921, 

 Kristensen 1922, and Fig. 167). 



The inability of H. influenzcB to grow on serum agar indicates that some con- 

 stituent of the red cells is essential for growth ; and it was at first assumed that 

 this constituent was haemoglobin itself. More detailed study, however, showed 

 that the addition to an agar medium, prepared with water or with peptone solution, 

 of pure crystallized haemoglobin does not suffice to ensure growth (Ghon and von 

 Preyss 1904, Thalimer 1914, Davis 1917, Olsen 1920, Fildes 1921). It would appear 

 (Fildes 1921) that the growth-promoting substance derived from the blood pigment 

 is methsemoglobin, or haematin, rather than haemoglobin itself. Haematin is more 

 active in this respect than the other derivatives which have been tested ; while 

 haematoporphyrin is inactive (Fildes 1921). There is, however, another factor 

 which comes into play, besides the presence of some suitable iron-containing pig- 

 ment. Davis (1917) showed that H. influenzce required for its growth the presence 

 of two distinct substances : one contained in, or derived from, haemoglobin ; the 

 other present in the tissues of various plants and animals, and synthesized by 

 most bacterial species other than H. influenzce. This second factor he likened to 

 a vitamin. This substance, as compared with the factor provided by blood pigment, 



