CULTIVATION 481 



of carbol-fuchsin applied for five to ten minutes. They lose 

 the stain in Gram's method. They are non-motile, and do not 

 form spores. 



In many cases of the disease, especially in the early stages of 

 the more acute, influenza bacilli are present in large numbers 

 and may be easily found. On the other hand, it is often 

 difficult or impossible to find them, even when the symptoms 

 are severe; this may be due to the restriction of the organisms 

 to some part not readily accessible, or it may be that they 

 actually die out in great part while the effects of their toxins 

 persist. It has also been observed in recent epidemics, in which 

 the disease has been less widespread and on the whole less 

 severe, that the period during which the bacilli have been readily 

 demonstrable in the secretions has been on the average shorter 

 than in the previous epidemics. 



Cultivation. The best medium for the growth of the 

 influenza bacillus is blood-smeared agar (see p. 43), which 

 was introduced by Pfeiffer for this purpose. He obtained 

 growths of the bacilli on agar which had been smeared with 

 influenza sputum, but he failed to get any 5^6-cultures on the 

 agar media or on serum. The growth in the first cultures he 

 considered to be probably .due 'to the presence of certain organic 

 substances in the sputum, and accordingly he tried the expedient 

 of smearing the agar with drops of blood before making the in- 

 oculations. In this way he completely succeeded in attaining 

 his object. The blood of the lower animals is suitable, as well 

 as human blood; and the favouring influences of the blood 

 would appear to be due to the haemoglobin, as a solution of this 

 substance is equally effective. The colonies of the influenza 

 bacilli on blood -agar, incubated at 37 C., appear within twenty- 

 four hours, in the form of minute circular dots almost trans- 

 parent, like drops of dew. When numerous, the colonies are 

 scarcely visible to the naked eye, but when sparsely arranged 

 they may reach the size of a small pin's head. This size is 

 generally reached on the second day. In cultures the bacilli 

 may show considerable variations in size and in shape ; they die 

 out somewhat quickly, and in order to keep them alive sub- 

 cultures should be made every four or five days. By this 

 method the cultures may be maintained for an indefinite period. 

 Even in sub-cultures growth on the ordinary agar media is slight 

 and somewhat uncertain ; there is, however, evidence that growth 

 is more marked when other organisms are present that is, is 

 favoured by symbiosis. Neisser, for example, was able to 

 cultivate the influenza bacillus on plain agar through several 



