CULTIVATION OF BACILLI 421 



somewhat feebly, and are best stained by a weak solution (1 : 10) 

 of carbol-fuchsin applied for 5 to 10 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 agar (see page 38), which was intro- 

 duced 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 subcultures 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 inoculations. 

 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 transparent, 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. The bacilli die out somewhat quickly in cultures, 

 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. 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 bacfllus on plain agar 

 through several generations by growing the xerosis bacillus 



