CULTIVATION OF B. INFLUENZA. 431 



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 diffi- 

 cult 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 the most recent epidemic, 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 influ- 

 enza bacillus is blood agar (see p. 42), 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 ^w^-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 inoculations. In this 

 way he completely succeeded in attaining his object. The blood 

 of the lower animals is suitable, as well as human blood. 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 completely transparent. 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. They also grow well on agar smeared with a solution 

 of haemoglobin ; growth on the ordinary agar media is slight 

 and somewhat uncertain. A very small amount of growth takes 

 place in bouillon, but it is more marked when a little fresh blood 

 is added. The growth forms a thin whitish deposit at the bot- 

 tom of the flask. The limits of growth are from 25 to 42 C., 



