Sept. 1, 1932 Commercial Honey and Spread of American Foulhrood 267 



MINIMUM NUMBER OF SPORES OF BACILLUS LARVAE PRODUCING. 

 VEGETATIVE GROWTH ON ARTIFICIAL CULTURE MEDIA 



Bacteria are known to pass tlirough a definite cycle of growth, par- 

 ticularly when cells from an old culture are transferred to fresh 

 culture media. The growth stages have been described by Buchanan 

 (3; IS, Ch. Tl, Henrici (12), and Winslow (IS, Ch. VI) somewhat as. 

 follows: The initial stationary phase during which no growth takes 

 place; the logarithmic phase when the organisms begin to divide, 

 slowly at first but gradually accelerating; and so on through the com- 

 plete cycle of growth. Henrici (IS, p. 21, 24) has observed that — 



Various factors, as temperature; the size, the age, and previous history of the? 

 inoculum; and the composition and nutrient value of the medium, influence the- 

 growth curves of bacteria. * * * Qf the various factors which influence the 

 rate of growth and form of the growth curve, the initial number of cells introduced! 

 into a unit volume of medium seems to be one of the most important. 



Robertson (25), in studies of cultures of certain protozoa, has 

 shown that growth seems to be stimulated by the presence of other 

 cells of the same type. This characteristic has been described at 

 various times as mass action or communal activity. 



Early in 1929, in conjunction with the spore-feeding experiments.-. 

 in the apiary, an investigation was started to determine whether 

 there is a similar manifestation of mass action in the vegetative 

 growth of spores of Bacillus larvae on artificial culture media. In a^ 

 preliminary paper on this subject the writer (27, p. 4-56) made the 

 following observations : Starting with a seeding of 5,000,000,000 spores^ 

 of B. larvae on a suitable slanted solid culture medium, it was found 

 at the end of 48 hours' incubation at 37° C. that growth had occurred, 

 in the original and in a diluted seeding containing 60,000,000 spores, 

 but not in one containing 50,000,000 spores. Growth occurred in a 

 diluted seeding containing only 5,000,000 spores after six days' 

 incubation, and in one containing 700,000 spores after 10 days' incu- 

 bation. (Table 4, Group 1.) These observations indicated that a. 

 certain initial mass of spores is necessary to start vegetative growth.. 

 Furthermore, although the growth results were rather irregular owin^ 

 to the comparatively small number of cultures made, they seemed to 

 show that, within certain limits, the smaller the seeding the longer 

 the incubation period necessary to obtain germination of the spores 

 and vegetative growth. From this preliminary work it was assumed 

 that the lower limits of dilution of the stock suspension that would 

 give growth on longer incubation had not been reached. 



Ahrens (1) has observed, in cultural studies of scales treated with- 

 formalin solution for different lengths of time, that growth may: 

 occur in cultures from such scales after varying periods of incubation; 

 up to 30 days, depending on the length of treatment and the per- 

 centage of formalin in the solution. Burnside (7) states, in connec- 

 tion with studies of disinfection of American foulbrood combs by- 

 fumigation with formaldehyde gas, that "it is probable that if scales; 

 had been washed and the incubation period increased, growth of 

 Bacillus larvae would have been obtained in some instances whem 

 negative results were recorded." 



Therefore, a single trial series of cultures was run (No. 7, Table 4)^ 

 the total incubation period being 30 days. Results from this set of 

 cultures showed that in some cases growth was obtained aftier 30i 



