258 Journal oj Agricultural Research voi. 45, No. 5 



vidual species, in virulence and in ability to produce disease. Fur- 

 thermore, as stated by Zinsser {31, p. 188-189) — 



Whether or not infection occurs depends also upon the number of bacteria 

 which gain entrance to the animal tissues. A small number of bacteria, even 

 though of proper species and of sufficient virulence, may easily be overcome by 

 the first onslaught of the defensive forces of the body. Bacteria, therefore, must 

 be in sufficient number to overcome local defenses and to gain a definite foothold 

 and carry on their life processes, before they can give rise to an infection. The 

 more virulent the germ, other conditions being equal, the smaller the number 

 necessary for the production of disease. The introduction of a single individual 

 of the anthrax species, it is claimed, is often sufficient to cause fatal infection; 

 while forms less well adapted to the parasitic mode of life will gain a foothold in 

 the animal body only after the introduction of large numbers. 



In the case of American foulbrood the quantity of infectious mate- 

 rial that honey must carry in order to produce disease in a colony has 

 never been determined. White (SO, p. ^0, footnote 1) states, in con- 

 nection with inoculating healthy colonies experimentally with Bacillus 

 larvae: 



It was found that less than one scale is sufficient disease material to produce a 

 considerable amount of disease in the colony. In some experiments one scale, 

 therefore, might supply all the spores needed although the use of a somewhat 

 greater quantity of material is advisable in most instances. 



While infected honey no doubt does become mixed with disease-free 

 honey, it is probable in many cases that, because of the practice of 

 using large settling and storage tanks, infected honey would be so 

 diluted with spore-free honey as to make the spore content insufficient 

 to produce infection even if fed to healthy bee larvae. Therefore, one 

 object of these investigations was to determine the minimum number 

 of spores of Bacillus larvae in honey necessary to produce American 

 foulbrood in healthy colonies of bees as correlated with the infectivity 

 or spore content of the average commercial honey. 



In order to obtain information relative to this subject, experiments 

 were conducted in the apiary over a period of five years. In these 

 experiments honey or sugar sirup with a known content of spores of 

 Bacillus larvae was fed to healthy colonies and the minimum number 

 of spores that would produce infection was determined. At the same 

 time laboratory studies were carried on with cultures of spores of B. 

 larvae, concerning certain growth phases of the organism, particularly 

 the minimum number of spores that would produce vegetative growth 

 on artificial culture media. Methods for demonstrating the presence 

 or absence of spores of B. larvae in samples of commercial honeys were 

 also investigated, and these honeys were studied in relation to their 

 infectiousness as correlated with the spore-feeding experiments. 

 These three phases of the investigation will be discussed in the order 

 mentioned. 



MINIMUM NUMBER OF SPORES OF BACILLUS LARVAE NECES- 

 SARY TO PRODUCE DISEASE IN HEALTHY COLONIES OF BEES 



methods of proceduee 



Location of Experiments 



These investigations were started during the summer of 1926 in a 

 small experimental apiary located about half a mile from the bee 

 culture laboratory of the Bm-eau of Entomology at Somerset, Md. 

 The location at Somerset was undesirable, however, because of its 



