20 BULLETIN S09, U. S. DEPARTMENT OF AGRICULTURE. 



EXPERIMENTAL INOCULATIONS 



A very satisfactory colony for inoculation purposes is a queen- 

 right nucleus that can be accommodated comfortably on from 3 to 5 

 brood frames approximating the Langstroth size. The hive, the 

 arrangement of the apiary, the colony, the feeding, and the manipu- 

 lations in general are similar in the experimental study of American 

 foulbrood to those employed by the writer in the study of sacbrood 

 (25), Nosema-disease (26), and European foulbrood (27). The inocu- 

 lated colonies were, therefore, in the open. Precautions tending to 

 minimize the likelihood of robbing, swarming, absconding, and the 

 accidental straying or drifting of bees should not be overlooked. 



The inoculations are made by feeding a suspension of the spores of 

 Bacillus larvae in sirup or honey, the spores being obtained from pure 

 cultures or from brood dead of American foulbrood. Three or four 

 tubes of spore-containing cultures grown on brood-filtrate or egg- 

 yolk-suspension agar furnish a suitable quantity of the virus. Like- 

 wise the spores contained in three or four scales give satisfactory 

 results. In some experiments, feedings made on successive days are 

 desirable. Direct inoculation by means of a capillary pipette is 

 less satisfactory in experiments on American foulbrood than in those 

 on sacbrood.^ 



The first symptoms of American foulbrood to be observed following 

 the inoculation usually appear in the worker larvae by the end of the 

 first week and not earlier than the sixth day. The affected larvae 

 are in capped cells. Not infrequently the first evidence of disease 

 noticed is the remains in cells here and there of partially removed 



is convenient to break the sterilized shell about one end of the egg, remove the pieces with flamed forceps, 

 and, after making a small hole in the other end, pour off the white. The yolk remaining is poured into the 

 flask. By breaking the limiting membrane of the yolk at the time of pouring into the flask the process is 

 facilitated. Different degrees of dilution of the egg yolk have been employed, 60 c.c. to SO c.c. of water giv- 

 ing satisfactory results. Any ingredient, such as the sugars, desired in the final medium may be added to 

 the water of the flasks. The egg of the hen has been used in most of the studies reported here. Eggs of 

 the duck were also found to be suitable. Occasionally a contamination of the egg suspension will be found. 

 In these cases the suspension becomes Arm after incubation resembling then the consistency of coagulated 

 milk cultures. If the contamination is present it ■will be apparent in a few days after storing, or it may be 

 determined earlier at incubator temperature. The sterile egg-yolk suspension as an ingredient for 

 special media retains its efficiency for a long period. Indeed an aqueous suspension of the dry residue from 

 the yolk supension was found to be efficient after 3 years, although its value is then apparently somewhat 

 impaired. 



Maassen (15) used bee-larvae and also brain agar in studying Bacillus larvae. The egg-yolk suspension 

 agar, in the writer's experience, has advantages over the brain agar. 



1 Cultures on the surface of agar are readily suspended in water as is also the disease material in the decay- 

 ing larvEe and scales of American foulbrood. These aqueous suspensions are used in making the sirup or 

 honey suspensions. From 1 to 3 c.c. of water for each scale is a convenient proportion. Sirup is made by 

 bringing to the boiling point an aqueous suspension of granulated sugar in which the sugar used exceeds 

 that of the water. When honey is used for the spore suspension it is diluted with water, the amount of 

 water added being slightly less than that of honey. In most instances honey is less suitable than sirup 

 inasmuch as it tends to encourage robbing especially during a dearth of nectar, it needs to be sterilized before 

 use, and is more expensive. 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 

 of the spores needed although the use of a somewhat greater quantity of material is advisable in most 

 instances. 



