The Sterilization of American Foulbrood Combs 7 



they will do so within that period. When a few spores were found 

 to have germinated, but without evidence of any vegetative growth, 

 a subculture was generally made, using a generous quantity of mate- 

 rial from the surface of the original slant. When good vegetative 

 growth was observed it was so recorded as positive growth. In 

 some instances when there was doubt regarding a culture several 

 more cells of the same kind were cultured from the same piece of 

 comb. 



SOLUTIONS TESTED 



In the earlier part of this investigation an attempt was made to 

 find a substitute for alcohol as a carrier for the disinfectant, because 

 of the difficulty of purchasing pure grain alcohol and the compara- 

 tively high cost of the commercial alcohol-formalin solution. Since 

 certain substances are used in various insecticide sprays to increase 

 the wetting and spreading powers of the spray solutions (6, 19), it 

 was suggested that if a liquid could be found which would spread 

 easily over the surface of the wax comb and diseased remains, forma- 

 lin added to such a liquid would be carried by it and brought into 

 contact with those remains; and that it would penetrate sufficiently 

 to kill the spores, even though the cells, particularly sealed cells, 

 might not be completely filled with the liquid. This property in 

 insect sprays is often obtained by the addition of soaps of various 

 kinds to the solutions. The addition of soap also tends to lower the 

 surface tension of a water solution. Dilute solutions of various types 

 of soaps, both soft and hard, were used to form the carrier for the 

 20 per cent of formalin. By experimentation with dry pieces of 

 comb it was found that only a comparatively small quantity of soap 

 can be used in a solution, as too much causes the liquid to become 

 viscous and jelly-like, inhibiting its entrance into the cells. In the 

 tests with soap solutions varying quantities of half-normal sodium 

 oleate soap, cottonseed-oil soap, and a 10 per cent solution of one of 

 the common hard toilet soaps were used in making up the solutions 

 for the disinfection of diseased combs. From 0.5 to 2 cubic centi- 

 meters of each of the various soaps was used per 100 cubic centi- 

 meters of a 20 per cent solution of formalin in water; if much more 

 than 2 cubic centimeters was used the solution became too viscous. 



In a recent article King (16) describes experiments in sterilizing 

 American foulbrood combs by using a disinfectant solution consist- 

 ing of the 20 per cent water-formalin solution, with sufficient soap 

 to form suds (about 1 pound to 5 gallons of solution), the addition 

 of the soap causing the solution to enter the cells more readily. 

 After 48 hours' immersion, followed by extraction and a short drying 

 period, the combs were given to a healthy colony. Only one colony 

 was used in the experiment, but King reports that no disease ap- 

 peared in it when observed carefully at intervals for seven weeks 

 after it had been given the treated combs. 



Among substances similar in physical properties to ethyl alcohol, 

 a commercial methyl-ethyl ketone solution, acetone, and iso-propyl 

 alcohol were tried as carriers for formalin, but acetone and iso-propyl 

 alcohol were too expensive for practical use. 



The use of hydrochloric acid in the sterilization of imported 

 hides, as a preventive of anthrax (£0), a disease caused by a spore- 



