GLEANINGS IN BEE CULTURE 



July, 1922 



them again or place them where bees have 

 access to them. It was with this point in 

 mind that an experiment was made to de- 

 termine the effectiveness of boiling the 

 frames in a solution of lye. 



An Experiment With a Solution of Lye. 



The Entomology Dex^artment here recom- 

 mended a solution of lye for treating frames 

 of colonies infected with American foul 

 brood. No previous, work had been done 

 to determine whether the solution was ef- 

 fective in killing all the spores of Bacillus 

 larvae or not. Accordingly an e;q)eriment 

 was undertaken to determine the effective- 

 ness of the method. The method recom- 

 mended was boiling the infected frames in 

 a solution of lye (made by adding one can 

 — 12 V^ oz. — to eight gallons of water) for 

 five minutes. The lye used in this experi- 

 ment was the Red Seal brand, but any good, 

 high-test lye may be used. 



The above method was tried out in the 

 following way: Bacillus larvae, the organ- 

 ism which causes American foul brood, was 

 isolated by the method recommended by 

 White (1) by using egg-yolk-suspension 

 agar. The culture was tested for purity 

 by inoculating it into all the laboratory 

 media, by morphological studies and final- 

 ly by feeding it, with proper precautions, 

 to a colony of bees which were free from 

 the disease. The pure cultures were grown 

 for a week at 37 °C and examined for 

 spores, and an aqueous solution of the 

 spores was made. Then 100 c. c. of the lye 

 solution was brought to the boiling point 

 (101.5°C) and 10 c. c. of the aqueous sus- 

 pension of the spores was added. At in- 

 tervals of one, two, three, five and seven 

 minutes 0.5 e. c. portions of the material 

 were removed and inoculated immediately 

 into tubes of broth which had been pre- 

 viously acidified so that the addition of 0.5 

 c. c. of the alkali solution would give a re- 

 action of pH=6.8. To these tubes of broth 

 were added 1.2 c. c. of egg-yolk-suspension. 

 They were then incubated at 37 °C for five 

 days. 



Growth in tubes as determined by micro- 

 scopic examination. 



Time of boiling, 1; growth, none. 

 Time of boiling, 2 ; growth, none. 

 Time of boiling, 3 ; growth, none. 

 Time of boiling, 5; growth, none. 

 Time of boiling, 7; growth, none. 



Discussion. 



Tlie data in this table would indicate that 

 boiliug lye used in the concentration recom- 

 mended by the Entomology Department 

 was effective in killing the spores of the 

 organism in even less time than five min- 

 utes. This is to be expected since Wliite 

 (1) has found the thermal death point of 

 spores from American samples to be 96 °C 

 for 10 minutes and that of the most resist- 

 ant spores, those from Cuban samples, to be 

 100°C for 11 minutes. Here we have a 

 higher boiling point, viz.. 101.5 and in ad- 

 dition the caustic action of the lye. 



There is one factor, however, that should 



be taken into consideration and that is the 

 wax that might possibly be left on the 

 frames. We tried experiments with boiling 

 the frames in the solution of lye but failed 

 to get growth. Several factors might in- 

 fluence the results here, however. The 

 number of organisms that would be left on 

 the frames and get into the lye solution 

 would be so small that, in taking such a 

 small quantity as 0.5 c. c, it would be easy 

 to miss them. However, if the frames are 

 cleaned from wax and dirt before boiling, 

 what little remains will be dissolved by the 

 hot lye solution and the spores killed. We 

 were never able to obtain any spores from 

 the frames after treatment. 



Hydrogen-ion Concentration. 



During the experiment we became inter- 

 ested in the hydrogen-ion concentration of 

 the media and also in the hydrogen-ion con- 

 centration of the larvae. Normal bee lar- 

 vae were obtained just before capping, 

 crushed and the hydrogen-ion concentration 

 determined by the colorometric method of 

 Clark and Lubs (2). The hydrogen-ion con- 

 centration was found to be pH=6.6, using 

 brom thymol blue as indicator. 



We found the optimum hydrogen-ion con- 

 centration to be pH=6.8, for both the bee 

 larvae agar and the egg-yolk-suspension 

 agar. The organism grew well between the 

 ranges of pTI=6.6 and pH^7.0, but best at 

 pH=6.8. 



Conclusions. 



1. The temperature (101. 5°C) of a boil- 

 ing lye solution (12i/^ oz. to eight gallons of 

 water) is sufficient to kill the spores of 

 Bacillus larvae in five minutes. 



2. The hydrogen-ion concentration of un- 

 capped normal bee larvae is pH=6.6. 



3. The optimum hydrogen-ion concentra- 

 tion for the growth of B. larvae in bee lar- 

 vae agar or egg-yolk-suspension agar is 

 pH=6.6 to pH=7.d. 



The authors Avish to thank Professor R. 

 H. Kelty of the Entomology Department for 

 his interest in the work and for feeding the 

 organisms to the nucleus. 



East Lansing, Mich. 



[A boiling lye solution has been used by 

 beekeepers for many years in cleaning prop- 

 olis and wax from frames, separators or 

 other hive parts, the idea having been first 

 suggested by Miss Emma Wilson, sister-in- 

 law of the late Dr. C. C. Miller. In using 

 lye to clean the frames in the treatment of 

 American foul brood, the first consideration 

 in the minds of beekeepers has been that of 

 removing the propolis and wax; but, as the 

 autliors point out in this article, the lye 

 helps to destroy the spores of American foul 

 brood both by raising the boiling point of 

 the solution and by its caustic action. The 

 technical discussion of the optimum hydro- 

 gen-ion concentration is given here for those 

 who may be interested in this phase of the 

 subject, though this will not be of interest 

 to many. In popular language this means 

 that the authors were careful to have the 



