AMEKICAN FOULBROOD. 19 



needed. About 1 c.c. of the brood nitrate is added to each 5 c.c. 

 of agar liquefied and cooled to about 50° C. After being inclined the 

 medium is ready for use and may be stored. Plating for pure cul- 

 tures may be done by adding the brood filtrate to the agar at the 

 time the plates are made. 1 



When it is desired to obtain cultures from the spore form of 

 Bacillus larvae, brood-filtrate agar is not a suitable medium in itself. 

 The spores should be germinated first by inoculating bee-larvae agar 

 (p. 18) or egg-yolk-suspension agar. By plating in 2 or 3 days the 

 vegetative form thus obtained, using brood-filtrate agar, pure cultures 

 can be assured. Studies have shown, however, that Bacillus larvae 

 is present in practically pure cultures in brood dead of the disease, 

 and by heating the spore-containing material in aqueous suspension 

 at 100° C. for one or two minutes as suggested above the occasional 

 contaminating organisms are eliminated usually without plating. 



Bee-larvaa agar is limited in its usefulness on account of the large 

 amount of brood required in its preparation. A more suitable me- 

 dium, therefore, was sought. An unheated egg-yolk agar was found 

 by the writer (28) to be very satisfactory. This is prepared as 

 follows : Fresh eggs are used. After having been immersed in a dis- 

 infecting solution the shell is broken, the white of the egg is poured 

 off, and the yolk is dropped into a flask containing about 70 c.c. of 

 sterile water. With a sterile pipette the aqueous suspension of yolk, 

 resulting from agitating the flask, is transferred to sterile tubes and 

 stored until needed. In making an egg-yolk-suspension agar about 

 1 c.c. of the egg-yolk suspension is added to each 5 c.c. of agar in 

 tubes liquefied and cooled to about 50° C. The agar is inclined and 

 may be stored until needed. 2 



1 Larvae, prepupse, or young pupae may be used in obtaining the brood filtrate. The use of sterile 

 water is a good precaution in making the dilution of the crushed brood mass. A dilution of 1 to 10 up to 

 1 to 50 gives very satisfactory results, although lower and higher dilutions have been used with success, 

 while at incubator temperature an autodigestion apparenty takes place. The "autodigested" suspension 

 is passed through filter paper before the bacteria-proof filter is used. For the latter the Pasteur-Chamber- 

 land F is very satisfactory. (Fig. 7.) The B grade has been used but the filtering is slower. The 

 Berkefeld N has also been employed, but is less efficient. In using this latter type of filter gravity alone 

 should be employed. If the weather is warm, or if the filtering is not to be done for a few days, the aqueous 

 suspension may be allowed to remain at room temperature. This permits the changes which take place 

 to advance sufficiently to make the filtering process comparatively easy. The chloroform saturated sus- 

 pension of brood material has been kept at room temperature for more than three years without its useful- 

 ness being impaired to any appreciable extent. After a considerable period the suspension becomes 

 practically sterile and has been used without being filtered. When an early use of the filtrate is desiredits 

 sterility should be tested by placing the tubes containing it at incubator temperature for a few days. 

 Sometimes it is desirable to use other brood-filtrate media than the agar. In this event a small amount of 

 brood-filtrate may be added to any one of the media ordinarily used in the laboratory. Such special 

 media usually support a growth of Bacillus larvae when inoculated with the vegetative form of the 

 organism. 



2 Eggs obtained from the market labeled "strictly fresh" have been suitable as a rule for the egg-yolk 

 suspension. Eg^s known to have been recently produced are to be preferred, however. Almost any of 

 the more common eficlent disinfecting solutions may be emplo5 7 ed for sterilizing the eggshell. Aqueous 

 solution of mercuric chlorid 1:1000; carbolic acid 5 per cent; formalin 10 per cent; and alcohol each have 

 been used, mercuric chlorid being preferred. To insure sterility autoclaving of the flasks with the con- 

 tained water has been practiced. A wide-mouthed flask is preferred. In securing the yolk aseptically, it 



