18 



BULLETIN 800, U. S. DEPARTMENT OF AGRICULTURE. 



ropy f oulbrood. In this bee-larvas agar 1 the growth is always slow 

 and somewhat feeble. Spores are produced to a slight extent only 

 or not at all. Such cultures are unsuitable for 

 experimental purposes. 



A medium containing a sterile nitrate ob- 

 tained from the brood of bees was devised by 

 the writer (23) which meets the requirements 

 of experimental studies. It is prepared as 

 follows: Healthy larvae taken from the brood 

 comb are crushed and the crushed mass is 

 diluted with water to several times its volume. 

 This is placed in a flask, and after adding 

 a few c.c. of chloroform the flask is stoppered 

 and allowed to remain at incubator temper- 

 ature overnight. The suspension then can be 



m 



Fig. 7. — A convenient method for obtaining sterile filtrates. 



filtered easily by using any bacteria-proof filter 2 (fig. 7). 



The 



filtrate is pipetted aseptically into sterile tubes and stored until 



i Larvae, prepupae, or pupse recently transformed furnish satisfactory brood material for making the 

 bee-larvae media. These are picked from the brood combs, crushed, and used as meat in formulas ordinarily 

 followed in making bouillon and agar. Excess heating should be avoided. In making the inoculations an 

 aqueous suspension of the spore-containing material heated to 100° C for one or two minutes should be 

 used. A tube of the special agar liquefied is inoculated with a loopful of the heated suspension. After 

 cooling it is incubated at about 38° C Three days or more may be required to produce a visible growth 

 (PI. VIII, E, F, G, H,I). 



2 In caring for and using the filter cylinders in these studies the following course hasbeenpursued: The 

 cylinderis immersed in water for a few hours or over night and is then sterilized in the autoclave. After 

 beingused for filtering a brood suspension it is washedin water without scrubbing. To remove furtherthe 

 brood material water is drawn through the cylinder underpressure as in filtering. Asaruleitisagainim- 

 mersedin water and allowed to remain over night, after which, the filter being assembled, water is filtered 

 again. It is then ready for use and may be stored until needed. Cylinders which have been used repeat- 

 edly in this way have lost none of their efficiency. The vacuum chamber is made in two sections similar 

 to a Novy jar. Vaseline is used on the wide ground flanges. No rubber band or clamps are needed. 



The flasks usedin the vacuum chamber of the apparatus before being sterilized are stoppered withcotton 

 wrapped about a small-sized test tube. The cylinder having been immersed in water is sterilized together 

 with the rubber connections and glass tubing in place, but without the glass mantle. In assembling the 

 apparatussterilized connection between the cylinder and flask in the vacuum chamber is readily ob- 

 tained through the opening in the plug left by removing the test tube. After a vacuum has been estab- 

 lished in the chamber the stopcock may be closed and the pump turned off. 



