OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF GLANDERS. 5 



PREPARATION OF THE MALLEIN. 



The concentrated mallein which has been used for our work and 

 which gave such satisfactory results was prepared at the request of 

 the authors by and in cooperation with Mr. A. M. West, of the 

 Biochemic Division, as follows: 



The media consists of bouillon containing 5 per cent glycerin, 1 per cent peptone, 

 and 5 per cent NaCl. The reaction is that of the natural acidity of the meat, no acid 

 or alkali being added. The flasks of media are inoculated with virulent cultures of 

 Bacillus mallei and placed in the incubator at 37.5° C. for a period of two months or 

 more. The stock cultures of B. mallei are kept on agar, and their virulence is re- 

 newed when necessary by passage through a series of guinea pigs. 



The well-grown cultures show a heavy mass of organisms, which generally sinks to 

 the bottom of the flask. This growth is of a whitish color splotched with brown. The 

 cultures are then removed from the incubator and heated for one hour in the Arnold 

 sterilizer. Then they are stored for two weeks in a dark closet to settle. The clear 

 liquid is then carefully decanted and the growth proper is discarded. 



A measured amount of the decanted Liquid is concentrated over a steam bath to 

 one-third its volume. It is then filled into flasks and sterilized and again filtered 

 while hot, first through one then through three paper filters. Next the clear liquid 

 is passed through a Berkefeld filter. This is followed by a concentration to one-tenth 

 its original volume and by sterilization. 



To the raw mallein, concentrated to one-tenth its original volume, is added 0.5 per 

 cent carbolic acid and 20 per cent glycerin. Then the liquid is again concentrated 

 to one-tenth its original volume, filtered while hot through filter paper, and sterilized. 

 It is kept in a dark place for a week, and if upon inspection a precipitate is found the 

 mallein is again passed through paper filters and sterilized. The finished product is 

 a clear, sirupy, dark-brown liquid," with a disagreeable odor. The mallein is then 

 bottled, under aseptic conditions, in small vials and is ready for use. 



It is advisable to provide the mallein for the tests in small vials, 

 each containing about 1.5 c. c. of mallein, which is sufficient for testing 

 15 horses. After the vial has been opened and part of the contents 

 used for testing, especially if the mallein has been taken out with a 

 camel's-hair brush, the remainder should not be used for tests applied 

 on subsequent days, but should be discarded. 



THE USE OF DRY MALLEIN. 



Another form of mallein which has been used quite extensively for 

 the eye test is the mallein siccum, or dry mallein. This represents an 

 alcoholic precipitate of mallein. It is a fine gray powder and must 

 be dissolved in water before it is used. The solution loses its effective- 

 ness in a very short time and must be prepared fresh on the day of 

 the test. Dr. K. F. Meyer, formerly of the University of Pennsyl- 

 vania and now of the University of California, has used the dry 

 mallein extensively, and at the present time this preparation is em- 

 ployed in Pennsylvania for the application of the ophthalmic test. 

 For this purpose two vials are sent out from the laboratories of the 

 Pennsylvania Livestock Sanitary Board, one containing the pow- 



