PREPARATION OF BACTERIAL ANTIGENS II 



referred to, may not be profoundly changed in its structure 

 and methods which involve such possibilities are to be avoided. 



The method of choice for the preparation of a bacterial anti- 

 genie product is one which liberates the characteristic proteins 

 from the cell bodies in maximal amounts and with minimal 

 chemical modification. I have sought to accomplish this by 

 the use of physical means, and to this end have developed a 

 method in which the essential feature is the rapid freezing and 

 thawing of the bacteria in aqueous suspension. By this method 

 large amounts of the native antigenic proteins are obtained in 

 solution and the anticomplementary constituents of the cells 

 are removed. The method is simple, and as I have used it with 

 the colon- typhoid-intermediate group, is as follows: 



The organisms are grown for from forty-eight to seventy-two 

 hours in culture tubes containing the synthetic medium previ- 

 ously described, at the end of which time the entire contents 

 are transferred to silver plated metal tubes. (The tubes are 

 made in the following manner : A hole f inch is drilled 4! inches 

 deep in the center of a i inch diam. solid steel rod. The rod is 

 cut off at a length of 5 inches thus leaving a J inch wall. The 

 ends are rounded off and both the inside and outside are 

 polished. The tubes are then heavily silver plated. In order 

 to keep the tubes from bursting during the freezing process, a 

 tapering silver plated pin is placed in them. This pin is 4 inches 

 long and & inch in diameter at the top, through which 2 

 smaller pins are inserted at right angles in order to hold it in 

 the center of the tube. After the material is added to the 

 tubes, a cork is placed in the end and it is sealed tightly by 

 applying several layers of adhesive tape. A small metal handle 

 is inserted in the cork. The tubes are immersed in a flask of 

 liquid air to within i inch of their tops for from three to four 

 minutes. They are then removed and the contents thawed 

 quickly by dipping the tubes for a few seconds at a time in 

 boiling water. By shaking the tubes vigorously, the material 

 in the tubes, if carefully watched, may be very rapidly thawed 

 without the temperature of the contents rising above 40 C. 

 In this way, a change of temperature of more than 200 C. 

 is obtained in a period of from four to five minutes. 



