276 MANUAL OF MICROBIOLOGICAL METHODS 



amniotic fluids or chorioallantoic or amniotic membranes usually present 

 no major problems if a sufficient concentration of antigen can be obtained. 

 Membranes may be disintegrated to some extent by one of several 

 methods, e.g., grinding with an abrasive or using a mechanical blender 

 or sonic vibrator. After disintegration with or without the addition of 

 an extractive, e.g., saline or phosphate buffer solution, the material is 

 centrifuged at sufficient speed to remove the grosser particulate matter. 

 Antigen from allantoic or amniotic fluids can be concentrated by placing 

 the fluid in a viscose tube and hanging it in the air current from an 

 electric fan. Quite often the fluid should first be dialyzed against 

 20 vol of distilled water at 4°C overnight to prevent large precipitates 

 of salts from forming as the evaporation proceeds. Antigen may also 

 be concentrated sometimes by high-speed centrifugation and resuspension 

 of the sediment in a fraction of the amount of the original suspending 

 fluid. See discussion on concentration. 



Antigen propagated in yolk-sac membrane requires special attention. 

 After the membranes have been disintegrated, extracted, and centrifuged, 

 the fipoidal cake forming at the top of the centrifuge tube is usuaUy 

 discarded. Placing the preparation at 4°C until the lipoidal material 

 has become more solid, circling the cake with a wire, and removing it 

 with a curved spatula may aid in what otherwise can be a troublesome 

 procedure. 



Depending on the size of the organism and the speed at which the 

 preparation has been centrifuged, the desired portion of the extract may 

 be in the supernatant fluid or sediment. In some instances the super- 

 natant fluid may be used as a satisfactory antigen. Further centrifuga- 

 tion at higher speeds will concentrate the antigen if it is of sufficient 

 size, and this procedure may be necessary to obtain one of appropriate 

 strength. If the antigen is of adequate size to appear in the sediment 

 at slower centrifuge speeds, then it may be purified by repeated sediment- 

 ing and resuspending in fresh menstruum. 



Not infrequently it may be necessary to shake the yolk-sac preparations 

 with equal volumes of ethyl ether for the purpose of extracting sufficient 

 Upoid to render the antigen complementary. A separatory funnel aids 

 in separating the aqueous from the ether layer. This same proce- 

 dure may be necessary when dealing with mouse-brain virus antigen. 

 LyophiUzed preparations can be extracted with much more efficiency than 

 wet preparations. It is advisable to determine the effect of the lipid 

 extraction procedure on the stability of the antigen. 



Antibodies. While the complement-fixation procedure is invaluable 

 to the virologist, it must be rigidly controlled to prevent embarrassing 

 errors. It is a not uncommon occurrence to encounter human sera with 

 antibody titer against egg products. Hence, one must always include 



