282 MANUAL OF MICROBIOLOGICAL METHODS 



before and during the evaporation, the process can be halted when the 

 proper end point can be obtained. Carrying the evaporation sUghtly 

 beyond the desired amount allows one to dilute up to the correct volume 

 with distilled water. The bag is hung up slightly below eye level, and 

 the outside cleansed with cotton saturated with alcohol. A large syringe 

 and 20-gauge needle can be used to withdraw the concentrate. The 

 needle is inserted just above the fluid level with the point down in the 

 fluid. A small amount of distilled water may be used to rinse out the 

 remnant in the bottom of the bag. Some preparations, after dialysis, 

 may be concentrated 8-10 times without a precipitate developing. This 

 same method may be employed for concentrating soluble antigen from 

 virus preparations, i.e., the supernatant fluid following high-speed 

 centrifugation. Whether or not sodium chloride is added to physiological 

 concentration depends on the use to which the preparation is to be put. 



Adsorption and elution. Some viruses are known not only to adsorb 

 to red blood cells but also to be able to elute from them. The process 

 may occur too quickly at room or incubator temperatures; hence the 

 adsorption is accomplished at 4°C. This procedure can perhaps best be 

 illustrated by describing it for allantoic fluid containing influenza virus. 



A 3 per cent suspension of chicken red-blood cells and the virus prepara- 

 tion are chilled to 4°C. The two materials may then be mixed in equal 

 parts, or the red cells sedimented by centrifuging and then resuspended 

 in the virus preparation. The latter method introduces only insignificant 

 dilution of the virus preparation. After the red cells and virus have been 

 allowed to react for at least 2 hr at 4°C, the mixture is centrifuged, pref- 

 erably at the same low temperature and employing precooled receptacles. 

 The agglutinated cells are sedimented readily at low speeds, 5 min at 

 2,500 rpm in a bucket centrifuge, and the supernatant fluid is decanted 

 and saved. The sedimented red cells are resuspended in warm (37°C) 

 saline or other menstruums. The amount of concentration desired dic- 

 tates the amount of menstruum to use. Ten times concentration can 

 be realized quite efficiently. The resuspended cells are incubated at 

 37°C for at least 2 hr, and an extra 30 min to 1 hr at this temperature will 

 increase the yield. Occasionally, say once every 30 min throughout the 

 37°C incubation period, the mixture should be agitated gently. 



After the virus has eluted, the red blood cells do not pack so easily 

 by centrifugation. Employing higher speeds on the centrifuge or using 

 a conical-bottom tube will facilitate depositing the red cells securely so 

 the supernatant fluid can be decanted cell-free. Since inactive virus 

 may adsorb but not elute, yet can agglutinate red ceUs, it is important 

 to be aware of how much this might affect the computation of con- 

 centration. Determining the infectivity titer of each fraction aids in 

 determining this error. The adsorption-elution process can concentrate 



