270 MANUAL OF MICROBIOLOGICAL METHODS 



should be checked for sterility and stored at 4°C. Contaminated 

 batches should be discarded. Before use, the 3 per cent suspension is 

 diluted 1 : 12, giving a 0.25 per cent suspension. 

 5. Virus suspension. Sharp end points are most often obtained from 

 virus suspensions that are free from macroscopic particulate matter. 

 Usually a brief period of centrifugation will accomplish this. 



Methods. The method of performing the test will vary with the virus 

 tested and the menstruum suspending the virus. For illustrative pur- 

 poses chorioallantoic fluid containing influenza virus will be discussed. 



Fluids having a low hemagglutinin titer or harvested from older 

 embryos may possess sufficient inhibitor to obscure sharp end points. 

 In such instances it may be necessary to treat the preparation with 

 receptor-destroying enzyme or to adsorb the virus on red cells and 

 elute it therefrom before using it. These two methods are discussed 

 elsewhere. 



A suitable number of Wassermann tubes, e.g., 8-10, are placed in a 

 single row in the serology rack. One-half milliliter of saline is pipetted 

 into each tube. Virus fluids in v/hich a high concentration of virus is 

 suspected should be diluted considerably before they are serially diluted 

 in the tubes in the rack. A preliminary dilution of 1 : 100 is advisable. 

 If such is the case, the pipet used to transfer the potent virus preparation 

 to the diluting fluid should be discarded and a new pipet used for serial 

 dilution in the rack. One-half milliliter of the 1 : 100 virus fluid is added 

 to tube 1 in the rack, and after mixing, 0.5 ml is transferred to tube 2, 

 and so on, as in an ordinary agglutination test. The test fluid is omitted 

 from the last tube so it can serve as a control. 



One-half millihter of 0.25 per cent erythrocytes is then added to each 

 tube, and the rack shaken to ensure mixing of the ingredients. The test 

 should be left undisturbed for 2 hr at either room temperature or 4°C. 

 Some virus suspensions require 4°C to cause hemagglutination. Ade- 

 quate time has elapsed when the cells in the control tube have settled 

 out, usually 1-2 hr. 



The white background for the serology rack allows light to be reflected 

 up through the tubes, and by looking down into them the results may be 

 read. The control tube containing only saline and red cells should have 

 the cells settled out in a small, compact button. In the tubes showing 

 hemagglutination the cells will be spread out in an even film over the 

 whole of the round bottom of the tube. Tubes containing insufficient 

 virus to cause hemagglutination will resemble the control. The so-called 

 "bull's eye" or "doughnut" reaction, a mixture of positive and negative 

 results, will sometimes be found between the last positive and first 

 negative and is to be read as negative. The tube containing the least 



