• VIROLOGICAL METHODS 265 



ipated titer is 10~®, one could proceed as follows: Dilute the preparation 

 10"^ 10~S and 10~® using three 100-fold serial decreases and a separate 

 pipet for each dilution. The same pipet and 10-fold serial decreases 

 could be used to prepare 10"^ lO-s, 10-9, lO-^o, and lO-^^ dilutions. A 

 10-fold increase in titer is not uncommon if either of the above suggestions 

 are not followed. Whatever method is used, it is advisable to use it 

 consistently, especially if one wishes comparable results. 



At least 5 and preferably 10 embryos should be inoculated with an 

 equal volume from a single dilution. If the worker starts with the least 

 concentrated sample and progresses toward the most concentrated, 

 a single syringe may be used throughout. In certain instances it may be 

 desirable to use a separate syringe for each dilution in order to avoid 

 contamination. Any investigator does well to analyze his technic in 

 handling syringes, diluting virus, etc., so as to minimize the probability of 

 his introducing viral or bacterial contamination into his experiment. 



Embryos receiving the most dilute preparation are best harvested 

 first, progressing to those receiving the least dilute. If one is interested 

 in minimal infective amounts, material from embryos receiving the same 

 dilution may be pooled. If a 50 per cent egg infective dose (E.I.D. 50) 

 is desired, each embryo harvest is tested separately and the 50 per cent 

 end point calculated mathematically. Methods for calculating 50 per 

 cent end points are discussed below. It should be remembered, however, 

 that regardless of the method used, there is no substitute for employing 

 significant numbers in any experiment. Nor does mathematical pre- 

 ciseness overcome an inherent wide biological variation. 



Obviously the infectivity end points will be determined by several 

 methods because viruses vary in what they. can do. Some will produce 

 lesions, others kill embryos, still others agglutinate red cells, and probably 

 all produce a certain amount of detectable antigen. Any of these 

 methods can be used to determine the results of extinction dilution. 

 In some instances a combination may be employed, such as dilution 

 factor and lesion count. 



In any experiment it is well not to lose sight of other properties of the 

 virus when testing its infectivity. Under standard conditions the infec- 

 tivity might be directly proportional to other functions, yet these propor- 

 tions are easily upset, and too much should not be left to presumption. 

 For example, under standard conditions a ratio of 6,400 : 10"^ may exist 

 between hemagglutinin and infectivity. Yet because the stability of 

 hemagglutinin and infectivity may differ considerably, the ratio may be 

 changed to 6,400 : 10"^ if the specimen is allowed to stand at the proper 

 temperature. It follows that ratios of other functions should be watched 

 with equal care. 



Eggs can be deembryonated and used for propagating viruses. For 

 thjs technic the reader is advised to consult Bernkopf (1950). 



