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MANUAL OF MICROBIOLOGICAL METHODS 



Determination of the 50 per cent infectious dose for embryos {E.I.D, 

 50). It is generally agreed that the 50 per cent infective dose is less 

 subject to extremes than the minimal infective dose. Reed and Muench 

 (1938) have worked out a method of obtaining such an end point which 

 is in wide use. The method is illustrated in Table 27 on an actual 

 protocol wherein 10 embryos were inoculated in each dilution group. 



Table 27 



The 50 per cent end point lies between 10~^ and 10~^. The proposed 

 formula for determining the proportionate distance is: Number above 

 50 per cent or 72.7 — 50 = 22.7, divided by the number above 50 per cent 

 or 72.7 — the number below 50 per cent or 47.6 = 25.1. The fraction 

 22.7/25.1 = 0.904, hence the 50 per cent end point is lO-^-^o^ dilution- 

 wise, or the specimen contains 10^^°" E.I.D. 50. The cumulative totals 

 are obtained on the presumption that eggs infected with 10~^^ dilution 

 of virus would also have been infected with 10~^°, 10~^, and so on. Unin- 

 fected eggs are accumulated in reverse order. 



Several comments are in order concerning this method. It is con- 

 venient and therefore has become widely used. One obtains a false sense 

 of preciseness with a figure carried out to the third decimal. Although 

 from the cumulative totals it appears that 189 eggs were used in this 

 test, actually only 70 were used. One could question if IQ-^-^^^ is a 

 more accurate end point than 10~^ wherein actually 50 per cent of the 

 embryos were infected. Even using 10 eggs per dilution group it is 

 difficult to reproduce results consistently within 0.5 log units. This in 

 itself is not a criticism of the calculation procedure but a commentary 

 to remind one that mathematical preciseness does not alter an unprecise 

 technic. 



Needless to say the same method may be used for estimating a 50 per 

 cent infectious dose in animals and in determining neutralization titers 

 of antisera wherein log dilutions of the serum have been mixed with a 



