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An Inquiry into the Influence of the Constituents of a Bacterial 



Emulsion on the Opsonic Index. 

 By A. F. Hayden, M.B., B.S., F.B.C.S., I.M.S., and W. Parry Morgan, 

 M.A., B.Sc, M.B. 



(Communicated by Sir A. E. Wright, F.E.S. Eeceived August 24, — 

 Eead November 2, 1911.) 



(From the Inoculation Department, St. Mary's Hospital.) 



In preparing an emulsion of bacteria for opsonic estimations it is necessary 

 to break up the masses so far as possible into their constituent bacterial 

 elements and then to separate these from any clumps by centrifugalisation. 

 The rate at which the suspended particles of an emulsion settle depends not 

 only on the centrifugal force applied, but also on the fineness of the particles, 

 and therefore on the efficiency of the method of breaking up the masses. 

 If this is not efficient the suspended matter will fall in the form of coarse 

 particles, leaving a relatively clear supernatant fluid containing very little in 

 suspension. 



In the case of a tubercle emulsion we find that the best results are given 

 by triturating a small quantity of dried bacilli with a pestle and mortar of 

 which the grinding surfaces have the same curvature ; using these, 

 five minutes' grinding is ample. The mass of dried bacilli is first ground up 

 in the dry state and then made into a paste with a little 1-per-cent. saline 

 (the strength used in all our experiments). The crude emulsion is then 

 made by taking up the paste with 1 to 1| c.c. of saline. When this 

 emulsion is thoroughly centrifugalised it separates out into a deposit and an 

 opaque supernatant fluid which is practically free from bacilli but which 

 contains a considerable amount of bacterial detritus. If this supernatant 

 fluid be pipetted off and the deposit again mixed with fresh saline and 

 thoroughly centrifugalised, the second supernatant fluid will contain much 

 less detritus and will be correspondingly clearer. By repeating this process 

 several times it is possible to get a supernatant fluid which is almost clear 

 and free from detritus. The deposit will then consist wholly of washed 

 bacteria. 



It is thus seen that the usual bacterial emulsions which are employed for 

 measuring opsonic power consist of three elements, the saline used as a 

 menstruum, bacillary detritus, and intact bacilli. 



We have set ourselves the problem of determining the effect of the 

 bacillary detritus on phagocytosis and its influence in the estimation of the 



