62 STUDIES IN IMMUNITY. 



sive than Pfeiffer's method of diagnosis. Certain general rules of 

 technique should be followed to insure uniformity : 



A 24-hour culture of the vibrio to be examined is suspended in 

 from 5 to 7 c.c. of bouillon or normal salt solution. Two drops of 

 this suspension are then dropped from a fine capillary tube on a 

 slide or in a watch glass; a drop of preventive cholera serum is then 

 added (a serum supposedly as strong as our own) ; as a matter of 

 fact, this dose is considerably more than is necessary, but it is well 

 to use as much as this on the chance of dealing with a very resistant 

 vibrio. A small platinum loop of this mixture is then taken and 

 placed on a cover slip, and a small drop of fresh normal serum is 

 added by means of the same loop after sterilizing it. Defibrinated 

 blood, of course, may be used in place of serum or simply a freshly 

 drawn drop of human blood. The drop of normal serum is mixed 

 with the other drop and the cover slip applied to a hollow ground 

 slide and sealed with vaseline. The slide is then placed for two 

 hours in the incubator, which time is quite sufficient to obtain a 

 complete transformation. We emphasize these details because the 

 three factors vibrio, normal serum and preventive serum should 

 be used in rather exact proportions. It is well to make two control 

 preparations at the same time and by the same method. One of 

 these should contain a typical Koch vibrio that is known to give the 

 phenomenon rapidly with active serum. From this control we 

 learn whether enough of each serum has been used. A second con- 

 trol is made with the vibrio to be examined, normal serum, and 

 a drop of salt solution in the place of the preventive serum. This 

 second control will indicate whether the organism in question is 

 very attenuated and so liable to transformation by the normal 

 serum alone without any cholera serum. If the second control also 

 gives Pfeiffer's phenomenon, which is unlikely, no definite conclu- 

 sion may be drawn as to the pathogenic nature of the organism 

 examined. 



Since the preparations remain only two hours at body tempera- 

 ture, perfect asepsis to prevent growth of other contaminating 

 bacteria is unnecessary. Examination may be made either in the 

 fresh condition or by means of stained preparations. There is fre- 

 quently some difficulty in staining the granules, particularly when 

 the preparation has been left too long in the incubator and the 



