322 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



clear. The reaction is best observed by holding the tubes (without 

 shaking) against a dark background, when it will be seen that the 

 turbidity first appears as a faint opalescence at the bottom of the 

 tubes, but in the course of five minutes extends throughout the 

 specimen, becoming increasingly denser and ultimately settling to 

 the bottom as a precipitate. 



The desired titer is frequently obtained on the sixth day fol- 

 lowing the last injection. If an examination of a test specimen 

 taken from the ear does not indicate the desired strength at this 

 FTO 2i time, it may be necessary to give a fourth, a fifth, and 

 even a sixth injection, but it may also happen that the 

 particular animal cannot be brought to the titer that is 

 necessary, with any number of injections. If, however, 

 the examination shows that the serum can be used, the 

 animal is bled to death, the serum separated by cen- 

 trifugation, cleared by passing through a Berkefeld 

 filter, and finally stored in little glass beads or am- 

 poules in portions of 1 c.c. each. No preservative is 

 added, and it is accordingly necessary throughout to 

 observe aseptic precautions. 



All examinations are conducted in little test-tubes, such 

 as those used in the Wassermann work, which must, of 

 course, be scrupulously clean. Or one may use little tubes 

 drawn out like the one represented in Fig. 21, which shows 

 its natural size. Or, if very small amounts of material 

 only are available for the examination, this may be 

 conducted in glass capillaries. The turbidity then 

 develops at the zone of contact between the two fluids, 

 and may be advantageously observed with the aid of a 

 magnifying glass. 



PREPARATION OF THE SUSPECTED MATERIAL. This is brought 

 into solution with the aid of 0.85 per cent, saline, and is then further 

 diluted to such a degree that on boiling a small amount (1 c.c.) with 

 a drop of 25 per cent, nitric acid a slight opalescence develops. This 

 would correspond to a 1 to 1000 dilution of the blood in its original 

 state and represents the minimal degree of dilution (i. e. } the maximal 

 concentration) with which the actual test should be made. 



The solution of the suspected material should, of course, also 

 be perfectly clear, to which end it may be necessary to pass it 



