306 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



The desired titer is frequently obtained already on the sixth day 

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

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

 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 injec- 

 tions. If, however, the examination shows that the serum can be 

 used, the animal is bled to death, the serum separated by centrifu- 

 gation, cleared by passing through a Berkefeld filter, and finally 

 stored in little glass beads or ampoules 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, if very small amounts of material only are available 

 for the examination, this is 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 

 through a Berkefeld filter, or, if the quantity be small, through a 

 Silberschmidt microfilter. 



THE EXAMINATION PROPER. Six tubes are placed in a suitable 

 rack and labelled I, II, III, IV, V, and VI. Tube I and II receive 

 1 c.c. of the solution under investigation, III and IV 1 c.c. of two 

 control solutions made up from dried animal blood, i. e., from blood 

 which does not correspond to the antiserum that is used, e. g., cat or 

 dog blood, if the antiserum is antihuman in character, and which has 

 likewise been diluted so as to correspond to a 1 to 1000 solution (see 

 preceding section), V 1 c.c. of sterile 0.85 per cent, saline, and VI 

 1 c.c. of a 1 to 1000 solution of blood (made up of dried material) 



