Having proved the stain to be blood, a solution of it should be made in normal saline, sufficiently strong to give the HNO₃ reaction for albumin, or to foam when shaken up. If the amount of solution be small, the tests can be carried out in capillary tubes or pipettes. The solution of the stain must be cleared by filtration or the centrifuge.

The tests must be controlled by comparison with known human blood, and blood from several domestic animals. For this purpose the various antisera should be kept in stock.

Two sets, A and B, of six small tubes are used, into each of A is placed 0.05 c.c. of human antiserum from a sensitised rabbit. Into each tube of Set A is then added double the amount of diluted antigen, as follows:

Set A —1.Receivesextractof knownhumanblood-stain.
2.“““ox““
3.“““horse““
4.“““sheep““
5.“““pig““
6.“““stainunder observation.

Set B is now charged as follows:

Set B —1.Receivesnormalrabbit serum0.05c.c.
2.“humanantiserum0.05“
3.“ox“0.05“
4.“horse“0.05“
5.“sheep“0.05“
6.“pig“0.05“

To each tube in Set B is now added about 0.1 c.c. of the extract of the suspected stain.

It will thus be seen that in Set A known antigen is added to known precipitin, with the exception of tube 6, which receives suspected antigen, while in Set B the suspected antigen is added to known antisera, with the exception of No. 1, which contains normal rabbit serum.

The tubes are observed in an hour and the results noted. Should the stain be human blood, positive reactions will be present in tubes 1 and 6 of stand A, and tube 2 of B.

The negative reactions in the other tubes in stand A prove that the antiserum used is specific, and will only react with its own antigen; in stand B that the stain is composed of specific human antigen, reacting only with its own antiserum.