no THE PRINCIPLES OF IMMUNOLOGY 



iso-precipitins, and, as we have seen, their existence is irregular 

 and questionable. 



Practical Application. Wladimiroff first applied precipitation 

 practically in the diagnosis of glanders in horses, using the serum of 

 suspected horses against a filtrate from cultures of glanders bacilli. 

 Kraus employed the reaction to identify closely-related bacteria. At 

 the present time, however, agglutination is employed for the detection 

 of glanders and also for identification of bacteria rather than precipita- 

 tion, because the latter procedure introduces the more cumbersome 

 technic of obtaining filtrates. 



The Forensic Blood Test. Uhlenhuth and Beumer published their first re- 

 sults on the use of the precipitin reaction in legal medicine in 1903. Other 

 studies were rapidly contributed, and to-day the method has an established place 

 in the identification of stains by blood and other fluids such as seminal fluid. 

 If spots on clothing or other material are suspected of being blood, this must 

 be proven by chemical, microscopic or spectroscopic examination. Subsequently 

 the precipitin test is used to determine the species from which the blood orig- 

 inated. Before proceeding to this test it is necessary to have immune pre- 

 cipitating serum against the suspected species, usually man, an additional 

 immune precipitating serum against some other species and a normal rabbit serum. 

 The immune sera are prepared according to the method outlined on page 108. 

 The suspected material must be carefully guarded against possible substitution 

 or contamination until the immune sera are prepared. It is then dissolved in 

 physiological salt solution and a perfectly clear filtrate used. If the material is 

 on cloth the latter should be teased so as to permit of solution ; if on some solid 

 material, such as a knife blade, it should be scraped off, ground in a mortar and 

 a small amount of salt solution added. Cloth should be placed in a test-tube or 

 bottle, and it is well to have a control with unstained cloth. The time for 

 extraction depends to a certain extent on the freshness of the material, but it 

 is wise to allow it to extract in the refrigerator over night, adding a few drops 

 of chloroform to prevent bacterial growth. If extraction does not proceed well 

 in salt solution it may be necessary to extract with I per cent, potassium cyanide 

 solution, correcting the alkalinity after extraction, by means of tartaric acid. 

 To prove that the solution contains protein a small amount may be boiled and 

 treated with acetic or nitric acid as in the ordinary test for albuminuria. A 

 final solution of the suspected material in a dilution of i-iooo is usually em- 

 ployed, and this dilution may be approximately determined by the foam test. 

 For this purpose make a i-iooo solution of any convenient serum, blow air 

 through it in a test-tube and note the persistence of bubbles above the fluid. 

 Dilute the extract gradually and blow air through it, repeating until that dilution 

 is obtained which will produce a foam of about the same viscosity as that in 

 the control tube. If the solution is not perfectly clear it may be centrifuged or 

 passed through a filter of washed asbestos or cotton. On the assumption that 

 the spot is suspected of being human blood the test is set up as follows : 



Material Result 



Suspected extract -f- anti-human serum + 



Suspected extract 4- normal rabbit serum 

 Control extract T anti-human serum 



NaCl solution -(- anti-human serum 



Human serum (i-iooo)-f- anti-human serum -f 



Beef serum (i-iooo) + anti-human serum 

 Sheep serum (i-iooo) -(-anti-human serum 



The amounts throughout are o.i c.c. of each reagent, and the test is made 

 by the Fornet ring method. Inasmuch as acceptable antisera should titrate 

 1-10,000, the reaction in i-iooo dilution occurs within a few minutes, and the 

 above result would be interpreted as a clear-cut positive for human blood pro- 

 vided the preceding chemical and other tests for blood had been positive. It 

 should be remembered that the precipitin reaction in this instance simply iden- 

 tifies the material as human protein, and a similar result might be obtained 

 from human seminal fluid, albuminous urine, purulent sputum, exudates and 

 transudates, unless the preliminary tests had been carried out. 



