254 INFECTION AND IMMUNITY 



bered. A single injection of a large quantity has occasionally yielded 

 a precipitating serum of considerable strength, 1 but this method is 

 not usually successful. Injections are made at intervals of from five to 

 seven days. Seven to twelve days after the last injection the animals 

 may be bled from the ear, and a preliminary test made to ascertain the 

 precipitating value of the serum. If this is insufficient, more injections 

 may be made. Bleeding should be done 7 to 12 days after the last 

 injection. Such sera may be preserved in the dark and at a low tem- 

 perature. If a preservative is added, Nuttall prefers chloroform to 

 the phenols, because of occasional turbidity produced by these. 



Precipitating antisera for tests should be clear. If turbid, the sera 

 should be filtered through small Berkefeld or porcelain candles. 



PREPARATION OF BACTERIAL FILTRATES AND PROTEIN SOLUTIONS 

 FOR PRECIPITIN TESTS. Bacteria may be grown in nutrient broth 

 having an initial reaction of neutrality or five-tenths per cent acidity 

 to phenolophthalein. The cultures are incubated for times varying from 

 a week to several months, and are then filtered through porcelain or 

 Berkefeld candles until perfectly clear. Bacterial extracts may also 

 be made by eniulsifying agar cultures in salt solution, placing at 37.5 C. 

 in the incubator for a week or longer, and filtering. More rapid ex- 

 traction of bacteria may be accomplished by repeated, rapid freezing 

 and thawing -of salt-solution emulsions, by shaking in the shaking ma- 

 chine or by centrifugalizing, rubbing up the sediment with dry salt, 

 and the addition of distilled water to isotonicity. 



Protein solutions to be tested should be made in salt solution. When 

 dealing with blood stains, as in doing the test for forensic purposes, 

 the stains should be dissolved in salt solution, an approximate dilution 

 of one in five hundred being aimed at. This solution if turbid should 

 be filtered through a small porcelain filter. It should be clear and color- 

 less, show a faint cloud on boiling with dilute acetic acid, and show 

 distinct froth when shaken. 



When the reaction is to be done for determining the nature of meat 

 (detection of horse-meat substitution for beef, etc.), about 20 to 40 

 grams of the suspected meat are macerated in a flask, and covered with 

 100 c.c. of salt solution. This mixture is allowed to infuse at room 

 temperature for three to four hours, and is then placed in the refrigerator 

 for twelve hours or more. At the end of this time 2 c.c. are shaken 

 into a test tube. If profuse frothing 2 appears, the extract is ready for 



1 Michaelis, Deut. med. Woch,, 1902. 



2 P. Th. Mutter, "Technik. d. serodiagnos. Methoden." 



