DIPLOCOCCUS PNEUMONIA 363 



f ection confers immunity for only a short period. Two and three attacks 

 of lobar pneumonia in the same individual are not unusual, and it is 

 uncertain whether even a temporary immunity is acquired in such 

 infections. Active immunization of laboratory animals may be carried 

 out by various methods. The method usually followed is to begin by 

 injecting attenuated 1 or dead bacteria or bacterial extracts. Subse- 

 quent injections are then made with gradually increasing doses of living, 

 virulent microorganisms. Great care in increasing the dosage should 

 be exercised since the loss of an animal after two or three weeks' treat- 

 ment by a carelessly high dose of pneumococci is not unusual. Wads- 

 worth centrifugalizes freshly grown pneumococcus cultures and to the 

 pneumococcic sediment adds a definite quantity of concentrated salt 

 solution. At the end of 12 hours, the pneumococci are dead and con- 

 siderable destruction of the cell-bodies has taken place. Dilution with 

 water until the solution equals 0.85 per cent NaCl now prepares the 

 emulsion for inoculation. The sera of animals immunized with pneu- 

 mococci contain active bactericidal substances. 



Specific agglutinins in pneumococcus immune sera were first 

 thoroughly studied by Neufeld 2 and since then have been made the 

 subject of extensive studies by Wadsworth, 3 Hiss, 4 and many others. 

 For the sake of obtaining plentiful growth for agglutination purposes, 

 Hiss has recommended cultivation in 1% glucose broth with the addi- 

 tion of small amounts of sterile calcium carbonate to absorb acid formed 

 from the glucose. Pneumococci do not regularly agglutinate in diluted 

 immune sera and agglutinations are best studied in suspensions of more 

 concentrated immune serum. Agglutination begins at the end of about 

 15 minutes, and can be studied both by formation of clumps and by the 

 sediment. 



An entirely new turn has been given to studies of the pneumococcus 

 group by the observation by Neufeld and Haendel that as regards reac- 

 tions to immune serum several varieties of pneumococci exist. Studies 

 based upon this observation of Neufeld 5 and his associates have more 

 recently been made by Dochez and Gillespie. 6 These workers have 

 studied pneumococci isolated from many different human sources, both 

 by means of protection in mice and by agglutination. They have accord- 

 ingly divided pneumococci into four groups, in which they include, for 



1 Radziewsky, Zeit. f. Hyg., xxxvii, 1901; Neufeld, Zeit. f. Hyg., xi, 1902. 



2 Neufeld, loc. cit. 3 Wadsworth, loc. cit. 4 Hiss, Jour. Exp. Med., vii, 1905. 

 5 Neufeld and Haendel, Arb. a. d. Kais. Gesund., 1910, xxxiv. 293. 



e Dochez and Gillesme, Jour. A. M. A., 1913, Ixi. 727 



