VARIATIONS IN TYPHOID BACILLI 301 



3. Normal rabbit serum. Serum obtained from normal rabbits and 

 inactivated at 56°C. for half an hour. About 1 cc. of each medium was 

 placed in small test tubes used for Wassermann work under strictly 

 aseptic precautions, and kept in an ice-chest. 



4. The strains used were plated on plain plates three times, and 

 each time one single colony fished to 0.85 per cent sterile salt solution 

 and from this plated on another new plate. 



Technique 



Stock cultures or newly isolated cultures as described above were 

 suspended in 1 per cent pepton water, and transferred to serum broth 

 or broth; thence retransf erred from serum broth to new serum broth 

 or from plain broth to new plain broth, by using a small platinum loop; 

 then incubated at 37°C. Usually after twenty-four hours' growth the 

 culture was plated on plain agar or implanted on slant, using a platinum 

 needle or a very small platinum loop (in the use of the loop we took great 

 precautions against including any serum broth) ; after standing over- 

 night in the incubator at 37°C. the growth was evenly emulsified in 

 0.85 per cent salt solution (the bacterial growth always covered the 

 entire surface of the slant). Special care was taken to have the 

 emulsions of the cultures of as uniform a thickness as possible and for 

 this purpose a tube of typhoid bacterial emulsion was kept for 

 comparison. 



Graded dilutions of the serum were made with 0.85 per cent sodium 

 chloride solution and ranged from 1 : 50 to 1 : 24,300. Half a cubic centi- 

 meter of each dilution was transferred to small agglutination tubes 

 and an equal amount of bacihary emulsion was added to each tube 

 and also to a salt solution control. 



The results were recorded after two hours' incubation at 37°C. and 

 again after standing overnight in the ice-chest. The controls never 

 showed agglutination. By this method cultures which had been grown 

 on serum were allowed to develop for one generation on agar without 

 serum before their agglutinability was tested. Controls were treated 

 in the same way. 



The serum media were occasionally tested for loss of agglutinating 

 power, and were controlled for contamination by plating on plain plates 

 or Endo plates or by inoculation in sugar media, but results were 

 always negative. Such control cultures were made in sugar media 

 (namely, xylose, arabinose, glucose, maltose, mannitol, lactose, sucrose, 



