5 219 



The bacteria used in the preparation of the emulsions or suspensions were 

 grown at 37° on the surface of ordinary meat-peptone-agar in large Roux's bottles; 

 the growth was carefully scraped off in as dry a state as possible, and emulsified 

 in 0-85 "/o NaCl solution, which will be subsequently referred to as normal saline, 

 very thoroughly. The agar-culture suspension thus obtained was standardised to a 

 definite degree of opalescence or opacity (i. e. Vi normal emulsion), by diluting it with 

 normal saline until it presented the same degree of opacity as a certain stand- 

 ard emulsion of arbitrarily chosen strength, to which formaldehyde had been 

 added as a preservative: the comparison and standardisation were effected by the 

 observation of lines ruled on paper through two small test-tubes, filled, one with 

 the standard, and the other with the new emulsion, and held side by side. The 

 employment of some such standard emulsion as the starting point for all subsequent 

 experiments is of the greatest importance, and is really an absolute necessity for 

 exact work. 



It is well known that the agglutinating power of an immune-serum may vary 

 more than 100<'/o, according as the measurements are made with a denser or 

 a thinner bacterial emulsion (Sacquépée), and other allied phenomena, such as the 

 combining proportions, the "zones of inhibited agglutination", and so on — exhibit 

 similar wide variations. Eisenberg & Volk have endeavoured to avoid this source 

 of error by making up the bacteria grown in a given time on a definite area of 

 agar-surface to a definite volume of bacterial emulsion; but the nature of bacterial 

 growth renders such a method of procedure obviously inadequate, and our own 

 experiments have shown us that emulsions of bacteria made up in this manner 

 may vary from one another more than 100% in bacterial concentration. A fresh 

 emulsion in normal saline of a 24-hour agar-culture of our B. coli was always acid 

 in reaction; titrated against iVo OH solution, and using phenol phthalein as indicator, 

 its acidity was equivalent to that of an equal volume of Vio,ooo to '/2o,ooo normal 

 HCl solution. 



The agglutinating immune-sera, derived from goats or rabbits, were kept con- 

 stantly on ice and in darkness; in some cases with the addition of small amounts 

 of chloroform as an antiseptic, in other cases without it. We would here emphas- 

 ise the fact that we have never been able to observe any irregularities in our 

 experiments that could be attributed to the addition of these small amounts of 

 chloroform to the sera. 



The general technique of the experiments was as follows. Small test-tubes, 

 measuring 11 cm. x 7 cm. were set up in series, and into each tube were measured 

 15 c. cm. agar-culture suspension*) or bouillon-culture, together with such an amount 



■) Wherever an agar-culture suspension of any given age is mentioned in tliese pages, a standardised 

 suspension in sterile 085"'/o NaCl solution of bacteria grown on agar at 37° for the given time is meant. 

 When the age is not mentioned, it is always to be understood that the culture is of 24 hours growth, 

 whether on agar or in bouillon. 



