H. B. Hutchinson and J. Clayton 149 



Subsequent work with cellulose agar plates resulted in the production 

 of colonies which ajjreed niacroscopicall}' with those previously obtained, 

 but microscopically appeared to consist solely of the "thread" form. 

 Inoculation of cellulose agar slant tubes and incubation for five days 

 was, however, followed bv the occurrence of the coccus form again. 

 Hence it was assumed that isolated cells of the latter form had eluded 

 search under the microscope. 



Having occasion at a later date (1915) to prepare tubes and plates 

 of the so-called mixed culture, it was evident that growth in liquid 

 culture with filter paper was not only more vigorous but arose in much 

 less time than was the case on agar plates. It was accordingly decided 

 to prepare subcultures bv the dilution method, and to this end a set of 

 test tubes was prepared, each tube containing a piece of filter paper 

 15 X 60 mm. in size, and 5 c.c. of stock mineral salt solution containing 

 2-0 grm. of sodium ammonium phosphate per litre. In this as in all 

 subsequent sets the reaction of the mineral salt solution was first 

 brought to the neutral point with phenolphthalein and then to each 

 1(K» c.c. of solution 1-0 c.c. of N/IO sodium hydroxide solution was added. 



The requisite attenuations were made in physiological salt solution 

 and ranged down to 1 > lO'-*. By means of sterile pipettes one drop 

 (j'.^th c.c.) of a particular attenuation was introduced into each of six 

 tubes set aside to receive this attenuation. 



After incubation for four days at 30°, growth w^as evident in all 

 tubes which received attenuations up to 1 x 10^. Of the six tubes 

 receiving the next higher dilution, viz. 25-30 inclusive, tubes 26, 27, 28 

 and 29 showed the characteristic colour and growth on the filter paper, 

 while examination of tube 29 showed it to contain what was apparently 

 a pure growth of the thread form. As an alternative to incubating the 

 culture for a further period, thus giving opportunity for the develop- 

 ment of any occasional cell of the coccus form that might be present, it 

 was decided to prepare a fresh dilution set on similar lines. This was 

 completed in less than an hour and, in addition, the culture was trans- 

 ferred to ordinary filter paper tubes. Incubation for a further period 

 of four days served to induce growth in the dilution tubes as high as 

 tube 23 but it was then evident on microscopical examination that all 

 the tubes contained a mixture of the "thread" and "coccus" forms. 

 In view of the fact that the inoculant used for this set of cultures con- 

 tained apparently a pure thread form and could most certainly not have 

 contained anything approaching the number of coccus forms indicated 

 bv this second dilution set, viz. 40,000,000 per c.c, the conclusion was 



