36 
one egg for each liter of infusion. Heat gently for twenty minutes 
to coagulate the albumin, and then filter while hot through paper. 
W eigh the amount of filtrate obtained and add sufficient water to make 
up the loss to the original amount. Take the reaction again and 
neutralize with sodium hydrate to an acidity of 0.5 per cent, then 
add 1 per cent peptone and one-half per cent sodium chlorid and 0.1 
per cent dextrose. Heat again for twenty minutes in streaming steam 
in an open autoclay. Again take reaction, neutralize and repeat until 
the reaction of the infusion is just 0.5 per cent. The medium is now 
filtered through paper and filled into Fernbach flasks, test tubes, etc. 
These are sterilized in the autoclay at a temperature of 120° C. for 
twenty minutes. 
The fermentation tubes prepared and sterilized without the addition 
of the 0.1 per cent of dextrose are now planted with a young culture 
of the colon bacillus and incubated fortv-eight hours. If there is 
no muscle sugar present the growth stops sharply at the closed end 
without gas production and the reaction of the medium in the closed 
and open arms is about the same. 
Test tubes are also filled and the final reaction is taken from the 
medium in one of the test tubes which were sterilized. The reaction 
is always found to rise about 0.1, due perhaps to the formation of acid 
salts by the heat of sterilization. 
Our strongest toxines were obtained b}^ adding the dextrose after the 
final sterilization and at the time of inoculation, confirming the results 
obtained by Hitchens working with Kinyoun. 
The reaction of the bouillon used to grow the diphtheria culture is 
yery important if we desire to obtain a strong poison. The reaction 
of the bouillon we use, 0.5 per cent acid to phenol-phthalin, is dis- 
tinctH alkaline to litmus. 
The diphtheria bacillus during the first period of its growth always 
produces an increased acidity in the bouillon. The culture used in 
this laboratory causes a marked rise in acidit}^ during the first twenty- 
four or forH-eight hours and then a gradual change to alkali produc- 
tion, so that by the seyenth da}^ the reaction of the culture has returned 
to about its original point, 0.5 per cent. 
Park and AVilliams, and also Theobald Smith, haye shown that an 
excess of acid in the medium is detrimental to the production of 
strong poisons. The rise in acidity above noted amounts to 1 to 1.5 per 
cent. The reaction of the medium therefore neyer becomes more acid 
than 1.5 to 2 per cent, using phenol-phthalin as an indicator. A 
bouillon which is 1.5 per cent acid to phenol-phthalin is still distinctly 
alkaline to litmus. 
The method as aboye described has giyen the best results in this 
laboratory. With it we haye, in one instance, obtained a toxine so 
strong that 0.0008 c. c. was sufficient to kill standard weight guinea 
