DIPHTHERIA TOXIN. 475- 



manner described on p. 155. To the amount of normal alkali neces- 

 sary to neutralize the liquid an excess of alkali, corresponding- to 7 c.c. 

 of N NaOH per liter, is added. The mixture is then heated to 70 and- 

 filtered through paper, and finally through porcelain. The sterile fil- 

 trate is then transferred to sterile wide flasks, and inoculated with 

 the culture. 



The bouillon may be boiled, filtered and then sterilized by steam- 

 ing on each of 3 successive days, or by heating- in the autoclave at 

 110. Heating-, however, tends to alter the reaction of the medium, 

 and hence diminishes the toxicity of the liquid. Nevertheless, steril- 

 ization by heat is more convenient and hence is resorted to most 

 often. 



2. Instead of adding- commercial pepton which, as indicated 

 above, is liable to vary in composition, Martin adds an equal volume 

 of a pepton solution obtained by digesting- the stomach of a pig-. 



This is prepared as follows: A clean pig's stomach is freed from 

 fat, cut up into small pieces which are then passed through an Enter- 

 prise sausage machine. To 200 g. of the finely divided tissue, 1 liter 

 of water and 10 c.c. of concentrated HC1 are added, and the mixture 

 is set aside at 50 for about 12 hours. 



The digested liquid is then carefully decanted to a filter of ab- 

 sorbent cotton (Fig 1 . 35, p. 237). Inasmuch as the filtrate is intensely^ 

 acid it is advisable to neutralize the excess of acid by the addition of 

 a strong alkali. This can be done by adding 25 c.c. of a 16 per cent.. 

 NaOH solution. The liquid is then thoroughly mixed and the residual 

 acidity is determined by titration (see p. 155). To the amount of 

 N alkali necessary to neutralize the liquid, an excess of 7 c.c. per liter 

 should be added in order to impart the most favorable alkalinity. 



The mixture is then boiled or heated at 120 for 10 minutes. 

 When the precipitate settles, the liquid can be filtered through 

 paper. The filtrate will be perfectly clear, if the neutralization was 

 done in the cold. It may be used at once, or it may be placed in 

 flasks and sterilized by steam; or at 115 for 15 minutes. 



In the pepton solution thus prepared, the diphtheria bacillus 

 grows without producing any acidity. To make the culture medium 

 proper, Martin adds to this pepton solution an equal volume of the 

 fermented meat extract (p. 474) to which salt, but no Witte's pepton,. 

 has been added. The mixture is then titrated and given the alkalin- 

 ity mentioned above. It is then heated, filtered, filled into flasks and 

 sterilized by filtration through porcelain or by steam. This medium 

 as stated does not yield acid products, and it can give rise to a toxin, 

 such that 0.002 c.c. (^ c.c.) will kill a 500 g. guinea-pig. 



