47 
The Italian method is based entirely upon the work and theoretic 
considerations of Prof. Guido Tizzoni ° and Giuseppina Cattani. 
Tizzoni early abandoned the methods of standardizing tetanus 
antitoxin by injecting the serum and the poison separately into the 
animal with an interval of twenty-four hours. He soon found that 
the method of Elrrhch. Kossel, and TTassermann. which consists 
of injections of mixtures in vitro of toxine and antitoxin (of diph- 
theria'. gives more accurate and rehable results. In applying 
this method to tetanus. Tizzoni encountered certam difficulties, 
due more particularly to the unstable nature of the tetanus toxine. 
He correctly observes that ' Xothing more embarrassing can be 
hnagined than experimenting with a toxine wliich changes in the 
hands of the experimenter and the precise grade of the potency of 
which can not be known at a given moment. Researches pmsiied 
under these conditions are long, laborious, often contrachctory. 
uncertain, and confused: the conclusions derived from them are 
rather deductions than demonstrations ; it is impossible to institute 
a continuous series of experiments, and the fe.w certain results ol>- ^ 
tamed are arrived at almost by accident in the midst of a farrago 
- of observations which are of no service on account of the defects, 
in greater or less degree, in the dose of toxine used." A stable 
toxine from tetanus cultures can not be obtained. Cultures reach 
their maximum toxicity from the seventh to the fifteenth day and 
then gradually weaken, even when kept cold and protected from 
fight. Filtration weakens the poison. Tizzoni found that filtrates 
even when kept under oil. in vacuo, or imder gases lose their toxic 
value. 
Tizzoni and Efitasato independently found that if filtered cultures 
are reduced to dryness in vacuo at a low temperature '20~-22^ C. 
the resulting yeUow-brown scales gradually lose toxicity. Xo better 
success was had with Brieger and Frankefis method of precipitation 
with alcohol. It seems that Tizzoni was the first to obtain a pre- 
cipitated tetanus toxine with ammonium sulphate, following the 
method of Brieger and Frankel with other » toxin e>. Buchner and 
Rapp used the same method and obtained a stable poison. But 
Tizzoni found these precipitates not to be constant. He believes 
that these precipitates first decompose rapidly and then very slowly, 
so that they contain a large quantity of transformed toxine. capable 
of confusing the results of experiments. 
o The miormation in regard to the Italian nietliod of standardizing tetanus anti- 
toxin was gotten from "Sul Modo di Determinare la Potenza del Sero Antitetanico, 
col metodo della mescolanza in tntro" by Prof. Guido Tizzoni. in •Alemorie 
della r. Accaderoia delle Scienze dell" Instituto di Bologna. ■' Tomo S. Serie Quinta. 
Bologna. 1S99-1900. Professor Tizzoni's entire article was kindly translated for us 
by Miss E. B. Jones. 
