17 
killed. B} r a simple calculation the relative strengths of the un- 
known and the standard preparation can then he obtained. 
An important contribution to the subject of biological assay was 
furnished by Carl Buhrer, a who investigated the activity of some of 
the toxic fluid extracts, among them being digitalis and convallaria. 
He selected frogs and rabbits as experimental subjects. The frogs, 
either R. esculenta or temporaria, were chosen with great care and 
obtained fresh every two weeks, but he used only one species for one 
preparation and for digitalis only R. temporaria. He seems to be the 
first to note any effect of sex upon the results and says he finally 
employed females exclusively. The effect of season upon the reac- 
tion of the frogs was corrected by repeating his experiments at various 
times of the year. Btihrer seems not to have considered the size of 
the animal as being important, for while he weighed the frogs before 
the drug was injected, the weight was not used in calculating the 
dose or judging the results. The method he used was as follows: 
The frog was stretched out on its back and then, with as little hemor- 
rhage and other injury as possible, its heart was exposed. The rate 
was counted at once and again after five minutes when the drug was 
injected into the thigh lymph sac and the animal put into a moist 
chamber and observed at intervals. The dose of the drug to be 
tested was prepared by diluting the fluid extract with different 
amounts of salt solution and injecting one-half cubic centimeter. 
The end reaction or standard consisted in finding the smallest amount 
of drug which would cause systolic stoppage of the heart within 
twenty-four hours. The comparative strengths of the different speci- 
mens were then reckoned as so many milligrams, according to the 
dilution of the drug, without paying any attention to the weight of 
the animal. By this method he found the specimens of 1897 varied in 
the ratio of 1 to 4, while the 1898 fluid extracts were weaker, but 
more uniform in strength. He though it was possible that this 
apparent weakness was due to a difference in the reaction of the frogs, 
as the 1898 specimens were examined one and one-half months after 
those of 1897, and he had found that even in from two to four weeks 
the animals might alter very considerably in their reaction to one and 
the same drug. This variation he thought was due to their winter 
fast, but in this case his supposition was proved incorrect by subse- 
quent examination made in December, 1899. 
Buhrer further continued his experiments, employing rabbits which 
were arranged for blood pressure estimations. The diluted drug was 
injected intravenously at intervals of from five to ten minutes, until 
the lethal dose was reached. By carrying out several experiments 
a Buhrer, Untersuchungen liber die Wirksamkeit einiger toxischer Fluid-Extrackte, 
Inaug. Dissert., Basel, 1900. 
65051— Bull. 48—09 2 
