"3- As methods of biological assay, the following can at present be recommended as sufficiently accurate :
"(*') The frog method, with a period of observation of at least 4 hours.
'' (a) Preparation of an extract of digitalis leaves with absolute alcohol. I gramme of digitalis leaves, coarsely powdered (B. 20) and dried to constant weight over sulphuric acid, is allowed to stand for 24 hours at room temperature with 25 c.c. of absolute alcohol, with occasional shaking in a closed spherical flask of about 100 c.c. content. The mixture is then boiled for 30 minutes with a reflux condenser, on a sand-bath over the smallest possible flame, and, while still hot, is filtered through a plain filter of about 9 cm. diameter. The residue is washed with absolute alcohol on the filter until the filtrate becomes colourless. The combined filtrates are slowly evaporated in a thin-walled, tared watch-glass, on a boiling water bath to 5 c.c. (about 4.5 gr.), the drying of any portion being carefully avoided.
"The concentrated extract, while still hot, is transferred with the aid of distilled water to a graduated flask, and made up to 25 c.c. with distilled water.
"By this procedure one obtains an emulsiform, greenish solution in weak, watery alcohol. This must be used immediately for the test.
"(b) Assay of the extract, obtained as described under (a), on fro°s, by determination of the minimal lethal dose by the so-called unlimited-time method.
"For the test only healthy male frogs must be used (grass frogs, rana temporaria or rana pipiens], kept under constant conditions and weighing up to 40 grs. each. The body weight is determined to an accuracy of 0.5 gr., after drying the skin and expressing the urine.
"The extract prepared as above described is injected into frogs, through the mouth, into the breast lymph-sac, with a syringe graduated in hundredths of a c.c. Larger quantities than 0.3 c.c. ; or with weakly active preparations 0.5 c.c., should not be injected into the breast lymph sac ; if necessary, the injections are to be made, in such cases, also into one or both oi the lymph-sacs of the thighs. "The following signs of intoxication appear :
"Within i Y2 to 2 hours after the injection restlessness, air-hunger, formation of froth, paralysis and, in the course of four hours, stoppage of the heart. The criterion for the determination is that the stoppage is either systolic or rapidly transformed into systole.
"The orientating tests are carried out as follows : "Doses differing by 20 per cent per gramme of frog are injected, one or two frogs
being used for each dose.
"The final determination can be made by the following procedure : "The mean between the smallest active and the greatest inactive dose is the first approximation. By further more exact determination, with four to six frogs on each dose, the final value can be obtained with an accuracy of 10 per cent. The determination is completed when, of two doses differing by 10 per cent, the higher kills a majority of the frogs injected, the lower a smaller number.
"The value is expressed as a percentage of the standard preparation, which is tested at the same time and in the same manner. Only such leaves shall be passed for issue as differ from the standard preparation by not more than of 25 per cent. "The assay of digitalis tinctures is made in the following manner : "10 c.c. of the official tincture (= i gr. of leaves)i are concentrated on the water bath to 5 c.c. volume, washed into a measuring flask with distilled water, and made up to 25 c.c. The assay is made according to the same method as described above for digitalis leaves. "(n) The cat method as modified by Magnus from that of Hatcher. "For biological standardisation on the cat the % per cent infusion of the digitalis leaves is used, prepared according to the indications of the Dutch Pharmacopoeia, and then made isotonic by the addition of NaCl ; in preparing this infusion, the temperature of 90° C. is not to be exceeded, and the extraction is to be continued for 15 minutes after this temperature has been attained. Cats are used w7ith a body weight between 1.7 and 2.7 kilograms. The cat is anaesthetised with ether, a tracheal canula is inserted and, with the help of artificial respiration, a moderate anaesthesia with ether is maintained. The infusion runs at a regular rate from a graduated burette, arranged as a Mariotte's bottle, through a wide canula into the femoral vein. The rate of infusion is so adjusted that the duration of the experiment amounts to about forty minutes ; minimum 30 minutes, maximum 55 minutes. If, as a result of the first determination, it appears that the preparation is especially potent, the infusion is suitably diluted, and the first experiment is not included in the calculation.
;"The dose is determined which is necessary to produce stoppage of the heart ; this is recognised by/palpation of the thorax, by the asphyxial convulsions, and the interrupted """How of the fluid into the vein; it is further confirmed by opening the chest.
"If the animal is found to be ill (pneumonia) or pregnant, the result obtained with it is rejected.,___ "In this manner one determines the lethal dose of the 0.5 per cent infusion ori*cats i and continues the determination until the mean percentage deviation of the single / results, from the mean value of the whole scries, is smaller than 6.67 V~. The average (