Ezt-ra cting and Testing Procgdure 



lEie clama were opened with a siiacking knife while they were still 

 alive „ The shucked olaras were washed in fresh water to remove all foreign 

 par'iicles from the tissues, and then drair;.ed. The entire siphon (neck) was 

 severed from the Isody, and the siphons and bodies were treated separately. 

 These were minced using a hand operated mea's chopper. The ground material 

 was mixed ihoroughly, and to 100 grams of the minced material was added 

 100 milliliters of 0„I2 N hydrochloric acid (l part concentrated hydrochloric 

 acid to 99 parts distilled water). This mixture was boiled for 5 minutes 

 while baing stirred continuously. After cooling, the mixture was brought 

 to its original volume by the addition of distilled water. A portion of 

 the final mixture was bottled and stored in the ice box of the vessel until 

 the boat returned to Zetchikan, At the laboratory the pH of the extract 

 was determined with a pH meter, and, when necessary, it was adjusted to a 

 range of pH 4.0 to 4.5 by adding 5 N hydroch].oric acid or 0.1 H sodium 

 hydroxide. The extract was centrifuged, and the supernatant liquid was 

 placed in vials and stored at 0° to 3^ 0, until tests were performed. 



The extracts were tested on white mice which weighed between 15 

 aaid 25 grams. The mice, obtained from a supplier in California, were a 

 mixture of two strains. Thsy were shipped air espress and arrived at the 

 laboratory in good eonditioao 



The extracts were injected intraperitoneally by means of a two 

 milliliter insulin syringe. The smallest practicable needle (27 gauge) 

 was used to redu'je leakage from the puncture. The voliane of extract 

 injected into each ro.ouse was varied according to the wei^t of the mouse, 

 One^twentieth of a milliliter of the extract - or diluted extract - for each 

 gram of live weight was used» This procedure was found to be more satis- 

 factoiy than the injection of a standard amount of extract and the ajjplication 

 of a correction factor based on the weight of the mouse. 



The lethal time was computed, in seconds, from the time that one- 

 half of the volume of extract had been injected until the mouse took its 

 final normal, rhythmic breath. This time of death, in almost every case, 

 was accoarpanied by a complete relaxation of the body, followed by a series 

 of reflex motions varying in intensity and duration. When necessary, with 

 the highly toxic materials, the extracts were diluted so that the lethal 

 time exceeded four minutes. 



A table based on the toxicity curve of Sommer and Meyer (1937) was 

 used to convert the lethal time to toxicity of the injected solution. The 

 tonicity of the liaw material, expressed as mouse units per 100 grams of raw 

 materia],, was calcolated by applying the appropriate dilution factors, A 

 morise unit (MU) has been defined as that amount of injected shellfish poison 

 that kills a 20 gram mouse in 15 minutes (Sommer, et al., 1948), Since 

 Sommer and Meyer (1937) constructed their cui've from data obtained from 

 mussels, a 3im3.1ar cur^e Tas developed from data obtained using butter clam 

 extracts. A conrparison of the two carves demonstrated that the data 



