250 BACTERIOLOGICAL METHODS 



Test Animals. The animals upon which the substance in 

 question is to be tested shall be white mice of not less than 15 

 nor more than 30 grams weight. 



Dilutions and Dosage. The dose is to be calculated per gram 

 of body weight and should when diluted equal between 0.03 and 

 0.04 cc. per gram weight; that is, 0.6 to 0.8 cc. for a 20 gram mouse. 

 The diluent is to be distilled water and primary dilutions are to 

 be made of such strength that the dose is easily measured with 

 a i cc. pipette graduated into hundredths. This is most easily 

 accomplished by the use of the substance in greater concentra- 

 tion than is required to kill in the above volume doses. 



Administration of the Test Solutions. After the required 

 dose of the diluted disinfectant has been estimated it is meas- 

 ured into a suitable dish and is then diluted further to the re- 

 quired volume by adding sterile distilled water in sufficient quan- 

 tity. A series of mice are then injected subcutaneously with 

 varying amounts of the substance until the least fatal dose (L. 

 F. D.) is determined, the mice being kept under observation. 



Time Limit of the Observation. After the animals have been 

 inoculated they are kept under observation for a period of 24 hr. 

 unless death results in a shorter period of time. 



Phenol Comparative Test. Mice of the same lot are similarly 

 injected with pure phenol properly diluted to make the meas- 

 urements of the dose easy and then further diluted in a small 

 dish to equal a volume dose of 0.03 to 0.04 cc. per gram of body 

 weight and the fatal dose determined as above. This least fatal 

 dose (L. F. D.) of phenol is unity and the least fatal dose of 

 the substance in question is estimated in per cent, of this. 



Determining the Comparative Toxicity. The phenol toxicity 

 of the disinfectant tested is to the toxicity of phenol as x is to 100. 

 The example given below would be represented in the following 

 proportions: 4.5 : 18 ::x : 100 = 25 per cent., that is disinfectant 

 "A" is one-fourth as toxic as is pure phenol. 



