and Laboratory Methods. 1939 



diluted one-half with distilled water before using. Then these are rinsed in 

 water, dried in the air and mounted in Canada balsam. To bring out the gran- 

 ule stain most clearly, Mandybrer earlier recommended putting the preparations 

 in a 3 per cent, solution of copper sulphate to which has been added enough 

 ammonia to dissolve the incipient precipitate. 



Investigations were carried out chiefly on the edge of the liver of the animals 

 used (frog, dog, cat, ram, guinea pig, rabbit, rat, monkey). All experimental 

 work was carried out on dogs. To obtain bone marrow of warm-blooded 

 animals, the following procedure was adopted : The animal was cut rapidly 

 across the throat (severe anaemia of marrow takes place in stabbed animals), the 

 thigh bone is freed at one cut from all its soft and hard bonds, and the upper 

 epiphysis sawed off at about the level of the spongy bone tissue together with 

 two succeeding pieces of about a finger's breadth in length. The bone seg- 

 ments are so pried off these, by means of a knife, that the tissue of the marrow 

 remains unhurt. Cylindrical pieces of marrow are thus obtained which are cut 

 at the ends with a knife or razor to remove the bone sawdust earlier made. 

 Frog tissue was obtained in the same way. Practice renders the process very 

 rapid and free from injury to tissue. The following fixatives were used : 

 1. Nikiforow's modification of Foa's liquid — equal parts of a five per cent, solu- 

 tion of potassium dichromate and a saturated solution of sublimate in a .6 per 

 cent, salt solution. 2. Osmic mixture for fat ; with warm-blooded animals, Her- 

 mann's stronger solution; with cold-blooded, either solution, but preferably the 

 weaker. Preparations were left in the liquids for 14-16 hours and then passed 

 through the alcohols (45, 60, 80, 95 per cent.) to absolute in the dark. From 60 per 

 cent, upwards enough iodine is used to make the liquid tea-colored. The whole 

 alcohol treatment lasts three days, of which absolute alcohol requires only 12 hours. 

 The preparation 2-3 hours in Flemming's mixture, in Hermann's 8-10 days, then 

 24 hours in running water followed by increasing alcohols (as above) for 5-6 

 days. All preparations are put in a mixture of 8 parts absolute alcohol and 1 

 part chloroform, then into half and half of each, then in absolute 1 part and 

 chloroform 3 parts, finally into pure chloroform. The whole procedure is con- 

 ducted in the dark and 12 hours are allowed for each mixture. The next 

 liquid is chloroform saturated with paraffin at the ordinary temperature (12 

 hours), then chloroform saturate at 25°-27° (24 hours); then 32° saturate (12 

 hours) ; lastly pure paraffin (45°-46° melting point) for 2-3 hours. Imbedding 

 takes place either in this or in paraffin of 52° melting point. Microtome sections 

 bjJ. in thickness are fgistened by the glycerine albumen process. 



Stains — Hermann and Flemming are stained in the usual 1 per cent, alco- 

 holic safranin solution left on the slide in a moist chamber for 24 hours, 

 washed in water and differentiated in acid alcohol (1-1000). After Nikiforow's 

 mixture haematoxylin-eosin is used with Ehrlich-Biondi ; also Bordeaux-iron- 

 haematoxylin as follows : Preparations remain in the mordant for 5-6 hours, in 

 haematoxylin 20-24 hours. The form used was Weigert's or a .5 per cent, aque- 

 ous. These solutions can be used repeatedly. Isolation preparations of marrow 

 may be made of marrow previously hardened in Nikiforow's fluid (12-24 hours), 

 washed very thoroughly and stained. Often such tissue may be put into 1 per 



