558 A GUIDE FOR STUDENTS OF MICROTOMY. 



In order to obviate the differentiation stage, one may dilute both the 

 borax carmine and the acid hsemalum till they are about one-third or 

 one-half as strong ; dilution of the borax carmine may be carried out 

 with 50 per cent, alcohol (not methylated spirit) and with distilled water 

 in the case of haemalum. In these solutions the animals remain till 

 sufficiently stained. But the best results are got by the overstaining 

 and differentiation method. 



1047. Example II. From a frog remove a large leg or thigh muscle, 

 and cut it into two pieces about as big as the nail of the little 

 finger. If desired, the liver, a halved testis, or a kidney may also 

 be used. 



Transfer the material to a capsule containing at least 20 c.c. of 

 Zenker's or Helly's fluids ( 73, 684). Leave till next morning, and wash 

 in running water under the tap for at least three hours, preferably over- 

 night, then transfer to 50 per cent, alcohol for an hour ; then to 70 per 

 cent, alcohol containing enough tincture of iodine to give the solution 

 a light port -wine shade. Add more iodine as the colour disappears, 

 prolonging the treatment overnight for large pieces. Pour away the 

 alcohol, and add pure 70 per cent., in which the material is washed at 

 least three hours. Transfer to 90 per cent, for several hours and leave 

 in absolute alcohol overnight. Next morning it is safest to give the 

 material another hour in a fresh change of alcohol absolute. Pour away 

 a good deal of the alcohol and add about the same quantity of xylol or 

 cedar oil. Shake, leave half an hour, and then transfer the material 

 to pure xylol or cedar oil ; leave half an hour. Pour away some 

 of the xylol, either add chips of hard wax to cover the tissue, 

 or add some of the stock xylol and wax mixture. Leave an hour 

 in thermostat on the upper shelf, pour off, and add molten pure 

 wax ; leave one or two hours on the bottom shelf. Embed blocks 

 ( 142, 143). 



1048. Example III. Preparation of an Embryo for Serial Sections. 

 Fix in Bourn's fluid corrosive acetic or picro-nitric, overnight ( 110, 

 63, 97). In the case of the first and last mentioned fixatives, the 

 embryo is afterwards transferred to 30 per cent, alcohol (half-hour), 

 50 per cent, (two hours), and then washed for a day in several changes 

 of 70 per cent. The corrosive acetic fixed specimens are similarly 

 treated except that at this stage iodine solution is added to the 70 per 

 cent, (or this may be done in 90 per cent.) alcohol till the corrosive 

 sublimate is removed. Leave overnight in 90 per cent, alcohol (or 

 at least three or four hours), and at least six hours in two changes of 

 absolute alcohol (preferably overnight). De-alcoholisation and clearing 

 must be done carefully as directed in 591, p. 269. It is a good 

 plan to bring embryos from absolute alcohol, through several 

 gradually strengthening mixtures of alcohol and cedarwood oil to 

 pure cedar-wood oil, and then wash out in benzole. Embed in wax 

 as described in 591, generaUy about one hour in benzol and wax, 

 and two hours in pure wax. Embed blocks ( 142, 143). Now read 

 144 to 151. 



