2662 .Journal of Applied Microscopy 



An Imbedding Medium for Brittle Objects. 



I have tried various means of securing good serial sections of amphibian 

 embryos. Aside from the collodion method, which is too tedious to be practi- 

 cable, double imbedding in collodion and paraffin has been the most efficient of 

 the methods known to me. The object is infiltrated in thick collodion and is 

 then cleared in bergamot oil and imbedded in paraffin. It is better not to 

 allow the collodion to harden by evaporation before clearing. When this method 

 works at its best it gives good sections. The objections to it are that it is a slow 

 method, that the hardening of the collodion in the bergamot oil is uncertain, 

 that the cutting must be done with a very slow movement to get the best results, 

 that the sections tend to roll or to wrinkle, and that ribbons are not easily made. 

 At best the yolk cracks, breaks out, or crumbles away in a part of the sections. 



In order to obviate these difficulties I have sought to mix directly with paraffin 

 some substance which will play the part taken by collodion in the above process. 

 It is necessary to have a tough, elastic substance, readily soluable in or miscible 

 with paraffin, whose melting point is so different from that of paraffin that it 

 will harden at a different time and so form a network throughout the tissue, the 

 interstices of the network being filled with paraffin. The substance must at the 

 same time be harmless to delicate protoplasmic structures and be adaptable to 

 all the processes of paraffin work. 



These conditions seem to be filled by ordinary caoutchouc or crude India 

 rubber. The method is very simple. Mix with hard paraffin about one per cent, 

 of India rubber cut into very small pieces. Dissolve by heating to 100° C. for 

 twenty-four to forty-eight hours. Higher temperatures are to be avoided. Several 

 days in the paraffin bath at 55° or 00° will serve the purpose. Undissolved rub- 

 ber and impurities remain at the bottom of the vessel. Filter or use the super- 

 natant fluid. Keep a stock of the prepared mixture cold, as the rubber separates 

 out after a few weeks if the mixture is kept melted. Use exactly as ordinary 

 paraffin, except that xylol and not cedar oil must he used for clearing. 



The mixture (solution) has a melting point a little lower than that of the 

 paraffin used, in accordance with well known chemical laws. It is satisfactory 

 for all kinds of paraffin work. It may be cut at very low temperatures and 

 makes ribbons better than paraffin. I have used it for amphibian and chick 

 embryos and for brains and have been unable to discover any detrimental effect 

 on the tissues. I have obtained better sections through the yolk of amphibian 

 embryos imbedded in this medium than by the collodion-paraffin process. 

 Wrinkled sections straighten well on water, as collodion-paraffin sections 

 do not ; but there is also less tendency to wrinkle. Tissues fixed in any way 

 can be cut. It is not necessary to use a fixing fluid that will leave the yolk soft. 

 Such fluids often do not fix other tissues well. 



The hardened block is slightly brown, but does not differ perceptibly from 

 paraffin in transparency. The melted solution is murky. This murkiness may 

 be prevented by dissolving in the paraffin before the rubber is added enough 

 " mineral rubber " (asphalt) to give the paraffin a light amber color. This paraffin- 



