102 The Preparation of Microscope Slides 



The technique of clearing specimens in a hydrocarbon or in chloroform 

 before embedding in wax is exactly the same as dehydrating, and no 

 special precautions need be observed. In the case of delicate objects in- 

 tended for wholemounting, however, it is necessary to provide some 

 gradient between the alcohol and the essential oil if one is to avoid dis- 

 tortion. The simplest way of doing this is to use a flotation method; that 

 is, one first of all pours a layer of the oil on the bottom of the tube and 

 then very carefully, with the utmost precautions against mixing, floats a 

 layer of alcohol on top of this. The object is transferred from the alcohol 

 to the upper layer of alcohol in the tube through which it drops to the 

 junction of the two fluids. As soon, however, as it has become partially 

 impregnated with the clearing agent, it sinks to the bottom of the tube. 

 As it lies there, columns of alcohol will be seen rising from it. When the 

 alcohol has ceased to rise, the object is extracted with a pipette with 

 care so as not to get any alcohol into the pipette, and then transferred 

 to a tube or dish of the pure essential oil where clearing will be com- 

 pleted in a few minutes. 



There is no more common cause of failure in the preparation of micro- 

 scope slides than imperfect dehydration and clearing. It is a sheer waste 

 of time to endeavor to embed an object in paraffin unless all the water 

 and all the alcohol have been removed from it, and it is a waste of time 

 to endeavor to impregnate it with xylene unless all the water has been 

 removed from it by alcohol. It is quite impossible to give any particular 

 time schedule for any particular object; experience is the only guide. 

 It is easy to see, however, when an object has not been dehydrated or 

 cleared perfectly. The least trace of milkiness— as distinct from opales- 

 cence— is an indication that the water has been imperfectly removed in 

 alcohol, so that the object cannot be cleared properly. If this slight milki- 

 ness is observed, the object must be returned to absolute alcohol until 

 such time as all the water has been removed. There is no simple method 

 of determining when all the alcohol has been removed by xylene. It is 

 usually safer to use three changes, allowing ample time in each, than to 

 embed an object that has been cleared imperfectly and that will be 

 impossible to section subsequently. 



Perfect clearing is just as essential in objects intended for wholemounts 

 and is much easier to determine, since the essential oils are of sufficiently 

 high refractive index to make a properly cleared object appear glass-clear. 

 No further clearing will take place in Canada balsam, so that, unless the 

 object appears perfect in oil, it is a waste of time to mount it. 



A final point to remember is that dehydrating agents must of necessity 

 be hygroscopic, and that they will dehydrate the air as readily as they 

 will dehydrate the specimen. It is desirable, therefore, either to use fresh 

 absolute alcohol from an unopened bottle or, if one is not using the whole 



