on 



THE ART OF MAKING MICROSCOPE SLIDES 



Dehydration 



of fixatives: first, that adequate volumes 

 (at least 100 times the volume of the part 

 to be fixed) be employed; second, that 

 mixtures containing either chromic acid or 

 potassium dichromate with formaldehyde 

 be used in the dark. After fixation, tissues 

 should be thoroughly washed in water if 

 this is the solvent for the fixative, or in 

 alcohol if the fixative is based on the lat- 

 ter. Objects are usually stored, after fixa- 

 tion and washing, in 70 % alcohol ; though 

 if they are to be kept a long time before 

 dehydration, it is recommended that 5% 

 of glycerol be added to the alcohol. This 

 glycerol musi,, however, be very thor- 

 oughly washed out before dehydration 

 commences. 



Choice of a Dehydrating Agent 



Chapter 25 discusses the numerous or- 

 ganic solvents wliich from time to time 

 have been proposed for the dehydration 

 of biological specimens and the selection 

 between them is not usually of great im- 

 portance. The classic method of dehydra- 

 tion is to soak the object in a graded series 

 of alcohols, usually 10 or 15% apart. De- 

 hydration through gradually increasing 

 strengths of alcohol may be vital when 

 one is dealing with delicate objects con- 

 taining easily collapsible cavities, such as 

 chicken and pig embryos, but a block of 

 tissue may be taken from water to 95% 

 alcohol without any apparent damage. 

 Even though one uses increasing strengths 

 of alcohol, the series normally in employ- 

 ment at the present time is by no means 

 satisfactory. It is customary, for example, 

 to pass from water to 30% alcohol at one 

 end of the series and to pass from 85% to 

 95% alcohol at the other. The diffusion 

 currents between water and 30% alcohol 

 are far greater and far more intense than 

 those between 85 and 95%, and an 

 inteUigently graded series for delicate ob- 

 jects should run from water to 10% to 

 20% to 50% to 95% alcohol rather than 

 through the conventionally spaced grada- 

 tions. This is not at all in accordance with 

 the recommendations in most textbooks 

 but is based on the author's experience 

 over a long time. In using this classic 

 method of dehydration, it is not necessary 

 to confine the technique to ethanol. Meth- 



anol or acetone will dehj-drate just as 

 effectively, though they are rather more 

 volatile. 



There is a considerable vogue nowadays 

 for the substitution for a straight dehy- 

 drating agent of some solvent which is 

 both miscible with water and also with 

 molten wax. The best known of these is 

 dioxane, though n-butanol has also been 

 recommended. The writer is not in love 

 with these methods for, though the sol- 

 vents involved are excellent dehydrating 

 agents, they are relatively poor solvents 

 of paraffin and frequently cause great 

 shrinkage of delicate objects in the final 

 transition between the solvent and the 

 wax. For such purposes as the routine ex- 

 amination of the tissue blocks in a patho- 

 logical laboratory, or for sectioning rela- 

 tively sturdy plant materials, they may 

 justifiably be employed. For sections in- 

 tended, however, to retain intact struc- 

 tures on which research is subsequently to 

 be conducted, it is most stronglj^ recom- 

 mended that the standard routine of pass- 

 ing from a dehydrating to a clearing 

 reagent be retained. 



Selection of a Clearing Agent 



Reference should again be made to 

 Chapter 25 for a list of the materials which 

 have from time to time been recommended 

 for de-alcohohzing, or clearing, biological 

 specimens. The choice of a clearing agent 

 in section cutting is of far more impor- 

 tance than the choice of a dehj^drant, for 

 there is not the shghtest doubt that pro- 

 longed immersion in some of the volatile 

 hydrocarbons, particularly xylene, leads 

 to a hardening of the tissue with subse- 

 quent difficulty in sectioning. The classic 

 method is to pass from alcohol to xylene, 

 but the only apparent reason for the 

 choice of xylene over toluene or benzene 

 lies in the work of Squire (1892, page 80) 

 who timed the evaporation rate of these 

 three solvents from an open watch glass 

 and found xylene to evaporate the most 

 slowly. There is little choice in the solvent 

 power of any of these three hydrocarbons 

 on wax; the writer's preference is for ben- 

 zene, though it seems impossible to shake 

 the faith of the conventional that the more 

 expensive xylene is a necessity both as a 



