Dehydration for Embedding 29 



by rinsing in 50% alcohol, are transferred through the following 

 series at 4- to 12-hr. intervals. For small root tips the intervals need 

 not be over one hour. 



(1) 1/2 dioxan 

 i/, water 



(2) 2/^ dioxan 

 1/^ water 



(3) Two changes of anhydrous dioxan as in the previous schedule. 



Infiltrate in paraffin as descibed later. 



The following five-grade series is recommended for delicate or 

 easily plasmolyzed material: 10% "commercial" dioxan in water, 

 25%, 50%, 75% dioxan at 1- to 4-hr. intervals. Change corks and 

 make two or three changes of anhydrous dioxan at intervals of 1 to 

 12 hr., depending on the size of the pieces. The time intervals in 

 this series are not critical. Anhydrous dioxan is a solvent of wax, but 

 the rate of dissolving and infiltration can be increased by the addi- 

 tion of 5 to 10% xylene or chloroform to the last change of dioxan. 



If anhydrous dioxan is difficult to obtain, a dioxan-normal butyl 

 alcohol series may be used. The method has been tested extensively 

 and is highly recommended. Dehydrate in the foregoing dioxan series 

 to the commercial grade. Transfer to equal volumes of commercial 

 dioxan and commercial butyl alcohol for 1 to 12 hr. Make two or 

 three changes of anhydrous butyl alcohol and proceed with infiltra- 

 tion. A similar dioxan-tertiary butyl alcohol series also is satisfactory. 



Regardless of the dehydrating agent and wax solvent that were 

 used, it is desirable to evacuate any residual air that may remain in 

 the tissues at this point. Place the uncorked specimen bottle into a 

 dry jar, use a safety bottle between this jar and the aspirator, and 

 evacuate until no more bubbles come out of the specimens (Fig. 3.1) . 



During the experimental period following the introduction of 

 dioxan, unsatisfactory results were reported by many workers. Some 

 lots of dioxan produced severe shrinkage; other purchases yielded 

 acceptable, though variable, results. An inexpensive and satisfactory 

 commercial grade dioxan can now be obtained. 



Dehydrating agents can be re-claimed after they have been used 

 and have absorbed some water. Commercial grades that contain a 

 low percentage of water can be made anhydrous. It is rarely profit- 

 able to re-claim ethyl alcohol, or any other dehydrating agent that 

 contains more than 5% water. If the water content is not over 5%, 

 remove part of the water with anhydrous calcium chloride. Use a 

 large jar with a layer of CaClo at least one-third of the volume of 



