Tech. D:^ 



Keep dish uncovered during daytime to promote evaporation. Cover at 

 night or when preparation cannot be watched. Depending on atmospheric 

 conditions, adjust the cover to increase or decrease evaporation and 

 differentiation, so that after the U to 7 day period the roots lie in 

 a pure acetic-glycerine solution. 



Roots may now be mounted in pure gylcerine. Nematodes within roots 

 should be black, root tissue pale yellow and clear. 



Roots treated with Flemiriing strong fixative 



Flamming Fixative, strong formula, consists of: 



1% chromic acid 1$ parts 



2% osmic acid k parts 



Hoots are washed free of soil and immersed in water heated to 70-80° C 

 for 1-2 minutes in order to kill the nematodes within. The roots are 

 then transferred to the fixative for 10- 1^ minutes, the degree of 

 staining can be controlled by observation under the binocular stereo- 

 scopic microscope. Wash roots in running water for several hours or 

 overnight. Dehydrate by passage through a graded alcohol series to 

 absolute. Clear in clove oil and, if permanent slides are desired, 

 mount in Canada balsam. 



Foliar parts treated with Flemming strong fixative 



In order to get satisfactory in situ staining of tissues containing 

 chlorophyll it is necessary to pretreat before staining. Godfrey (1935) 

 recommended treatment with hot acetone prior to soaking in the Flemming 

 fixative. 



Drop small pieces of the plant material into boiling acetone in a small 

 conical flask held in a water bath. Boil for a few minutes and leave 

 in the slowly cooling acetone until the green color is removed. Wash 

 in several changes of water and then transfer into the Flemming fixa- 

 tive. Observe the process under the microscope and remove from the 

 stain when the nematodes are sufficiently blackened. Wash in running 

 water several hours or overnight. Dehydrate, clear, and mount in balsam, 

 euparal, or diaphane, if permanent preparations are desired. 



Osmic acid is quite expensive, and although it gives excellent results 

 in the methods cited, other techniques of in situ staining have been 

 developed. 



Rapid method of staining for nematodes in roots 

 A modification of the original lactophenol plus cotton blue or acid 



