Toch. D:7 



NZMTODE STAINING IN D2/iD LEAVES (MINDERMN 19^6) 



A Ketiiod whereby nematodes even in opaque bro^^n leaves can be observed. 

 The leaves are bleached with hydrogen peroxide and made transparent by 

 immersion in lactophenol. Nematodes ?nd other organisms are stained 

 by appropriate stains dissolved in lactophenol. 



Damp leaves or pieces of them are covered with the following bleaching 

 mixture : 



Water 3 parts by volume 



20/0 airononia solution 1 part " " 



30^ hydrogen peroxide 1 " " " 



Leave from 1 to 2U hours, depending upon the material to be bleached. 

 Wash in water. Stain by pouring a hot stain solution (± 65° C.) over 

 the leaves. After about 5 m:mutes, pour off dye and replace with pure 

 lactophenol until no appreciable ajnount of dye comes out of the leaves. 

 The specimens are mounted in lactophenol and sealed xd.th "Zut" if 

 permanent slides are desired. Acid Fuchsin and Cottonblue in 0.05^ 

 solutions xjere found most suitable for nematodes from a wide selection 

 of stains tested for this purpose. 



ACETO-OSMIUK IN SITU METHOD FOR ROOTS (TARJAN AND FORD 195?) 



This method was developed for observing nematodes in citrus feeder 

 roots which do not stain satisfactorily using conventional methods 

 because of the presence of suberin and other unsaturated compounds. 



The staining schedule found most effective was as follows: Insert 

 washed roots into the fixing and staining solution at 52° C. for 2 

 hours. (V/ater, 16 parts; 10% acetic acid, 10 parts; and 2% aqueous 

 osmium tetroxide, 2 parts.) Was.h stained roots for 1 hour in running 

 x-irater. Bleach in 10-30$ hydrogen peroxide at 32° C. until the dark- 

 ened tissues lighten perceptibly, then wash several times i-jlth water. 

 Pass through 70$, 90$, and 100$ ethanol at 52° C., 1/2 hour in each. 

 Immerse in methyl salicylate at 52° 0. until tissues clear, after 

 which the roots may be stored indefinitely in this liquid at room 

 temperatures. Although other clearing agents such as clove oil are 

 just as effective, methyl salicylate (synthetic oil of xidntergreen) 

 may be preferred due to its inoffensive odor. 



Tissues that have been treated but still are opaque can be transferred 

 back through 95$ ethanol directly into the bleach and then passed up 

 through the alcohol series to the clearing agent again. Fxxcess expo- 

 sure to hydrogen peroxide will result in excessive destaining of the 

 .nematodes and, in some caset;, will cause disintegration of the roots. 



