(Nob included in ropyripht) Tech. D:8 



BROMT'UFWI, OR BROITTHYMOL IN SITU STAIN FOR NEMATOTIFS 

 J. D. Kirkpq trick and W. F. Mai 

 Cornell University, Ithaca, New York 



Clearing is by means of a bleach which makes possible the use of a 

 wide variety of stains. In the procedure described using pH indicator 

 dyes the color development and contrast obtained between plaiit and 

 nematode tissue apparently is due to the greater buffer capacity of 

 the nematode tissue. These maintain the absorbed dye in the pH range 

 in which it is most highly colored. The nematode tissues apparently 

 absorb considerable ajmounts of the dye. These two factors combined 

 result in good color contrast. 



Bromphenol blue gives a deep blue to green color to the nematodes and 

 eggs contrasted with a very light yellow or clear background of plant 

 tissue. Orange or light green color contrasted with a very light 

 yellow background are obtained when using bromthymol blue or bromcresol 

 green. 



Kill fresh material with F.P.A. (formalin, 5 ml.', propionic acid, 5 ml.; 

 and 5'0^ ethanol, 90 ml.). Rinse in ^0% ethanol and then place in water. 

 In the above steps higher alcohol concentrations dehydrate, twist, and 

 deform the nematodes; xylol destroys them. Drain water fiom the mate- 

 rial and then transfer to 1% sodium hypochlorite until "cleared." Drain, 

 rinse 3-h times with water to remove excess NaOCl2, rinse in ^0% ethanol 

 in which it can be stored. Transfer to 1% bromphenol blue in ^0% eth- 

 anol. and leave for at least h hours (0.0^-0.1^ in ^0% ethanol for 12-16 

 hours if more detail is required). Staining time is not critical and 

 can be extended; some destaining is possible by leaving material in S0% 

 ethanol for several days. 



Drain off stain, rinse with ^0% ethanol until wash is faintly yellow, 

 drain briefly and transfer to a petri dish containing 0,2^ v/v acetic 

 acid in 50^ ethanol. (Too much acid will destroy the buffer capacity 

 of the tissues.) Observe the material for nematodes and eggs while 

 the material is in the petri dish. For semi -permanent mounts the 

 tissues can be transferred to water for several minutes and then 

 mounted in glycerol. For permanent mounts the tissues can be trans- 

 ferred through a series consisting of 50^ ethanol, 70/S ethanol, and 

 70% isobutanol (1-2 minutes in each) and mounted in diaphane (euparal). 

 If greater detail is desired of the plant cell walls, the tissue seg- 

 ments are placed in an aqueous solution of 1:50,000 safranin for 10 

 minutes, or until the desired degree of staining is obtained. Do this 

 after removal of the tissue from the acetic acid step and before pro- 

 cessing into semi -permanent or permanent mounts. Store all mounts away 

 from bright light. 



Smaller rootlets may be ruined by the "clearing" times required for 

 the larger roots. The bleach appears to disintegrate nematodes that 

 were dead prior to sarm^ling and fixing. Prolonged immersion in the 

 bleach will dissolve swollen female root-knot and cyst nematodes, but 

 the eggs persist much longer. The stain, which is expensive, can be 

 used repeatedly when saved and filtered through cloth. 



