622 Journal of Agricultural Research voi. xv. No. 



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thoroughly washed in running water. Certain generally accepted prin- 

 ciples of killing and fixing were considerably modified by greatly shorten- 

 ing the time the material was left in the different reagents. The time 

 the material remained in the grade alcohols was cut to 5 minutes for 10 

 per cent alcohol, 10 minutes for 15 per cent, 20 minutes for 20 per cent, 

 and 30 minutes each for 30, 40, 50, 70, 80, 90, and 95 per cent. Absolute 

 alcohol was used for 30 minutes with one change. 



Xylol-alcohol was used both in short steps of 5, 10, 15, 20, 25, 50, and 

 75 per cent, or in a 25, 50, apd 75 per cent concentration. P'or the short 

 jumps the schedule of 5 minutes for 5 per cent, 10 minutes for 10 per cent, 

 etc., was adopted; for all the longer jumps 30 minutes were allowed. 

 The longer jumps apparently did not injure the material or its staining 

 qualities. Pure xylol was used for one hour, with one change. The 

 infiltration with paraffin was carried out as rapidly as was feasible, start- 

 ing with shavings of 45° paraffin in xylol in cold solution, for 12 to 15 

 hours (overnight), then at the water-bath temperature, gradually adding 

 more soft paraffin, for 24 hours. The mixture of xylol and paraffin was 

 replaced by pure 45° paraffin for two hours, the latter by 55° paraffin for 

 four hours, with one change and the material then embedded in the 

 harder paraffin. 



.^^ciospores and telial columns with promycelia and sporidia attached 

 were killed in Flemming's fluid and handled with the aid of a centrifuge. 

 By using a short spinning at moderate speed to send the small objects to 

 the bottom of the centrifuge tube little material was lost in decanting or 

 pipetting off the different reagents. When the material had been brought 

 to pure paraffin, it was transferred to a glass tube 2 inches long with 

 X-inch bore, which had been previously well coated inside with glycerin 

 and stopfiered at one end with a close-fitting cork. This piece of tubing 

 was then placed in the centrifuge while hot and the centrifuge started 

 rather rapidly. After about a minute of rapid spinning the centrifuge 

 was slowed down and kept going at a modera.te rate until the paraffin was 

 completely cooled. Experience has shown that if the small objects are 

 simply allowed to settle and the tube then cooled in water, the paraffin 

 will solidify first next to the glass and remain fluid in the center with the 

 result that the core of the plug will be hollov/ when the final contraction 

 and cooling has taken place. The hollow may extend quite down to the 

 cork, making it impossible to cut a clean, well-shaped block for sectioning. 

 The use of the centrifuge completely overcomes this trouble and also 

 crowds the minute objects closer to the bottom of the tube against the 

 cork. When the paraffin has cooled, the cork is pulled out and the par- 

 affin plug pushed out of the tube. 



The paraffin method was used exclusively for embedding bark and leaf 

 tissues. It was found that the bark tissue could be easily cut down to 

 5 M without resorting to hydrofluoric-acid treatment. Wood was treated 



