ISOLATION OF CHLOROPLASTS 469 



Granick Procedure 



Weigh rapidly on a torsion balance 3 g. fresh leaf tissue, exclud- 

 ing mid ribs and main veins. Place the tissue between wet paper 

 toweling until ready for use. In this manner the cells absorb water, 

 become turgid, and are more easily torn apart. After removing from 

 the paper, wash the tissue with distilled water, dry superficially, 

 and place some of the material in a 150 ml. porcelain mortar con- 

 taining 1 g. sand and 25 ml. 0.5 M glucose soln., cooled to about 5°. 

 Rub gently until the liquid becomes dark green. Add more tissue and 

 continue the cellular disruption. Pour the suspension into a 50 ml. 

 centrifuge tube. Add 20 ml. cold glucose soln. to the residue in the 

 mortar, and after further grinding add the liquid to that in the 

 centrifuge tube. The time and rate of centrifugation depends on the 

 leaf material employed. The process and the number of centrifuga- 

 tions required must be controlled by microscopic examination of the 

 centrifugates under an oil immersion objective. 



Neish Procedure 



Remove as much of the fibrous material as possible, wash the leaf 

 tissue with distilled water, squeeze out excess water by hand, and cut 

 about 20 g. of the compressed mass with scissors into an 8 in. porce- 

 lain mortar. ]\Iash to a pulp, add about 200 ml. distilled water in 

 three portions, grinding after each addition, and filter the mixture 

 through 200 mesh bolting silk. Centrifuge the filtrate in 250 ml. tubes 

 at 2000 R.P.M. (With material prepared from sensitive fern a lower 

 speed is required since the larger chloroplasts in this species sediment 

 more rapidly.) The starch granules settle faster than the chloro- 

 plasts and hence may be separated from them at this point. Decant 

 the supernatant fluid containing the chloroplasts into all. gradu- 

 ated cylinder until 950 ml. is obtained, then add 2 M calcium chlo- 

 ride soln. to the 1 1. mark and mix the whole. After 30 min. the floc- 

 culated chloroplasts settle to about the 200 ml. level. Discard the 

 supernatant fluid and centrifuge the flocculated material at the same 

 speed as before. Remove the supernatant fluid and triturate the 

 chloroplasts with a glass rod fitted wdth a rubber policeman. Repeat 

 the centrifugations and washings until the concentration of the cal- 

 cium chloride is reduced to the point at which the chloroplasts again 

 begin to disperse in the liquid. The material is collected after a final 



