108 ALEXANDER L. BOUNCE 



mm. objective, to a point where the cells become so rare that they have to be hunted 

 for. Excessive homogenization is to be avoided, since it tends to damage nuclei and 

 lower the yield. If a small homogenizer is used, it may be necessary to homogenize the 

 material in two or three batches. 



The final homogenate after being divided into two portions is underlaid with ap- 

 proximately equal volumes of 0.34 M sucrose solution containing CaClj in 0.0002 M 

 concentration, in two 60-ml. centrifuge tubes. Centrifugation is carried out for 10 

 min. at about 2900 r.p.m. 



The supernatant is discarded up to, but not including, the loosely packed sedi- 

 ment. The sediment is suspended in about 30 ml. of 0.25 M sucrose without CaCl2 and 

 after complete suspension by homogenizing with 3 strokes in the loosely fitting 

 homogenizer, the pH is carefully adjusted to 6.2 by the addition of 0.01 M citric acid. 

 The suspension is divided into two equal portions, each of which is underlaid with an 

 equal volume of 0.34 M sucrose without calcium chloride, again using 60-ml. cen- 

 trifuge tubes. Centrifugation is carried out for 10 min. at 2100 r.p.m. 



The supernatant is poured off and the nuclei are again carefully suspended (using 

 the loosely fitting homogenizer with 3 strokes of the plunger) in about 25 ml. of 0.25 M 

 sucrose without calcium chloride. No adjustment of pH is necessary. The resulting 

 suspension is underlaid by an approximately equal volume of 0.34 M sucrose without 

 calcium chloride, using one 60-ml. centrifuge tube. Centrifugation is carried out for 

 10 min. at 1800 r.p.m. 



The supernatant is discarded, leaving nuclei which are now almost free from 

 mitochondria but which contain a relatively large admixture of erythrocytes. If an 

 appreciable number of mitochondria persist, another one or two centrifugations can 

 be carried out in sucrose solution. Usually this is not necessary, and, since the nuclei 

 must be suspended in 0.25 M sucrose to search for mitochondria, this extra centrifuga- 

 tion is omitted except in cases where very exacting work is required. 



The sediment of nuclei and erythrocytes is next suspended in about 50 ml. of 1% 

 gum arable solution previously adjusted to pH 6.2. Centrifugation is carried out for 

 15 min. at 1800 r.p.m., and the supernatant is checked microscopically to be sure 

 that most of the nuclei have been sedimented. The supernatant is discarded and the 

 nuclei are washed once more in about 15 ml. of 1% gum arable solution in a 15-ml. 

 centrifuge tube, the centrifuge being run for 15 min. at 1200 r.p.m. The gum should 

 be subsequently removed by washing the nuclei with distilled water if its absence is 

 essential for the work in hand. If the livers are perfused as described previously, the 

 use of gum arable can be dispensed with entirely. 



It is also possible to isolate nuclei from rat liver by a procedure nearly identical 

 with the above but in which 0.88 M (30%) sucrose is substituted for isotonic sucrose. 

 In this case the mitochondria maintain their usual shape and possibly suffer less 

 physical damage than when isotonic sucrose is used. If should be kept in mind that 

 the less the damage to mitochondria, the less is the likelihood of loss of mitochondrial 

 enzymes to the supernatant with possible subsequent transfer to nuclei. Since nuclei 

 isolated in this way from 0.88 M sucrose are more deeply colored than those isolated 

 in 0.25 M sucrose, it is possible that the concentrated sucrose causes adsorption of 

 colored material, e.g., hemoglobin from the supernatant. Perhaps some intermediate 

 concentration of sucrose might prove more suitable than either 0.25 M or 0.88 M. 



(4) Methods Involving Aqueous Solutions of Ethylene Glycol, Glycerol, or Poly- 

 ethylene Glycol. Nuclei can be isolated in solutions containing ethylene glycol or glyc- 

 erol at final concentrations of about 70%. The use of glycerol was first reported by R. 



