CENTRIFUGATION OF 

 PARTICULATES 



101 



DIGITONINE 



denser by addition of sucrose, if layer- 

 ing is employed. Temperature should 

 be kept at 5°C. or below throughout. 

 No attempt at completeness has been 

 made, and some interesting methods, 

 including ultrasonic and enzymatic cell 

 disruption, are omitted. 



1) Citric acid, low pH (Bounce, Ibid; 

 Cunningham, L., Griffin, A. C. and 

 Luck, J. M., J. Gen. Physiol., 1950, 

 34, 59-63). 



Procedure: Tissue homogenized sev- 

 eral minutes in cold 2% citric acid in 

 a Waring Blendor. The latter should 

 be equipped with a cooling jacket. 



Particulates obtained: Nuclei. 



Morphology: Reasonably good. 

 Nuclei plump and unwrinkled, but 

 with fine internal granulation. No 

 clumping or cytoplasmic tags. 



Value: Simplicity and use of large 

 volumes of tissue, if desired. Nuclei 

 readily counted and DNA per nucleus 

 determined by the Dische or other 

 suitable analytical technique. The 

 method of choice for this purpose, 

 but of little other value. 



Limitations: Water soluble sub- 

 stances, including much protein and 

 probably RNA, are lost; enzymes 

 inactivated. 



2) Citric acid, controlled pH 

 (Bounce, Ibid.). 



Procedure: Enough O.IM citric acid 

 is added dropwise to partially homog- 

 enized tissue in ice water to give a 

 pH of 6.0. Used with a low speed 

 Waring Blendor or glass homogenizer. 



Particulates obtained: Nuclei. 



Morphology: Similar to procedure 1. 



Value: Suitable for BNA and many 

 enzyme studies. 



Limitations: Similar to 1, except 

 that apoenzj'mes are retained. Co- 

 enzymes must often be added in de- 

 terminations of their activities, how- 

 ever. Adsorption effects must be 

 controlled by repeated washing of nu- 

 clei. 



3) Sucrose, near pH 7 (Schneider and 

 Hogeboom, Ibid.). 



Procedure: Small amounts of tissue 

 homogenized by ground glass appara- 

 tus or other "gentle" mechanical 

 means, in a medium of neutral but 

 unbuffered iso- or hypertonic (usually 

 0.88 M) sucrose. iProbably best to 

 avoid NaCl and other electrolytes for 

 general purposes. 



Particulates obtained: Nuclei, mito- 

 chondria, microsomes, supernate. 

 Also melanin granules and particulate 

 glycogen. 



Morphology: Excellent, with pres- 

 ervation of all particulates in a 

 form resembling that in living cells. 



Mitochondria filamentous, at least 

 in hypertonic sucrose, and stainable 

 with Janus Green B; nuclei homo- 

 geneous with several nucleoli. 



Value: Versatility, due to the large 

 number of fractions obtained. Suit- 

 able for determinations of enzymes, 

 nucleic acids, proteins and lipids of 

 these fractions. The method of 

 choice for microspectrophotometry of 

 stained nuclei, as optical homogeneity 

 is a definite prerequisite for valid 

 results with this method. 



Limitations: Solution and adsorp- 

 tion effects again come into play, but 

 many artefacts can be partially ruled 

 out. Nuclei probably not so readily 

 obtained in bulk as with methods 1) 

 and 2). 



4) Behrens technique (Behrens, 

 Bounce; Schneider and Hogeboom, 

 Ibid.). 



Procedure: Tissue frozen, dried in 

 vacuo, ground in ball mill to disrupt 

 cells, and centrifuged in non-aqueous 

 media, usually hydrocarbons. 

 Particulates obtained: Nuclei. 

 Morphology: More seriously dis- 

 torted than with any other proce- 

 dure; nuclear membranes wrinkled. 

 Contamination with cytoplasm must 

 be appraised from smears stained 

 with both acid and basic dyes. 



Value: Probably the most widely 

 applicable method for nuclei. Pro- 

 cedure of choice for water soluble 

 substances, i.e., coenzymes, vitamins, 

 free amino acids, etc. Results with 

 total protein and RNA also appear 

 the most reliable of any method. 

 Suitable also for BNA and many 

 enzyme studies. 



Limitations: Procedure is laborious. 

 Lipids, lipases, and many other en- 

 zymes removed or destroyed. 

 This brief summary can only suggest 

 the variety of applications of cell frac- 

 tionation to cytology, as new ones are 

 continauUy uncovered. For example, 

 B. L. Opdyke has recently described a 

 valuable technique for the isolation of 

 keratohyaline granules of epidermis, 

 using isotonic saline and a Waring 

 Blendor for homogenization. The par- 

 ticles are sedimented at 25,000 x g and 

 appear to have all the morphological 

 and staining characteristics of these 

 granules (See Keratohyaline Granules, 

 procedure for isolation). 

 Differential Leucocyte Count, statistical 

 study of uniformity in (Klotz, L. F., 

 J. Lab. & Clin. Med., 1939, 25, 424^34). 

 Diffraction Methods for measuring diameter 

 of red blood cells (Haden, R. L., J. 

 Lab. & Clin. Med., 1937-38, 23, 508-518). 

 Digitonine reaction of Winaaus for free 



