PAPER CHROMATOGRAPHY 



259 



PARAFFIN IMBEDDING 



Too much emphasis cannot be placed 

 on the importance of good technique 

 in the preparation and staining of 

 smears which is absolutely essential 

 for their correct evaluation. Regard- 

 less of the amount of training and ex- 

 perience one has had in cytology, it is 

 most difficult and often impossible to 

 interpret cells which have been poorly 

 preserved or improperly stained. 

 Paper Chromatography — Written by Eugene 

 Roberts, Division of Cancer Research, 

 Washington University, St. Louis 10. 

 July 15, 1951 — This technique makes 

 possible the identification and, in some 

 cases, the quantitative determination 

 of minute amounts of numerous sub- 

 stances of biological interest. Impetus 

 was given to the development of these 

 methods by their classical application 

 to the separation of amino acids (Cons- 

 den, R., Gordon, A. H. and Martin, 

 A. J. P., Biochem. J., 1944, 38, 224- 

 232). Subsequent studies using paper 

 chromatographic procedures have been 

 made of the chemistry and metabolism 

 of such diverse materials as inorganic 

 ions, amino acids, proteins and en- 

 zymes, carbohydrates, fats, vitamins, 

 purines and pyrimidines, pigments, 

 growth factors, and antibiotics. The 

 extensive literature dealing with the 

 theoretical and practical aspects of 

 this rapidly expanding subject has been 

 summarized in recent reviews (Clegg, 

 D. L., Analytical Chemistry, 1950, 

 22, 48-59; Strain, H. H., Analytical 

 Chemistry, 1951, 23, 2^38) and in an 

 excellent symposium (Biochemical So- 

 ciety Symposia No. 3, Partition Chro- 

 matography, Cambridge University 

 Press). 



Basically, the technique consists of 

 applying a small amount of solution 

 containing the sample near the edge of 

 a strip or square of filter paper and al- 

 lowing a suitable solvent to flow along 

 the paper past the sample either by 

 downward or upward migration. The 

 paper is removed and dried. When 

 conditions are properly chosen, the 

 components of the mixture are sepa- 

 rated in such a manner that each one 

 occupies a discrete spot on the paper. 

 When the substances of interest are 

 colorless, numerous optical and chemi- 

 cal methods may be applied in localizing 

 them. Radioautography has proven 

 to be an extremely useful adjunct in 

 work with isotopically labeled com- 

 pounds. The separation of components 

 in mixtures containing substances of 

 similar chemical properties is often 

 facilitated by the use of two solvents 

 (two dimensional chromatography). 

 The sample is placed 6 cm. from one 



corner of a large sheet of filter paper 

 (i.e., 18 X 22"). It is developed with 

 one solvent, taken out, and dried. The 

 sheet is then rotated 90° and placed 

 into a solvent of different properties. 

 After drying, the spots are visualized 

 by an appropriate method. For any 

 given pair of solvent systems the sub- 

 stances will occujJy characteristic posi- 

 tions on the chromatograms. The 

 preparation of reference chromatograms 

 with known compounds of a high degree 

 of purity is made under the same condi- 

 tions. The spots on the unknown can 

 then be identified by comparison with 

 the reference "map". Paper chro- 

 matography can be performed success- 

 fully in equipment usually found in 

 biological laboratories (test tubes, bell 

 jars, crocks, petri dishes) and does not 

 require training in complicated chemi- 

 cal methodology. 



Results of interest to the cytologist 

 have been obtained in the examination 

 of the free (or loosely bound) amino 

 acids in normal rat tissues (Awapara, 

 J., J. Biol. Chem., 1949, 178, 113-116), 

 in normal and neoplastic mouse tissues 

 (Roberts, E. and Frankel, S., Cancer 

 Res., 1949, 9, 645-648), and in isolated 

 hepatic cell nuclei of the rat (Dounce, 

 A. L., Tishkoff, G. H., Barnett, S. R. 

 and Freer, R. M., J. Gen. Physiol., 

 1950, 33, 629-642). Amino acids in 

 acid hydrolysates of mitochondria iso- 

 lated from various tissues have also 

 been studied by this method (Li, C. 

 and Roberts, E., Science, 1949, 110, 

 559-560). 



Pappenheim, see Panchrome, Kardos-Pap- 

 penheim, Methyl Green-Pyronin and 

 May-Giemsa Stains. 



Para Red (CI, 44) is useless as a stain (Emig, 



P-30). , . , . 



Parabenzoquinone, as a fixative for mito- 

 chondria (Baker, J. R., Nature, 1932, 

 130, 134; Sircar, S. M., J. Roy. Micr. 

 Soc, 1935,55, 238-244). 



Paracarmine (Mayer). Dissolve 1 gm. car- 

 minic acid, 0.5 gm. aluminium chloride 

 and 4 gms. calcium chloride in 100 cc. 

 70% alcohol. Warm slightly, if required. 

 Allow to settle and filter. Tissues to be 

 stained should not be alkaline or con- 

 tain much lime (Lee, p. 147). 



Paraffin Imbedding. For routine it is more 

 convenient than celloidin imbedding. 

 Thinner sections can be cut and it is 

 easier to make them in series. Paraffin 

 imbedding is quicker and the blocks 

 being dry are easily stored in a smaller 

 space. 



After the specimen has been cleared 

 (see Clearing) it is placed in paraffin 

 held at a temperature just sufficiently 

 high to keep it melted. For ordinary 



