POLYSACCHARIDES 



282 



POLYSACCHARIDES 



1 gm. Na thiosulfate hydrate in 20 cc. 

 aq. dest. Add, with stirring, 30 cc. 95% 

 alcohol, and then 0.5 cc. 2 N hydro- 

 chloric acid. Precipitated sulfur is 

 allowed to settle out, but the mixture 

 can also be used immediately. Schiff's 

 reagent: fuchsin-sulfite as used in the 

 reaction for Thymonucleic Acid, or the 

 Bauer-Feulgen reaction for Glycogen; 

 maximum sensitivity and stability are 

 obtained by adjusting the acidity to 

 the minimum amount which allows the 

 reagent to dry in a thin film on a glass 

 slide without becoming spontaneously 

 colored. Preserved in the cold. Sul- 

 fite wash: Aqueous bisulfite, e.g. 0.2 

 gm. K metabisulfite and 0.5 cc. cone, 

 hydrochloric acid to 50 cc. water. 



Procedure: Fix in usual fixatives; 

 alcoholic or picric acid fixatives are 

 suitable if water soluble polysaccharides 

 such as glycogen are to be demon- 

 strated. Bring to 70% alcohol. Leave 

 5 min. in periodic acid, rinse with 70% 

 alcohol, leave in reducing rinse 5 min., 

 rinse with 70% alcohol, leave in Schiff's 

 reagent 15 to 45 min., wash 2 to 3 times 

 in sulfite water. Examine or dehydrate 

 and mount as desired. Counterstain- 

 ing with a basic dye such as malachite 

 green (2 mg. per 100 cc. aq. dest.) is 

 satisfactory. Control sections treated 

 same except not exposed to periodic 

 acid. 



Modifications: Aqueous, instead of 

 alcoholic, periodic acid and reducing 

 rinse may be used, with increased likeli- 

 hood of removing soluble polysaccha- 

 ride such as glycogen or dextrins from 

 the section. McManus uses 0.5% 

 aqueous periodic acid without acetate 

 buffering (Stain Technology, 1948, 23, 

 99-108) ; Lillie uses aqueous sodium 

 periodate containing nitric acid (ibid., 

 1951, 26, 123-136). Periodate or iodate 

 entrapped, or bound to such com- 

 ponents as calcium in the sections, will 

 color the Schiff reagent; the reducing 

 rinse is intended to destroy these 

 iodates chemically. Ordinary washing 

 is used in the McManus and Lillie pro- 

 cedures, and is adequate whenever 

 iodate retention does not prove to be 

 a difficulty in the particular tissue under 

 investigation. The periodate-Schiff re- 

 action may be used subsequent to treat- 

 ment with enzymes such as diastase 

 (Lillie) or hyaluronidase to further 

 identify the material being stained. 

 Isolated substances can be tested for 

 stainability by "spot tests" with the 

 same reagents to help predict their be- 

 havior in tissue sections (see Hotchkiss, 

 loc. cit.). 



Mechanism of reaction and specificity: 

 Aldehyde groups newly formed by 



periodic acid and reacting with Schiff's 

 reagent are almost certainly produced 

 by the breakage of 1,2-glycols or equiv- 

 alent 1,2-amino alcohols or 1,2-dia- 

 mines. Biologically occurring sub- 

 stances containing such groups are the 

 simple sugars, polj'saccharides, cere- 

 brosides, inositol compounds, certain 

 hydroxy-amino acids, adrenaline. The 

 specificity of the stain further depends 

 in part upon the removal of low mole- 

 cular compounds in fixation and wash- 

 ing, leaving the polysaccharides (and 

 mucins, glycoproteins) as probably the 

 only known naturally occurring sub- 

 stances which remain to be stained. 

 The sugar residues contained in nucleic 

 acids and the hydroxyamino acids of the 

 proteins (except perhaps terminal resi- 

 dues or hydroxj'lysine) are chemically 

 so substituted that they do not react 

 with the periodic acid. A number of 

 other substances which reduce the oxi- 

 dant (such as tryptophan) do not yield 

 aldehyde groups. 



Not all sugars or polysaccharides 

 give equallj' intense coloration (Hotch- 

 kiss, loc. cit.: Jeanloz, R., Science 

 1950, 111, 289) and the presence or 

 amount of color cannot in every single 

 case be predicted from the known struc- 

 ture. A further difficulty is that in 

 some cases the structure of a complex 

 biological material (e.g. hyaluronic 

 acid) is inferred from the chemical 

 study of a highly purified component 

 isolated from only one or two special 

 sources. In such cases the spot tests 

 mentioned maj- be used to investigate 

 empirically the behavior of crude, or 

 purified, fractions from the tissue under 

 consideration. 



A method of acetjdating tissue sec- 

 tions has been described (McManus, 

 J. F. A. and Cason, J. E., J. Exp. Med., 

 1950, 91, 651-654) by which the 1,2- 

 glycol structures are blocked with 

 acetyl groups and no longer are able 

 to be oxidized by periodic acid. De- 

 acetylation with cold alkali introduces 

 an element of greater specificity, since 

 glycol substances such as polj'saccha- 

 rides can now again be stained, but the 

 amino derivatives of glycols are prob- 

 ably not released. 



The mechanism of the Bauer-Feulgen 

 and Casella reactions, in which chromic 

 acid and permanganate are the oxidants 

 is probably similar, but these oxidants 

 tend to destro}'- the aldehyde groups 

 which they first produce. Lillie (loc. 

 cit. 1951) has made a critical study of 

 the various Schiff reactions for carbo- 

 hydrates and should be consulted for 

 a review of this subject (see also review 

 by McManus, loc. cit. 1948). C. P. 



