Hale (1953a, b) notes that certain mucins which colour weakly with the periodic acid-Schiff methods, can be increased in staining ability by exposing them to a solution of sodium hydroxide. More of the susceptible linkages are thought to be made available to periodate oxidation by the manoeuvre, perhaps by protein extraction but not by de-acetylation or de-sulphation. This is an interesting observation which may furnish a means of identification further of PAS-positive materials.
Hale (1955) investigates the role of formalin fixation in reduction of colouring with PAS of rectal mucus, while gastric mucus has shown no such effect. Sodium hydroxide hydrolysis abolishes the formalin effect, perhaps due to polymerization and de-polymerization.
Lhotka (1952c) reports observations on gelatin blocks impregnated with isomers of cyclohexane glycols, and on sections of rat, rabbit and human intestine. Cis isomers in the blocks were oxidized at 10-60 sec while trans isomers were oxidized in 30-120 sec. The observations on sections were interpreted as showing cis isomers after 2 min oxidation, while trans isomers were shown after that time. It seems to the reviewer that it is to be remembered that some cis isomers are so oriented that rapid cleavage is possible, but that all cis 1,2-glycol do require longer oxidation. Two minutes' oxidation was recommended in the first histochemical use of periodic acid (McManus, 1946), but 5 min oxidation was found more
7
194 J . F . A . M C M A N U S
satisfactory (McManus, 1948). I t may be the difference is on the basis which Lhotka suggests.
Dempsey et al., (1950) observed increased basophilia of tissue proteins after periodate oxidation for 1 hr of a 1% aqueous solution at 37° C.
I t was observed that greatest basophilia was acquired by regions at high sulphur content—keratinized epidermis and hair—perhaps by the oxid
ation of sulphide or sulphydryl groups to corresponding sulphonic acids.
Pearse (1951) introduced performic acid as an oxidation agent for the demonstration of phosphatide lipoids and of keratin. I t was thought that alamine-beta-sulphinic acid coloured Schiff's reagent after formic oxi
dation of keratin, while the Schiff reaction was due to aldehyde materials produced from lipoid by performic acid. Lillie (1952) used peracetic acid for the study of ceroid and noted that it coloured hair matrix with Schiff's reagent, and blamed both reactions on ethylamic groups in a lipid present.
Lillie and Bangle ( 1954) did not believe a disulphide bond product such as supposed by Pearse, could give a recolouring of Schiff's reagent. Findlay (1955) was unable to block Schiff's colouring of hair after peracetic acid by aldehyde blocking reagents and other aldehyde reagents, six in all, did not block the Schiff colouring nor colour themselves.
Gomori (1950) introduced aldehyde-fuchsin as a stain for elastic tissue. The relationship of this dye material to Schiff-staining and to aldehyde reactions is obvious. The dye appears to stain a number of metachromatic and/or PAS-positive materials. Included are some of the basophiles of the anterior pituitary. Halmi (1952) and Halmi and Davies (1953) have investigated some of the staining reactions and sites of this interesting and potentially important dye. Benassi and Zini (1951) have described the relationships between metachromasia and PAS positive material in the kidney of several species.
Itikawa and Ogura (1954) studied optimum p H of Schiff's for colour
ing after periodate oxidation. This was found to be 2 · 4 while optimum colouring after Feulgen hydrolysis was at p H 3. Ihnuma and Saka (1952) observed colouring of cytoplasmic and nucleolar material, presumably including ribonucleic acid, in the liver of human and a fish, human spleen and lymph nodes, etc. They agree that a positive PAS reaction does not always mean the presence of carbohydrates. The question of 1,2-glycol linkages in ribonucleic acids is a familiar one. After observing PAS-posi
tive material in some human pancreatic cytoplasm, it was no surprise to the reviewer when Allen (1951) reported periodate susceptible linkages in ribonucleic acid.
The duration of periodate oxidation is important. Malaprade (1928) had described destruction of aldehyde by prolonged oxidation, and the
PERIODATE OXIDATION TECHNIQUES 195 personal communication of Lillie has confirmed the opinion that perio
date oxidation in aqueous solutions of known carbohydrates, mucin, glycogen, etc., is complete in 10 min in tissue sections. Dyer (1956) des
cribes dangers of over-oxidation in vitro, and Lfrotka ( 1953) has shown the appearance of Schiff colouration of abnormal situations after prolonged oxidation. I t does not seem reasonable to prolong oxidation past 10 min.
Recently the reviewer (unpublished studies) has decreased consider
ably the amount of periodic acid used in his histochemical technique.
While the 0 - 5 % aqueous periodate has a molarity of 0 · 022 and a p H of 2-1, well within the working optimum of Dyer (1956), it has been found that a 0 · 25% aqueous solution with corresponding decrease in molarity, still within the active range, and slight elevation of pH, presents the same oxidation effect for intestinal mucin. There seems no reason why 0-25% periodic acid in distilled water should not make an adequate oxidant for the PAS reaction.
Fats may become coloured with Schiff's reagent after periodate oxi
dation if of the type of glycolipids, as in Morrison and Hack's (1949) study of Gaucher's disease. Wolman (1950) found colouring of unsatu
rated fats with Schiff's reagent in smears and in sections after periodic acid for 10 min. The reaction was not blocked by acetylation and was thought due to oxidation of double bond carbon-carbon sites to 1,2-glycols. This PAS positivity of unsaturated lipid is still under discussion, but the acetylation procedure and Sudan Black staining should assist in the identification of lipids colouring with the PAS reaction. Wolman (1956) produces further evidence for lipid colouration by the PAS pro
cedure.
Chu et. al., (1955) describe in vivo control methods for the study of the PAS procedure.
Summary. These accessory observations represent areas in which investigation and analysis of techniques may prove productive. In some details, there is evidence that periodate oxidation in histochemistry may provide information unavailable or unrecognized by the methods of classical chemistry.
VI. APPENDIX
F U R T H E R DISCUSSION OF PROCEDURES
There is no point in having a satisfactory cytochemical method for a material if the tissue handling is such that all the substance which it is desired to study is lost before the test is applied.The most obvious example of this loss of material comes from the complete solution of ordinary storage fat, neutral or triglyceride fat, which occurs in the preparation of the paraffin section by the usual methods. A stain for fat, one of the
196 J. F . A . M C M A N U S
Sudan dyes, would be negative in the paraffin section of an ordinarily fixed, dehydrated and imbedded material. Osmium tetroxide, on the other hand, can fix neutral fat and carry it through paraffin imbedding, where it shows up in sectign by the black colour conferred by the reduced osmium.
The carbohydrates of animal and plant tissue resemble the neutral fat to some proportion, in that special precautions need to be taken to prevent their loss from tissue handling in the preparation of sections.
To begin with, and almost certainly by any methods, the simple sugars are lost by any technique, being soluble in nearly every solution. The more complex sugars, e.g. dextran molecules of molecular weight over 50,000 can be preserved into section (Mowry, personal communication) by avoidance of water. I t is possible then to preserve carbohydrates into section by :
(1) Avoidance of solvents in tissue handling, e.g. freeze dry or alcohol techniques,
(2) Special fixative methods, e.g. Holmgren and Wilander's (1937), subacetate of lead, ,
(3) Certain carbohydrates are so insoluble that solution is difficult or impossible, e.g. Chitin.
Before discussing the more conventional methods of tissue handling, some mention should be made of freeze dry techniques. The sections of glycogen-containing liver which are studied after good freeze dry pre
paration, are probably as reasonable a facsimile of the living appearance as can be obtained. However, the experience of many workers in the field (and this is shared by this reviewer) without the full-time services of a vacuum specialist or a capable gadgeteer, is that the freeze-dry machines at present on the market and in design are expensive, capricious, exas
perating and generally not worth the necessary effort to keep them work
ing ; all this apart from the fact that zones of degrees of preservation and morphology are present in the majority of freeze-dry blocks, just like the "Golgi" preparations, from which the investigator would choose an area most conforming to his idea of the real structure. Cytochemistry has by now advanced to the point that a freeze dry apparatus as a badge of guild membership is no longer necessary.
Fixation. The classical fixing solution for the water soluble carbo
hydrates is ethyl alcohol, either absolute or 95%. The cytological dis
advantages of this alcohol as a fixative have been outlined by Baker ( 1950). The other classical fixatives of the carbohydrates have been picric acid mixtures, Rossman's fluid and similar picric acid mixtures. Deane et al., (1947) used an ice-cold picro-alcohol formalin mixture. Lison and
PERIODATE OXIDATION TECHNIQUES 197 Vokaer (1949) used a similar mixture to which acetic acid was added, the whole kept at —73° by an acetone solid C 02 mixture. Some glyco
gens are readily preserved by formalinsaline, e.g. cervical squamous epithelium (McManus andFindley, 1949). Liver glycogen in most species and ground substance carbohydrate in all examined require non-aqueous fixatives, preferably left in the cold.
Lillie (1947) used a freezing mixture to freeze eyes immediately on removal, and then placed them in a cold picric acid alcohol mixture, or in cold alcohol, claiming that melting was associated with the penetration of the fixative and obtaining excellent preservation of cellular detail and carbohydrates. I have not used this method but it seems reasonable.
For the muco-proteins and muco-polysaccharides, with a high protein content, any of the classical protein precipitant fixatives—Zenker's, Helly's, Regaud's or Maxomov's solutions—are quite adequate.
Fixation by action on the carbohydrate itself probably occurs in Holmgren and Wilander's (1937) use of sub-acetate of lead, long used as a carbohydrate precipitant in classical chemistry. Dempsey and Wis-locki (1946) point out the shortcomings of this as a cellular fixative.
Couteaux-Bargeton (1950) has suggested osmium as a fixative for glyco
gen. McManus and Lupton (unpublished) have used a brief (4-6 hr), fixation in normal saline 50 c.c, bichloride of mercury 10 g, O s 04 1 g, dissolved in the order given. There is loss rapidly of PAS-positive material, mucin and basement membrane, after 6 hr fixation and blackened myelin sheaths become PAS-positive. This ready appearance of factitious staining diminishes the enthusiasm for O s 04 as a carbohyd
rate fixative.
Dehydration and imbedding can be carried out in the usual fashions for paraffin or celloidin techniques, although an occasional sample of cel-loidinwill be PAS-positive. If all the mucin or all the glycogen are desired to be preserved, water must be avoided completely, i.e. no washing out after an aqueous fixative, etc.
Sectioning and mounting. Sections when cut can be flattened on the slide with heat and finger pressure, one of the major contributions of the freeze-dry school. Alternatively, they can be floated on to 70% alcohol, which Leach (1938) showed did not harm very delicate mucin or mast cells. The staining can be carried on with the paraffin still on the section, or it can be carried out on the de-paraffinized collodionized slide which has been baked on in the oven, de-paraffinized and coated with collodion as originally recommended by Arnold (1908) for use of Best's (1906) carmine stain.
Reaction. Difficultly preservable material should be oxidized in alco
holic periodic acid, using 70% to 95% ethyl alcohol. The reaction time
198 J . F . A . M C M A N U S
must be lengthened to accomplish the equivalent of 5 min in aqueous periodic, sometimes up to 2 hr or more. In most instances the products of periodate oxidation in tissue—glycogen, mucin, etc.—are less soluble than the original material (Mowry and Millican, 1953) and aqueous Schiff's reagent can be used. When alcoholic Schiff's is felt necessary, it is hard to make since aldehyde-free alcohol is a troublesome preparation and the sulphurous acid of the Schiff's reagent quickly produces aldehyde from the alcohol. A solution usable for a few hours can be made by quickly dumping one part of Schiff's reagent into four parts of 96% alcohol and clearing with charcoal. The sequence is critical.
Dehydration, clearing and cover-slipping can be carried out in the usual fashions. Frozen sections do not allow demonstration of glycogen (McManus, 1948), probably due to water solubility as Pearse (1953) points out. Otherwise they can be handled like paraffin sections, and are preferable when glycolipids are being studied, although many of these will resist dehydration and paraffin imbedding as Gersh (1949), Morrison and Hack (1949) and Black-Schaffer (1949) have shown. Many of the complicating and simplifying procedures in the PAS technique are des
cribed and illustrated in McManus and Mo wry (1958).
REFERENCES
A l l e n , F . W . ( 1 9 5 1 ) . Federation Proc. 1 0 , 1 5 5 .
A r n o l d , J . ( 1 9 0 8 ) . Arch. Pathol. Anat. u Physiol, 1 9 3 , 1 7 4 . A r z a c , J . P . ( 1 9 4 7 ) . An. Méd., Mexico 8 , 9.
A r z a c , J . P . ( 1 9 4 8 ) . An. Méd., Mexico 9 , 1 5 . A r z a c , J . P . ( 1 9 5 0 ) . ' J . Clin. Endocrinol. 1 0 , 1 4 6 5 .
A r z a c , J . P . , a n d F l o r e s , L . G . ( 1 9 4 9 ) . Stain Technol. 2 4 , 2 5 . A s h b e l , R . , a n d S e l i g m a n , A . M . ( 1 9 4 9 ) . Endocrinol. 4 4 , 5 6 5 .
B a k e r , J o h n R . ( 1 9 5 0 ) . " C y t o l o g i c T e c h n i q u e " , 3 r d e d . W i l e y , N e w Y o r k . B a u e r , H . ( 1 9 3 3 ) . Z. mdkroscop. anat. Forsch. 3 3 , 1 4 3 .
B e n a s s i , G . , a n d Z i n i , F . ( 1 9 5 1 ) . Arch. sci. Biol. 3 5 , 5 7 7 . B e n n e t t , H . S . ( 1 9 4 0 ) . Amer. J. Anat. 6 7 , 1 5 1 .
B e n s l e y , C. M . ( 1 9 3 9 ) . Stain Technol. 1 4 , 4 7 . B e s t , F . ( 1 9 0 6 ) . Z. wiss Mikroscop. 2 3 , 3 1 9 . B i g n a r d i , C. ( 1 9 4 0 ) . Boll. soc. med. Chir. Pavia 5 4 .
B l a c k - S c h a f f e r , B . ( 1 9 4 9 ) . Proc. Soc. Exptl. Biol. Med. 7 2 , 2 2 5 . B r a d e n , A . W . H . ( 1 9 5 5 ) . Stain Technol. 3 0 , 1 9 .
C a m b e r , B . ( 1 9 4 9 ) . Nature 1 6 3 , 2 8 5 . C a s e l l a , C e s a r e ( 1 9 4 2 ) . Anat. Anz. 9 3 , 2 8 9 . C a t c h p o l e , H . R . ( 1 9 4 7 ) . Federation Proc. 6, 8 8 . C h o u d h o u r i , H . C. ( 1 9 4 3 ) . Nature 1 5 2 , 4 7 5 .
C h u , C. H . , S w i n y a r d , C. Α . , a n d B r i z z e e , K . R . ( 1 9 5 5 ) . Stain Technol. 3 0 , 2 8 5 . C l u t t e r b u c k , P . W . , a n d R e u t e r , F . ( 1 9 3 5 ) . J. Chem. Soc. 1 4 6 7 .
C o u t e a u x - B a r g e t o n , M . ( 1 9 5 0 ) . Compt. rend. soc. biol. 1 4 4 , 8 8 0 . C r i p p a , A . ( 1 9 5 1 ) . Boll. soc. ital. biol. sper. 2 7 , 5 9 9 .
P E R I O D A T E O X I D A T I O N T E C H N I Q U E S 199
200 J . F . A . M C M A N U S
P E R I O D A T E O X I D A T I O N T E C H N I Q U E S 201
M o w r y , R . W . ( 1 9 5 4 ) . J. Histochem. 2 , 4 7 0 .
M o w r y , R . W . , a n d M i l l i c a n , R . C. ( 1 9 5 3 ) . Amer. J. Pathol. 2 9 , 5 2 3 . N i c o l e t , Β . H . , a n d S h i n n , L . ( 1 9 3 9 ) . J. Amer. Chem. Soc. 6 1 , 1 6 1 5 . N i c o l e t , Β . H . , a n d S h i n n , L . A . ( 1 9 4 1 ) J. Biol. Chem., 1 3 8 , 9 1 .
N o v i k o f f , A . B . ( 1 9 5 5 ) . " H i s t o c h e m i c a l a n d C y t o c h e m i c a l S t a i n i n g M e t h o d s " , i n M e l l o r s , R . C , " A n a l y t i c a l C y t o l o g y " . B l a k i s t o n , N e w Y o r k .
O s t e r g r e n , G . ( 1 9 4 8 ) . Hereditas 3 4 , 5 1 0 .
P e a r s e , A . G . E . ( 1 9 5 1 ) . Quart. J. Microscop. Sci. 9 2 , 3 9 3 . P e a r s e , A . G . E . ( 1 9 5 3 ) . " H i s t o c h e m i s t r y . " C h u r c h i l l , L o n d o n .
P i g m a n , W . W . , a n d G o e p p , R . M . ( 1 9 4 8 ) . " C h e m i s t r y o f t h e C a r b o h y d r a t e s . "
A c a d e m i c P r e s s , N e w Y o r k .
R i c e , W . G . ( 1 9 5 4 ) . J. Lab. Clin. Med. 4 4 , 5 4 4 . Sehiff, H . ( 1 8 6 5 ) . Compt. rend. soc. chem. 6 1 , 4 5 .
S e l i g m a n , A . M . , G o f s t e i n , R . , a n d R u t e n b e r g , A . M . ( 1 9 4 9 ) . Cancer Research 9 , 3 6 6 .
S h i m i z u , N . , a n d K u m a m o t o , T . ( 1 9 5 2 ) . Anat. Record 1 1 4 , 4 7 9 . S h i n n , L . Α . , a n d N i c o l e t , Β . H . ( 1 9 4 1 ) . J. Biol. Chem. 1 3 8 , 9 1 . S t a p l e , P . H . ( 1 9 5 5 ) . Nature 1 7 6 , 1 1 2 5 .
S t a p l e , P . H . ( 1 9 5 7 a ) . J. Histochem. 5, 4 7 2 . S t a p l e , P . H . ( 1 9 5 7 b ) . Nature 1 7 6 , 1 1 2 5 . S t a p l e , P . H . ( 1 9 5 8 ) . Nature 1 8 1 , 2 8 8 .
S t a p l e , P . H . ( 1 9 5 9 ) . P e r s o n a l c o m m u n i c a t i o n .
V a n D u i j n , P . , O o s t r o m , J . , a n d W e h b e r g , B . J . ( 1 9 5 4 ) . B.J. Nederlotijdschr.
geneesk. 9 8 , 1 0 7 5 .
V a n D u i j n , P . ( 1 9 5 6 ) . J. Histochem. 4 , 5 5 .
V a n S l y k e , D . D . , H i l l e r , Α . , a n d M a c F a y d e n , D . A . ( 1 9 4 1 ) . J . Biol. Chem. 1 4 1 , 6 8 1 . V a n S l y k e , D . D . , H i l l e r , Α . , M a c F a y d e n , D . Α . , H a s t i n g s , A . B . , a n d K l e m p e r e r ,
F . W . ( 1 9 4 0 ) . J. Biol. Chem. 1 3 3 , 2 8 7 .
W a l l r a f f , J . , a n d B e c k e r t , H . ( 1 9 3 9 ) . Z. mikroscop. anat. Forsch. 4 5 , 5 1 0 . W i e l a n d , H . , a n d S c h e u i n g , G . ( 1 9 2 1 ) . Ber. deut. chem. Ces. 5 4 B , 2 5 2 7 .
W o l f o r m , M . L . , K o n i g s b e r g , M . , a n d S o l t z b e r g , S. ( 1 9 3 6 ) . J. Amer. Chem. Soc. 5 8 , 4 9 0 .
W o l m a n , M . ( 1 9 5 0 ) . Proc. Soc. Exptl. Biol. Med. 7 5 , 5 8 3 . W o l m a n , M . ( 1 9 5 6 ) . Stain Technol. 3 1 , 2 4 1 .