132 W . HOLMES

An antiserum produced againit a hzmolytic strain agglutinated suspen- sions of hzmolytic strains readily, while qhowing little 01- no cross agglutination with non-hamolytic strains, and v1ce versa.

All strains were non-pathogenic to laboratory animttli, even after admixture with gastric mucin.

I wish to acknowledge my indcbtedness t o Dr A. J. Rhodcs of thii Department for the freshly isolated strains, and to the Curator, National Collection of Type Culture\, London, for the two stock stmins. Animal expenses werc drfray-d by a grant from the l h r l of Moray Fund.

REFERENCE8

ANDERSON, C. G., AND OAG, 1939. Brit. .I. Exp. Path., xx, 25.

EYRE, J. W. . . . . . . 1899-1900. This Journal, vi, 1 . HARVEY, W. F. . . . . . 1921-22. I n d . J . Med. Res., ix, 66. HERROLD, R. D. . . . . 1931. J . In,. Dis., xlviii, 236.

TOPLEY, W. W. C., AND WIL-

R. K.

MILLER, c. P., AND CASTldGS. It. 1936. Ibid., lviii, 263. 1936. The principles of bactwiology and

SON, G. s. immunity, 2nd od., Lordon.

578 * 65 A NEW METHOD FOR THE IMPREGNATION O F NER17E

RXONS IN MOTJNI'ED PARAFFIN SECTIONS

WILLIAM HOLMES

Reit Memorial Research Fellow

From, the Deportnzen,t of Zooloyy ccnd Comparntive Anatomy, Oxford

(PLATE XX)

Histological methods for the demonstration of nerve axons which can bo applied to mounted paraffin sections have many advantages over those applicable only to blocks of tissue or frozen sections. Thin serial sections can be cut, and stains for the demonstration of tissue elements other than axons can be applied either to the impregnated sections or to alternate slides in the series.

Several " silver-on-the-slide " methods have been devised, but dis- advantages such as the requirement that the tissue be fixed in a special fixative have hindered their routine use. The method of Bodian (1936) gives excellent results with material fmed in various alcoholic katives (Bodian, 1937 ; Davenport and Kline, 1938), but results with formol-fixed material are less satisfactory, and difficulties have arisen through variations in the composition of the impregnating agent, Protargol. Of two substances marketed under this name in Great Britain one, of German manufacture and now no longer available, gives excellent results with the alcohol-fixed material ; the other, of American manufacture, gives very poor results. Several other silver protein compounds available in this country havr proved equally unsatisfactory.

A SILVER METHOD FOR PARAFFIN SECTIONS 133

Form01 h e t i o n is oommonly used for nervous tissurs for t l i r b good reSson that it allows subsequent treatment by a variety of neurohistologioal methods. Myelin sheaths, axom and most other elements can be demonstrated in formol-fixed materiel.

The following is E desoription of a '' silver-on-the-slide " method developed for application to mammalian peripheral nervous tissue, noimal and patho- logical, after fixation in saline formaldehyde solutions. It givus a uniform impregnation of the axons of medullated and non-medullated nerve fibres (figs. 3 and 4), and it has given good results in some preliminary trials on material from the central nervous system (figs. 1 and 2).

The extent to which all the finest non-medullatod fibres are demonstrated has not been tested quantitatively; nor has the completeness of the im- pregnation of sensory and motor endings been fully studied. As with all silver method., there is a possibility that some of tho finest nerve fibres esoapc impregnation, nnd modifications may be necessary to render tho method applicable to all parts of thc nervous system.

Technique Biopry specimens should bo fixed immediately on removal

and autopfiy material as soon as possible after death, as post-mortem ohanges may upset the impregnation. The fixative used hm been :

1. Fhaticni.

40 per cent. neutralised formaldehyde . . 15 C.O.

0.8 per cent. NaCl in water . 85 5 ,

It seems likely that formaldehyde solutioiis of different coilcentration.; might be used with equal success.

I t ha.; bren suggested by Mann (1902) and others that formnldehydc is most vcoessfiil as a fixative if material is transforred from it diroct to 95 per oent. alcohol for dehydration. Thore is some evidence that if this procedure i- udoptcAd for nervous tissue the material is proserved from distortion bettctr than if dohydration is carried out gradually through asconding grades of alcohol.

3. After find dehydration in absolute alcohol the material is clearotl and embedded in wax. Sections are cut and attached to the slides by albnmini3ation. For peripheral nerve scctions 15 p is a eonvnnicnt thickness if no countomtain i to be applied : otherwise the sections should be thinner. For albuminisation E solution containing three or four drops of n glycerin- albumin mixture to 10 C.O. of distilled water is sufficiently strong for most material. The slides a m dried for 24 hours a t 37" C. and then placed for half-an-hour in the p a r e oven to melt the wax around the wotions.

The slides are tlicn incubated a t 37°C. for 48 hours in the following reagent :

2. Dehydration. It is unnecessaxy to wttsh tho tissue aftar fixation.

4. XyloZ-acetic treatment.

Xylol . . 30 C.C.

Glacial acetic acid . - 70 9s

This rwgent may be wed repeatedly for successive batches of didos, but it is advisable to renew it B t intervals.

5. The slides are then taken down to distilled water through the graded alcohols.

6 . Impregiuition. The impregnating solution is made up thus :

0.01 per cont. silver nitrate in distilled water . . 100 C.C.

0-880 ammonia (Liq. nmmon. fort.. B.P.) . . l&op

134 W. HOLMES

It is desirable to make up the solution fresh for each impregnation by diluting down a stock 1 per cent,. solution of silver nitrate and adding the ammonia, for the dilute ammoniacal solution if kept may lose its silver by reduction and its ammonia by evaporation. The slides are incubated in this solution, in any convenient covered vessel, for from 6 to 24 hours a t 37' C. These time limits are elastic, for the impregnation often seems to be completo in less than five hours, and the only disadvantage of the longer impregnatioas is the danger of the sections becoming detached from the slides. The im- pregnating solution can only be used once.

Aftsr impregnation the slides should be treated individually through the succeeding stages.

7. The slides are washed by agitation in distilled water for 5 seconds. 8. Reduetion. This may be brought about by either a stronger or a weaker

Reasons for the choice of one or the other are discussed reducing solution. later.

For the strong reducer the following stock solution is prepared :

Sodium sulphite (anhydrous) . Sodium bisulphite . . 2.5 g. Water . . 100 C.C.

5 g. (10 g. of t,he hydrated crystals)

To this is added immediately before ucje : Amidol . . 0-5 g .

Thc weak reducer, which should be freshly prepared, coiisists of: Hydroquinone . . 1 g. Sodium sulphite . . 6 g. (10 g . of the hydrated crystals) Water . . 100 C.C.

Whichever reducing solut,ion is used the slides are placed in it for 30 scconds. 9. The slides are washed first by violent agitation for 10-15 seconds in it

bowl or sink of running tap water, a.nd then for 5 seconds in distilled water. 10. Toning. The sections are toned for 3-5 minutes in a 0.2 per cent,

solution of gold chloride, yellow or brown. If t,he yellmv chloride is used the solution should be acidified in the usual way by the addition of about 4 drops of glacial acetic acid to each 100 C.C. of the solution.

11. The slides me thoroughly washed by agitation in distilled water for 5 seconds.

12. The second reduction. The slides are placed in a 2 per cent. aqueous solution of oxalic acid. They should remain in this for not more than 10 minutes. When unfamiliar material is being treated it is advisable to watch the sections under the microscope with daylight illumination while they are in the oxalic acid, as the nerve fibres soon become visible and it is often desirable to stop the oxalic treatment before the 10 minutes have elapsed, in order to prevent too deep a colouration of other tissue elements,

13. The slides are briefly rinsed in distilled water and placed in a 5 per cent. aqueous solution of sodium hyposulphite for 5 minutes. If this solution becomes cloudy, all the oxalic acid has not been removed from the slides by the rinsing.

14. The slidas are washed thoroughly in distilled water, counterstained if required, dehydrated, cleared and mounted.

Discuasion 1. Fixation. A few preliminary tests of the method have been made on

material hardened in fixatives other than formaldehyde. Experiments with material fixed in bichromte-containing fixatives such as Zenker and Helly

FIG. 1.-Cerebellum of bird (GuZZua), showing Purkinje cells and their dendrites.

FIG. %.--Cerebellum of bird, showing a Purkinje cell and the fibres associated with it.

FIG. 3.-Fine nerve fibres amongst scar tissue in man.

Formol fixation, Amidol reduction.

Pormol fixation, Amidol reduction.

The exons have grown out from the central stump of the external popliteal nerve after a traction lesion. The outlines of the nuclei of Schwann cells and fibroblasts me just visible, and their nucleoli can be seen as black dots. Formol fixation, hydroquinone reduction.

FIG. &--Same material as fig. 3. normal peripheral trunk containing large myelinatod axons. hydroquinone reduction.

Part of a nerve bundle with the appearance of a Formol fixation,

Magnification as fig. 3.

JOURNAL OF PATHOLOGY-VOL. LIV

A SILVER METHOD FOR PARAFFIN SECTIONS

FIG. 1

FIG. 3

PLATE XX

FIG. 2

A SILVER METHOD FOR PARAFFIN SECTIONS 135

suggest that the impregnation is hindered by the presence in the sections of bichromate or compounds formed by it with the tissues (the mercuric chloride precipitate is removed in the usual way by iodine treatment of the sections). Treatment of the sections with the xylol-acetic mixture a t stage 4 for several days seems at least partially to remove these compounds, and impregnation of the axons is then to some extent possible. It seems that methods may be found of making chrome-fixed material amenable to the method, and this would be an advantage for neurocytological studies.

Promising results have been given with material fixed in Bouin’s fluid, all the picric acid being carefully washed from the sections before impregnation.

One alcohol-formol-acetic fixative mixture has been tried, and gave reasonably good results, though the impregnation of collagen was more dense than in formol-fixed material. In one case in which some spinal cord and cerebellar material was transferred from form01 to Carnoy’s alcohol- acetic-chloroform mixture before absolute alcohol a particularly good impregnation resulted.

The function of this treatment is twofold. Firstly the acid xylol removes most of the fat from the sheaths of the myelinated fibres. A certain amount of fat is left behind in formol-fixed material after embedding, unless the material has lain in the fixative for many months or years, and unless this is removed it tends to take up the silver. Secondly, the reagent appears to have some effect on the connective tiesues which diminishes their affinity for the silver and increases the sharpness of differentiation of the axons.

6. Impregnation. The primary requirement for successful impregnation is that the silver solution should be very dilute. After the addition of the ammonia the solution has an extremely low concentration of free silvcr ioiis. A concentration of 0.01 per cent. silver nitrate has been chosen because tho amount of ammonia it is necessary to add to it is not so cjmnll as to be inconvenient to measure, and not large enough to make the solution so alkaline that the sections become detached from the slides. The silver ion concentration is such that the solution gives a precipitate with chloride but not with chromate. A silver solution as dilute as 0.001 per cent. has been used with success.

By varying the amount of ammonia, added to the silver nitrate, it is possible to influence the impregnation of the nerve fibres and the depth of colouration of other elements. Thus it may be found that in different parts of the nervous system the best result is given by the addition of different amounts of ammonia. For peripheral nerve the amount of added ammonia may be increased up to 4 drops per 100 c.c., in which case the silver ion concentration is reduoed so that a precipitate is given with sulphide but not with chloride, and as the ammonia concentration is increased the depth of impregnation of nuclei, collagen and other elements is decreased, giving a sharper differentiation of the axons. The result given by the one drop mixture is however more useful in many cases, as nuclei are very lightly demonstrated, with their nucleoli a sharp black (fig. 3), and the relations of the axons to other structures can be studied without counterstaining. There is also a possibility that with the impregnating solutions containing more ammonia fewer of the finest axom are demonstrated, though this has not been proved.

The stronger reducing solution is similar to that recom- mended by Davenport and Kline (1938) in their modifications of Bodian’s method, and the weaker solution is that used by Bodian (1936) in his original method. It will be found that one or the other is preferable for different purposes and different material. The stronger reducer brings out more of the

4. Xy~l-ace t ic tmm%ent.

8. Reduction.

JOWUE. OF PATE.-VQL IIP I 3

136 W. HOLMES

finest axons, but a deeper staining of cytoplasm and connective tissue also takes place. The weaker reducer gives a better result if the demonstration of the finest axons is not essential, as the stain is lighter and the contrasts are more sharp. In some cerebellar material it has been found that the use of the stronger reducer is essential for the full demonstration of Purkinje neurons and the “ climbing fibres ” on their dendrites (fig. 2).

This must be thorough, as otherwise traces of the alkaline reducer are carried over to the gold bath. If this happens the gold is deposited in a form which gives a red colour to the sections after oxalic treatment and a precipitate may form on the slides. On the other hand the washing must not be unduly prolonged, as there is evidence that some of the silver in the tissue may to some extent be removed thereby.

Toning for longer than the time suggested tends to give a deeper impregnation of structures other than axons : in some cases it may be advisable to shorten the toning time.

The oxalic acid solution is a relatively mild reducer for the gold in the toned preparations. In some cases i t may be found advisable to use a stronger reducer, such as a 0.6 per cent. solution of Amidol, as recommended in the rapid “ silver-on-the-slide ” method of Davenport et al. (1939). I n this case the Amidol solution should be dropped on to the slide after toning. The section immediately turns deep blue, and the slide should then be washed and treated as in the routine given. Stronger reducing agents give a much darker preparation than does oxalic acid, but something may be gained in the completeness of the impregnation. The reducing agent must, however, be in neutral or acid solution, as otherwisp the resulting preparation is red in colour and without differentiation of the nerve fibres.

9. Washing after reduction.

10. Toning.

12. The second reduction.

SUllMnQTy

A new method is described for the impregnation of nerve axons in mounted paraffin sections of formol-fixed material. The method depends on (a) a preliminary treatment of the sections with a mixture of xylol and acetic acid and ( b ) impregnation with a dilute ammoniacal silver nitrate solution containing a very low concentration of free silver ions, followed by reduction, toning and a second reduction of the toned preparation. Preliminary experiments suggest that the method may be applicable to all parts of the nervous system and to material fixed in other fixatives.

1 am very much indebted to Mr P. B. Medawar for chemical m d hio- chemical information and to Dr A. H. T. Robb-Smith for criticising my preparationq and the manuscript.

REFERENCES

BODIAN, D. . . . . . . 1936. Anat. Rec., lxv, 89.

DAVENPORT, H. A., AND KLINE, 1938. Stain Technol., xiii, 147.

DAVENPORT, H. A., MCARTWR, 1939. Ibid., xiv, 21.

MA”, G. . . . . . . 1902. Physiological histology, Oxfwc7.

I, . . . . . . 1937. Ibid. , lxix, 153.

C. L.

J., AND BRUESCH, S. R.