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A Method for Staining Whole Brains for Gross and Macroscopic Study by W

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A Method for Staining Whole Brains for Gross and Macroscopic Study by W [ 134 ] A METHOD FOR STAINING WHOLE BRAINS FOR GROSS AND MACROSCOPIC STUDY BY W. HEWITT Department of Anatomy, St Thomas's Hospital Medical School, London A number of staining techniques are available for the macroscopic examination of thick slices of brain. Examples are those of Mulligan (1931) or Tompsett (1955), which stain the grey matter; or those of Waldman & Michaels (1954) or Brody & Wirth (1957), which colour the white matter with sudan stains. In all of these only the surface of a previously cut slice is stained and surface staining has a number of disadvantages. The techniques of Mulligan (1931) and Tompsett (1955) require a painstaking removal of the membranes and blood vessels, before sections are cut, to prevent smearing of the grey and white matter. Even then success is by no means always assured. Surface-stained brain slices will also be spoilt, by exposure ofunstained areas underneath, if accidentally knocked or chipped. Moreover, the section originally made may not be the one desired and it is not possible to pare away the surface to the desired level. Apart from these disadvantages, it can be useful to stain a whole brain so that when it is sectioned the grey and white matter can be clearly distinguished. For these reasons a technique has been developed for staining a whole brain using sudan stains. This was devised before the writer was aware of the staining methods of Waldman & Michaels (1954) and Brody & Wirth (1957); it is in fact based on a different principle. METHOD The brains can be fixed in either 70 % alcohol or, for preference, 10 % formaldehyde. The membranes and blood vessels need not be removed beforehand. The staining fluid used consists of a saturated solution of Sudan III and Sudan 1TV in a mixture of equal volumes of 70 % alcohol and acetone (prepared by adding a mixture of 0 5 g. of Sudan III and 0 5 g. of Sudan IV to every 100 ml. of alcohol acetone mixture). The brain is immersed in ten times its own volume of this fluid. A smaller volume than this can, however, be used provided it is resaturated when- ever the fluid becomes noticeably paler and clearer in colour. When staining is complete the brain will be a uniform red colour. The time re- quired for this is difficult to judge and is governed by the size of the brain. A cat's brain requires at least a month; a human brain not less than 6 months. The com- pleteness can be assessed by cutting through the brain stem about 5 mm. from its lower end, and if staining is complete at this level it may safely be assumed to be complete elsewhere. When staining is completed, differentiation is commenced by immersing the brain in 70 % alcohol. This fluid is changed at intervals when it becomes deep red in colour, and a quantity ten times the volume of the brain at first requires changing at weekly intervals, but later the time between changes lengthens. Differentiation Staining whole brain for gross and macroWcopic 8tudy 135 is complete when the alcohol only slowly changes to an orange red colour and does not deepen in colour beyond this. In general the period of differentiation equals the time required for staining. The stained brain can be stored in 70 % alcohol or 10 % formaldehyde, but once it has been dissected or sliced it must be stored in formaldehyde. If a cut surface is exposed to alcohol it becomes pale in colour. When the cut brain is stored in formaldehyde the fluid slowly becomes tinged pink. This is probably derived from stain present in the membranes, ventricles, choroid plexuses or the substance of the brain. After several changes, the formaldehyde usually remains clear. DISCUSSION This technique has been applied successfully to several animal brains as well as a human brain. When stained and differentiated the white matter is red in colour and can be clearly distinguished from the grey matter, stained a pale pink (PI. 1). The principle of the technique depends upon the affinity of sudan stains for lipids and the myelin of the white matter is thereby coloured. The staining is presumably effected because the alcohol acetone mixture is used as a solvent for the stain and alcohol alone is used for differentiation. The alcohol acetone solution of the sudan gives a higher concentration of the stain than when the solvent used is 70 % alcohol in which sudan is less soluble. On the other hand, the lower solubility of sudan in 70 % alcohol makes the latter a suitable agent for differentiation and the sudan is selectively removed from the brain tissue other than the myelin for which it has a greater affinity. Very effective results are obtained with this technique when sections of the brain are made. These are no trouble to prepare and may be of any thickness from 15,I upwards. The thinner sections can be cut on a freezing microtome from thicker slices of the brain (about 1-2 cm. thick) which have been previously washed for 24 hr. These are much enhanced by counterstaining with haematoxylin. Ehrlich's acid haematoxylin, differentiated with acid alcohol, has been successfully used for this and the sections mounted in glycerine jelly. Thicker sections can be made with a knife and stored in the manner already described. This method could prove a useful aid to dissecting a brain if a successful counter- stain for the grey matter could be devised. A number of experiments with this aim in view have been carried out but none have given entirely satisfactory results. Very dilute methylene blue is an adequate counterstain but washes out when stored in aqueous solutions, and even the use of the alcohol soluble dye has failed to prevent this. Bouin's fluid, which colours the grey matter yellow, has the same defect unless the brains are stored permanently in this fluid. Up to the present, the most suc- cessful counterstain has been a 01 % solution of Methasol Fast Yellow R.S. (Im- perial Chemical Industries Ltd.) in 70 % alcohol. This penetrates a whole brain imparting an orange yellow colour to the grey matter and tinging the red of the white matter a faint orange. There is a slight tendency for the dye to pass into solution when the brain is first transferred to formaldehyde but after this the colour remains fixed. The disadvantage of all the counterstains used has been that in addition to the cell bodies the axons are also stained and the red colour of the myelin becomes affected. 136 W. Hewitt SUMMARY A method is described whereby the white matter of whole brains is stained red with Sudan III and Sudan IV. The use of counterstains for the grey matter is discussed. I am indebted to the Dyestuffs Division of Imperial Chemical Industries Ltd. for their help by suggesting the use of Methasol Fast Yellow R.S. as a counterstain. My thanks are also due to Mr G. A. Langridge, of the Photographic Department of St Thomas's Hospital Medical School, for technical assistance. REFERENCES BRODY, H. & WIRTH, J. E. (1957). A staining and plastic embedding technique for macroscopic brain sections. Anat. Rec. 127, 65-73. MULLIGAN, J. H. (1931). A method of staining the brain for macroscopic study. J. Anat., Lond., 65,468-472. TOMPSETT, D. H. (1955). Differential staining and mounting of human brain slices. Med. Biol. Jllbustr. 5, 29-34. WALDMAN, S. S. & MICHAELS, D. D. (1954). Surface dyeing of the white substance in gross speci- mens of the brain. Anat. Rec. 119, 549-552. EXPLANATION OF PLATE Thick slices made through various brains previously stained with Sudan III and Sudan IV mixture but not counterstained. All are actual size. A. Horizontal section through human cerebral hemisphere. B. Coronal section through human cerebral hemisphere. C. Transverse section through human medulla oblongata at the level of the olivary nuclei. D. Coronal section through the brain of a domestic cat. E. Horizontal section through cerebral hemisphere of a macaque monkey. Journal of Anatomy, Vol. 93, Part 1 Plate 1 B c A D E HEWITT-STAINING WHOLE BRAINS FOR GROSS AND MACROSCOPIC STUDY (Facing p. 136).
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