J Clin Pathol: first published as 10.1136/jcp.26.3.241 on 1 March 1973. Downloaded from

Technical methods 241 4 Differentiate in 0-2% potassium hydroxide in Screening for neurofibrillary tangles 80% alcohol for one minute. and argyrophilic plaques with 5 Wash in water for 10 minutes. Congo Red and polarized light 6 Counterstain in solution B at room temperature overnight. 7 MOLLIE I. STOKES1 AND RODERICK J. TRICKEY2 From Wash in distilled water. The National Hospital for Nervous Diseases, Queen 8 Dehydrate, clear, and mount in Xam or DePex. Square, London. 9 View by standard light microscope incorporating polarizing equipment and a Lambda (2nd The capricious nature of silver impregnation tech- order red) interference filter. niques renders them unsuitable for bulk staining and The red congophilic staining of fresh amyloid is screening sections for the presence of argyrophilic usually adequate for its detection by conventional (senile) plaques and neurofibrillary tangles. For this transmitted light microscopy. But recognition of purpose, we describe a simple, consistent and highly plaques and tangles by this method can be difficult sensitive bulk staining method. It depends on the possibly because of the small content of orientated congophilia and resultant anomalous colour or protein fibrils. Congo Red binds to amyloid and to polarization (Ladewig, 1945) that characterizes the protein fibrils of tangles in such a way that an amyloid and other fibrillary protein components of optically active configuration is produced. When plaques and tangles (Terry and Wisniewski, 1970). viewed by conventional linearly polarized light, a green anomalous (interference) colour of polariz- Method ation is produced which is often misnamed dichroism. If a Lambda (2nd order red) interference filter is STOCK SOLUTIONS placed in the light path between the polarizer and analyser, correct orientation of the object in relation Solution A to the place of polarization, reveals the stained copyright. 0-5 % Congo Red in 50 % ethanol. material as bright green or yellow-blue structures standing out against a dark red background. Solution B 0-15 % Gallocyanin is added to a 5 % aqueous Comment solution potassium chromium sulphate. The solution is mixed, boiled for five minutes, cooled and filtered Unfortunately, the methods usually employed for the before use. demonstration of plaques and tangles, the silver impregnation techniques, are costly, capricious, time http://jcp.bmj.com/ PROCEDURE consuming, and not consistent when carried out in Formalin-fixed tissue is used. bulk. Batch impregnation oflarge numbersofsections to 1 Paraffin wax sections (8 ,u) are taken through can lead wide variation in the quality of the final xylene to 50% alcohol. preparations. Nor is the conventional use of meta- 2 Stain in solution A for five minutes. chromatic dyes satisfactory, as small deposits of 3 Wash rapidly in tap water. amyloid and similar fibrillary proteins may fail to

show red, despite being silver positive. Polarized on September 27, 2021 by guest. Protected 'Present address: Department of Pathology, University Hospital, light microscopy, however, can, by correct adjust- London, Western Ontario, Canada. ment of the lighting, be made extremely sensitive, the 'Department of Pathology, St George's HIospital Medical School, Hyde Park Corner, London, SWI. sought-for material standing out by both its bright- ness and colour from the background. Received for publication 1 I December 1972. Of the various techniques using Congo Red (Bennhold, 1922; Puchtler, Sweat, and Levine, 1962) for the demonstration of amyloid, we find that of Highman (Highman, 1946) is most suitable for our A simple lyophilizerfor direct line sublimation-continued. bulk staining. Easily prepared stock solutions can be used many times and there is little danger of over- When the preparation time was extended, the plate, differentiation. Background staining is minimal. H, was prechilled in order to prevent partial thawing As a nuclear stain, gallocyanin (Einarson, 1951) of the sample before the vacuum was applied. gives excellent results in conjunction with Congo References Red. It gives a precise nuclear stain on a clear background, avoiding the 'blueness' often associated Harris, R. J. C. (1954). Biological Applications ofFreezing and Drying. Academic Press, New York. with haematoxylin, and yet shows cytological detail. J Clin Pathol: first published as 10.1136/jcp.26.3.241 on 1 March 1973. Downloaded from 242 Technical methods Thanks to its stable progressive staining it requires basophilia: selective of exquisite progressivity. Acta path. scand., 28, 82-102. no differentiation. Highman, B. (1946). Improved methods for demonstrating amyloid in paraffin sections. Arch. Path., 41, 559-562. We are indebted to Dr A. D. Dayan for his en- Ladewig, P. (1945). Double-refringence of the amyloid Congo-Red- complex in histological sections. Nature (Lond.), 156, 81-82. couragement to publish this technique. Puchtler, H., Sweat, F., and Levine, M. (1962). On the binding of congo red by amyloid. J. Histochem. Cytochem., 10, 335-364. References Terry, R. D., and Wiiniewski, H. (1970). The ultrastructure of the Bennhold, H. (1922). Eine spezifische amyloidfarbung mit Kongorot. neurofibrillary tangle and the senile plaque. In Alzheimer's Muinch. med. Wschr., 2, 1537. Disease and Related Conditions: a Ciba Foundation Symposium Einarson, L. (1951). On the theory of gallocyanin-chromalum, edited by G. E. W. Wolstenholme and M. O'Connor, pp. staining and its application for quantitative estimation of 145-168. Churchill, London.

reveal that the key word is "communi- Letter to the Editor Book review cate". To that end I have dedicated it.' Alas, the proof reader was inadequately Resistance of Staphylococcus aureus to The Bare Facts of Systemic Pathology dedicated and the misprints may not Sulphamethoxazole and Trimethoprim By Joseph A. Sisson. (Pp. xii + 366. always be recognized by the students, eg, £5-25.) Philadelphia and Toronto: J. B. 'Cophorectomy prevents cancer' in Nakhla in 'Resistance of Staphylococcus Lippincott Co. Oxford and Edinburgh: Turner's syndrome; 'Urinary Bladder aureus to sulphamethoxazole and tri- Blackwell Scientific Publications. 1971. Carcinoma Etiology: key words: Analine methoprim' (J. clin. Path., 1972, 25, dye workers and schistosomiasis in 708-712) states: 'It is not known whether To quote from the Preface might suggest, Egypt'; Fungi 'often cause supperation trimethoprim-resistant strains of staphy- incorrectly, that your reviewer had not with draining sinuses'. lococci existed naturally before the drug read this book. Under carcinoma of cervix we find, came into use, or whether the resistance 'Students will find that this book meets aetiology: SEX (too early? too often? has arisen spontaneously and been their needs in several ways. First, it gives etc). This recalls an old school master's selected since.' succinct, clear, no-nonsense, up-to-date definition of et cetera as a confession of In December 1963, through the kind- definitions and descriptions of essential, ignorance; it can here scarcely be saidcopyright. to ness of Dr E. W. Witherspoon, a medical important concepts of pathology. It communicate. Slang, the language of a director of Burroughs Wellcome & Co contains much relevant, current infor- group, is similarly non-communicating, (Australia) Ltd, we received a supply of mation not available in any other single eg, under Grave's (sic) disease. . . 'patients BW56-72 trimethoprim. In the first textbook, particularly in the areas of are hyperthyroid with "bug" eyes'. His quarter of 1964 we tested some hundreds molecular pathology, lysosomal diseases contraction Rx presumably stands for of strains of organisms isolated in this and immunopathology.' therapy and under benign prostatic laboratory against trimethoprim by a These are bold assumptions, and I also hypertrophy is 'Rx: TUR'. 'N. Gonor- plate-dilution method. Four hundred and doubt if concepts can possibly be trans- rhoea is MOST common cause http://jcp.bmj.com/ of sixty-six strains of Staph. aureus were mitted in the staccato telegraphese the PID in USA'. Contraction-slang is the inhibited by a concentration of 1 jsg/ml author employs. Admittedly the last three craziest. trimethoprim; 40 grew in the presence of areas occupy a lot of space, but they There are many quaint bits of infor- 1 j&g/ml but were inhibited by 2 ltg/ml; certainly flout Sir Robert Hutchison's mation. 'Scar = collagenised granulation eight strains tolerated 2 sg/ml but were advice, 'It is always well, before handing tissue with few or no fibroblasts and (sic- inhibited by 5 ug/ml and two strains were the cup of knowledge to the young, to reviewer's sic) blood vessels 'A scar is a not inhibited by 5 jig/mI. wait until the froth has settled' (Brit. med. scar, is a scar, is a scar'. Presumably The medium used was blood agar J., 1925). Stein; here is another: 'Lesch-Nyham on September 27, 2021 by guest. Protected containing lysed horse red blood cells. 'Students themselves suggested the syndrome is a "touchstone disease".' It was satisfactory for the determination novel layout of the book with the text 'Everybody harbors 20 or more "chronic" of sensitivity to sulphonamides, but appearing on the left and blank pages for viruses.' 'Granuloma inguinale has Pund- probably was not entirely free of sub- notes on the right. They take lecture notes Greenblott macrophages'. 'Legally in- stances that interfered with trimethoprim. on the right-hand pages and expand on the intoxicated-0 15 % in blood (3 highballs) However, I believe that these results show "bare facts" with pertinent notes from some states now 0-10%.' His last entry that trimethoprim-resistant strains of classic pathology texts, literary articles and under AGING KEY WORDS No. 6 is Staph. aureus existed here in those days. other sources.' One real disadvantage of a 'Sisson's rule: There are at least two Prior to that time Cooper and Wald telegraphic skeleton, as of a railway time- pathological diagnoses for every ten years had used trimethoprim at the Royal table, is the monotonous uniformity of of age', and so ends this book without a Perth Hospital in Western Australia but each gobbet of information. There is full stop. I do not know of any being used in this neither chiaroscuro nor perspective. It is I would not commend this book to vicinity, certainly not in this hospital. to be hoped that the students do indeed anyone with less than five years' experience G. BUCKLE seek truly literary articles, because this in pathology, or with a taste for style in Director ofBacteriology, type of textbook indubitably imperils their communication. Alfred Hospital, Prahran, ability to use the English language. Victoria, Australia 'A glance at the pages of this book A. C. LENDRUM