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A staining technique for the identification of trypanosomes in thick blood films
LABORATORY M E E T I N G
301
Dr. J o h n McArthur : Advances in microscopy. Several new accessories were shown for use with the miniature microscope which has been demonstrated at previous laboratory meetings. These included a 16 ram. cine camera, which can be used as a bench stand for the microscope, and allows either still or cine pictures to be taken, in monochrome or colour, of what is seen focused in the eyepiece, without interrupting the image. T h e clamping together of the microscope and cine camera to form a compact and portable unit interferes neither with the use of the microscope for routine examinations, nor of the cine camera for making ordinary films even with the microscope attached. T o i!1ustrate the possibilities of a 35 ram. camera built into this microscope, a photomicrograph was shown of trypanosomes in a blood_ film, at a magnification of x 2,000, the exposure having been made at night on the top of a crowded bus in Piccadilly.
Dr. D. G. j a m i s o n and Dr. R. G. C o c h r a n e : Histopathological findings in a case of tuberculoid leprosy. Biopsies were taken from the skin at the centre, from the margin and from an area adjacent to the lesion in a case of tuberculoid leprosy ; a segment of the nerve supplying the affected skin was aiso removed. T h e material was prepared for examination under the microscope as (1) Sections impregnated with silver, and (2) Sections stained with haematoxylin and eosin. In the specimens impregnated with silver, the number of nerve fibres diminished relatively abruptly towards the affected zone. In the skin adjacent to the lesion there was a full complement of nerve fibres. At the margin of the lesion there was marked reduction in the number of axis cylinders impregnated with silver and in the centre of the lesion no axis cylinders were seen. Empty and infiltrated Schwann cell pathways were still to be seen both at the margin and in the centre of the lesion. Sections from the parent cutaneous nerve supplying the affected zone showed destruction of nerves comparable to that seen in the centre of the lesion. In the sections of the skin from the centre of the lesion stained with haematoxylin and eosin, the epidermis was flattened and in the underlying dermis there was an extensive cellular infiltration which included round cells, epithelial ceils and multinucleated giant cells. T h e picture was in fact typical of that associated with tuberculoid leprosy. In the section of the nerve stained with haematoxylin and eosin the fascicular pattern of the nerve fibres was grossly distorted ; within the epineurium there was a central necrotic zone surrounded by a cellular infiltration similar to that seen in the dermis at the centre of the skin lesion. T h e skin overlying the nerve and the surrounding tissues appeared normal at the time of biopsy. Sensory tests* paralleled the neurohistological findings fairly closely. T h e sensory acuity of the skin adjacent to the lesion was non-hal, whilst the centre of the lesion was anaesthetic to touch ; at the margin of the lesion sensory acuity was diminished and localization was impaired. * L~LE, SINCLAm & W~DDELL (1954)
J. physiol.,
123, 187.
Mr. K. J. R. M a c l e n n a n ( d e m o n s t r a t e d by Dr. C. A. Hoare) : A staining technique for the identification of trypanosomes in thick blood films. T h e staining of thick blood films by various modifications of the Romanovsky method is widely used for the parasitological diagnosis of malaria parasites. However, in the case of trypanosomiasis the standard methods for staining thick films usually produce unsatisfactory results, owing to poor staining and distortion undergone by the trypanosomes, with the result that these flagellates can only be detected but their specific identification may be uncertain. On the other hand, for mass field diagnosis of trypanosomiasis, carried out by the Veterinary Departments in Africa, the preparation of thin blood films from thousands of animals is out of question. T o overcome this difficulty a new technique for staining thick films was devised some years ago by LAWS (1931) in East Africa, which I have further developed in Nigeria. T h e method described below', derived from those described by FIELD and LE FLEMINO (1939)
302
LABORATORY MEETING
and by FIELD (1940) for the examination of malaria parasites, has been used successfully for the examination of 13,000 blood films, mainly from Zebu cattle. Apart from the fact that a thick film technique was essential to enable this volume of material from the field to be examined, it has been shown that the thick film revealed in a comparatively short period of examination trypanosomes that were apparent in thin films only after prolonged searching. Furthermore the species of trypanosome could be readily determined in the thick films. From examination of material in bulk from the field there is no doubt that the method has demonstrated infections that would otherwise have remained undetected. Method of preparation : the centre of a clean slide is touched on a drop of blood that has welled up on the ear of the animal after pricking an ear vein (any excess can easily be removed by shaking the slide laterally). T h e drop is spread with the corner of another slide to over an area about ½" in diameter. The smear is not too thick unless it runs easily if the slide is tilted, or, when dry, if the hands of a watch cannot be seen through it. T h e smear is thoroughly dried in a level position protected from dust and flies and preferably shaded from direct sunlight. Staining : (a) Dip for one second in a 0.5 per cent. aqueous solution of methylene blue. (b) Place in a trough of tap water until dehaemoglobinization is complete (this may take up to 30 minutes or more depending on the climatic conditions to which the smear has been exposed ; at Kaduna the water has a p H of 7.8). (c) Stain in Giemsa stain, diluted 1 part in 10 with buffered distilled water at pH 7.2. Staining is carried out for 30 minutes in a staining trough. (d) The slide is removed and differentiated by washing momentarily in tap water, and put to dry in a sloping or vertical rack. N O T E : It has been noticed that thick films from different hosts vary in the respon.se to this technique. Thus, it gives best results with Zebu cattle and goats, but in the case of dogs and horses, though still satisfactory, the capsules of the red cells show a denser background. T h e demonstration included thick films showing the following trypanosomes : Trypanosoma ~ivax, T. congolense, T. brucei. R E F E R E N C E S : FIELD, J. W. (1940). Trans. R. Soc. trop. Med. Hyg., 33, 635. - & LE FLEMI~C, H. (1939). Ibid., 32, 467. LAws, S. G. (1931). Lab. J., 7, 41.
301
Dr. J o h n McArthur : Advances in microscopy. Several new accessories were shown for use with the miniature microscope which has been demonstrated at previous laboratory meetings. These included a 16 ram. cine camera, which can be used as a bench stand for the microscope, and allows either still or cine pictures to be taken, in monochrome or colour, of what is seen focused in the eyepiece, without interrupting the image. T h e clamping together of the microscope and cine camera to form a compact and portable unit interferes neither with the use of the microscope for routine examinations, nor of the cine camera for making ordinary films even with the microscope attached. T o i!1ustrate the possibilities of a 35 ram. camera built into this microscope, a photomicrograph was shown of trypanosomes in a blood_ film, at a magnification of x 2,000, the exposure having been made at night on the top of a crowded bus in Piccadilly.
Dr. D. G. j a m i s o n and Dr. R. G. C o c h r a n e : Histopathological findings in a case of tuberculoid leprosy. Biopsies were taken from the skin at the centre, from the margin and from an area adjacent to the lesion in a case of tuberculoid leprosy ; a segment of the nerve supplying the affected skin was aiso removed. T h e material was prepared for examination under the microscope as (1) Sections impregnated with silver, and (2) Sections stained with haematoxylin and eosin. In the specimens impregnated with silver, the number of nerve fibres diminished relatively abruptly towards the affected zone. In the skin adjacent to the lesion there was a full complement of nerve fibres. At the margin of the lesion there was marked reduction in the number of axis cylinders impregnated with silver and in the centre of the lesion no axis cylinders were seen. Empty and infiltrated Schwann cell pathways were still to be seen both at the margin and in the centre of the lesion. Sections from the parent cutaneous nerve supplying the affected zone showed destruction of nerves comparable to that seen in the centre of the lesion. In the sections of the skin from the centre of the lesion stained with haematoxylin and eosin, the epidermis was flattened and in the underlying dermis there was an extensive cellular infiltration which included round cells, epithelial ceils and multinucleated giant cells. T h e picture was in fact typical of that associated with tuberculoid leprosy. In the section of the nerve stained with haematoxylin and eosin the fascicular pattern of the nerve fibres was grossly distorted ; within the epineurium there was a central necrotic zone surrounded by a cellular infiltration similar to that seen in the dermis at the centre of the skin lesion. T h e skin overlying the nerve and the surrounding tissues appeared normal at the time of biopsy. Sensory tests* paralleled the neurohistological findings fairly closely. T h e sensory acuity of the skin adjacent to the lesion was non-hal, whilst the centre of the lesion was anaesthetic to touch ; at the margin of the lesion sensory acuity was diminished and localization was impaired. * L~LE, SINCLAm & W~DDELL (1954)
J. physiol.,
123, 187.
Mr. K. J. R. M a c l e n n a n ( d e m o n s t r a t e d by Dr. C. A. Hoare) : A staining technique for the identification of trypanosomes in thick blood films. T h e staining of thick blood films by various modifications of the Romanovsky method is widely used for the parasitological diagnosis of malaria parasites. However, in the case of trypanosomiasis the standard methods for staining thick films usually produce unsatisfactory results, owing to poor staining and distortion undergone by the trypanosomes, with the result that these flagellates can only be detected but their specific identification may be uncertain. On the other hand, for mass field diagnosis of trypanosomiasis, carried out by the Veterinary Departments in Africa, the preparation of thin blood films from thousands of animals is out of question. T o overcome this difficulty a new technique for staining thick films was devised some years ago by LAWS (1931) in East Africa, which I have further developed in Nigeria. T h e method described below', derived from those described by FIELD and LE FLEMINO (1939)
302
LABORATORY MEETING
and by FIELD (1940) for the examination of malaria parasites, has been used successfully for the examination of 13,000 blood films, mainly from Zebu cattle. Apart from the fact that a thick film technique was essential to enable this volume of material from the field to be examined, it has been shown that the thick film revealed in a comparatively short period of examination trypanosomes that were apparent in thin films only after prolonged searching. Furthermore the species of trypanosome could be readily determined in the thick films. From examination of material in bulk from the field there is no doubt that the method has demonstrated infections that would otherwise have remained undetected. Method of preparation : the centre of a clean slide is touched on a drop of blood that has welled up on the ear of the animal after pricking an ear vein (any excess can easily be removed by shaking the slide laterally). T h e drop is spread with the corner of another slide to over an area about ½" in diameter. The smear is not too thick unless it runs easily if the slide is tilted, or, when dry, if the hands of a watch cannot be seen through it. T h e smear is thoroughly dried in a level position protected from dust and flies and preferably shaded from direct sunlight. Staining : (a) Dip for one second in a 0.5 per cent. aqueous solution of methylene blue. (b) Place in a trough of tap water until dehaemoglobinization is complete (this may take up to 30 minutes or more depending on the climatic conditions to which the smear has been exposed ; at Kaduna the water has a p H of 7.8). (c) Stain in Giemsa stain, diluted 1 part in 10 with buffered distilled water at pH 7.2. Staining is carried out for 30 minutes in a staining trough. (d) The slide is removed and differentiated by washing momentarily in tap water, and put to dry in a sloping or vertical rack. N O T E : It has been noticed that thick films from different hosts vary in the respon.se to this technique. Thus, it gives best results with Zebu cattle and goats, but in the case of dogs and horses, though still satisfactory, the capsules of the red cells show a denser background. T h e demonstration included thick films showing the following trypanosomes : Trypanosoma ~ivax, T. congolense, T. brucei. R E F E R E N C E S : FIELD, J. W. (1940). Trans. R. Soc. trop. Med. Hyg., 33, 635. - & LE FLEMI~C, H. (1939). Ibid., 32, 467. LAws, S. G. (1931). Lab. J., 7, 41.