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The preservation of arthropods for taxonomic and teaching purposes
(The previous number of these Transactions, Vol. 58, No. 3, was published on 28th May, 1964).
LABORATORY
MEETING
of the ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE held at the Royal Army M e d i c a l C o l l e g e , M i l l b a n k , S.W.,
on Thursday, 19th March, 1964 The President, CHARLES WILCOCKS, C.M.G., M.D., F.R.C.P., D.T.M. • H., in the Chair.
DEMONSTRATIONS
THE ROYAL ARMY MEDICAL COLLEGE DEPARTMENT OF PATHOLOGY
Colonel H. C. Jeffrey and Mr. R. M. Leach : Visual aids to teaching parasitology. Most text books on the subject contain a certain number of diagrammatic representations of the life cycle of various parasites, but only a certain number. As a visual aid to teaching, an attempt has been made to produce a systematic series of diagrams illustrating the protozoa and worms of medical importance. Charts used as museum exhibits and plates from these for issue to students were demonstrated; photomicrographs were included as an ancillary to the museum exhibit. DEPARTMENT OF ENTOMOLOGY
Mr. J. H. Grundy and Mr. N. R. H. Burgess: The preservation of arthropods for taxonomic and teaching purposes. The demonstration dealt with the practical preservation of arthropods, with special reference to those of medical importance, and was presented under nine headings: 1. A reminder that arthropods are composed of (a) a hard chitinous exoskeleton, and (b) soft interior parts, one or both of which may be required for study. 2. Protection from damage. The main methods were shown: i) Pinning, in collection boxes. ii) Immersion in spirit, or formalin mounts. iii) The removal of harmful interior parts. iv) Mounting on microscope slides. v) Mounting in transparent plastic. Each method was then dealt with in greater detail 3. Pinning. There are two reasons for pinning a specimen, (a) for taxonomic purposes, when speed of pinning, ease of examination, and correct label-data are all-important; (b) for teaching purposes, when the specimen is carefully set out to show definite features. 4. Immersion in spirit or formalin. Both hard and soft-bodied arthropods may be preserved in spirit (75-80 per cent. ethyl alcohol). 13 per cent. W/V gelatin may be used for sticking the specimen to an immersed mount. 2 per cent.-10 per cent. formalin liquid or its vapour preserves without clearing,
286
LABORATORY M E E T I N G
and tends to harden the specimen but not to distort it, and if not too strong will preserve the colour. The vapour technique is useful for anopheline mosquito eggs. Dried-out specimens may be soaked in 10 per cent. glycerin until suitably swollen. Methylated spirit (impure ethyl alcohol) should be avoided. 5. The removal of harmful interior parts. Often the specimen may deteriorate if its viscera are left intact. The soft body parts of larger specimens may be removed through a lateral slit between two abdominal segments. Moths may be emptied by slitting the ventral surface of the abdomen longitudinally. The cavity should then be filled with cottonwool soaked in preservative. 6. Mounting on slides for microscopic examination. Sclerotised specimens may first require maceration by boiling or steeping in 10 per cent. caustic potash solution before processing. All specimens must be dehydrated by passing through 70 per cent., 90 per cent. and absolute alcohol, cleared in xylene or cedar-wood oil, and mounted on the slide in resin such as DPX or Canada balsam. 7. Embedding in plastic. For demonstration purposes this is an excellent method. Several processes are available, and once the difficulties of "silvering" and the formation of air-bubbles have been overcome, durable and very presentable results may be achieved. 8. The Enemies of preserved specimens come under three groups: i) Pests: these include mites, moth larvae, beetles and their larvae, and psocids, which will feed on collections or on moulds formed on the specimens. These pests are discouraged by the use of paradichlorobenzene crystals which may be placed in cells or linen bags, or be mixed with molten paraffin wax evenly poured over the cork lining of store boxes. Dibutylphthalate repellent liquid may be squirted from a pipette along the joints of store boxes. ii) Moulds and fungi: species of Penicillium and Aspergillus will form on collections which are at all damp. Moulds may be inhibited or killed by the vapour of medicinal creosote applied to cottonwool pads. Only a small amount isrequired, otherwise celluloid stages etc. may become buckled. iii) Verdigris: where nickel plated brass pins are used, the silvery coating is frequently eaten into by certain dilute acids in the specimen: this allows the moisture in the atmosphere to attack the copper of the pin, forming a basic copper compound, verdigris. This appears as long green strands where the pin passes through the specimen. Verdigris will not form where stainless steel pins are used instead of brass. Damaged specimens may be cleaned by immersion in dilute ether. 9. Some actually used improvisations were shown: a petrol can cut up, soldered, and made into collecting boxes during the 1939-45 war; several methods for posting pinned mosquitoes, e.g., where the cork pinning sheet was fixed to the cork of a 3 in. x 1 in. glass tube and enclosed in it; and where a roll of celluloid plugged with corks in which specimens were pinned made a very strong postal package. A slide mountant was also shown of transparent plastic scrapings dissolved in chloroform.
L O N D O N S C H O O L OF H Y G I E N E A N D T R O P I C A L M E D I C I N E DEPARTMENT OF PARASITOLOGY
Professor P. C. C. G a r n h a m and Mr. R. Killiek-Kendrick: Plasmodium pinottii, a m a l a r i a parasite of the B r a z i l i a n toucan. P. pinottii is maintained in pigeons, which die of the infection in about a week.
If quinine is administered, the life of the bird is prolonged, and exoerythrocytic schizonts develop in the capillaries of the brain from the 21st to the 28th day of th~ disease. The mature schizonts occur in two forms, producing macro- and micro-merozoites respectively, with no intermediate stages. Such a phenomenon was thought to occur also in P. gaUinaceum, until Huff (1954, Exp. Parasit., 3, 433) questioned the theory by showing that there is a complete series connecting the two forms; now, the situation will have to be re-examined.
P r o f e s s o r P. C. C. G a r n h a m : D i s c o v e r y o f Besnoitia in the basilisk l i z a r d . Besnoitia spp. have been reported from cattle, rodents and other mammals. This is the first demonstration of the presence of these parasites in cold blooded animals. They were found in basilisk lizards in British Honduras. Free "spores" were seen in blood films and spleen smears, and typical cysts in the spleen, intestine, heart, skeletal muscle, lung and liver. Small collections of organisms were found in macrophage cells in the lung, which Dr. Jacobs suggests might be comparable to the proliferative ("pseudocyst") stage of Toxoplasma.
LABORATORY
MEETING
of the ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE held at the Royal Army M e d i c a l C o l l e g e , M i l l b a n k , S.W.,
on Thursday, 19th March, 1964 The President, CHARLES WILCOCKS, C.M.G., M.D., F.R.C.P., D.T.M. • H., in the Chair.
DEMONSTRATIONS
THE ROYAL ARMY MEDICAL COLLEGE DEPARTMENT OF PATHOLOGY
Colonel H. C. Jeffrey and Mr. R. M. Leach : Visual aids to teaching parasitology. Most text books on the subject contain a certain number of diagrammatic representations of the life cycle of various parasites, but only a certain number. As a visual aid to teaching, an attempt has been made to produce a systematic series of diagrams illustrating the protozoa and worms of medical importance. Charts used as museum exhibits and plates from these for issue to students were demonstrated; photomicrographs were included as an ancillary to the museum exhibit. DEPARTMENT OF ENTOMOLOGY
Mr. J. H. Grundy and Mr. N. R. H. Burgess: The preservation of arthropods for taxonomic and teaching purposes. The demonstration dealt with the practical preservation of arthropods, with special reference to those of medical importance, and was presented under nine headings: 1. A reminder that arthropods are composed of (a) a hard chitinous exoskeleton, and (b) soft interior parts, one or both of which may be required for study. 2. Protection from damage. The main methods were shown: i) Pinning, in collection boxes. ii) Immersion in spirit, or formalin mounts. iii) The removal of harmful interior parts. iv) Mounting on microscope slides. v) Mounting in transparent plastic. Each method was then dealt with in greater detail 3. Pinning. There are two reasons for pinning a specimen, (a) for taxonomic purposes, when speed of pinning, ease of examination, and correct label-data are all-important; (b) for teaching purposes, when the specimen is carefully set out to show definite features. 4. Immersion in spirit or formalin. Both hard and soft-bodied arthropods may be preserved in spirit (75-80 per cent. ethyl alcohol). 13 per cent. W/V gelatin may be used for sticking the specimen to an immersed mount. 2 per cent.-10 per cent. formalin liquid or its vapour preserves without clearing,
286
LABORATORY M E E T I N G
and tends to harden the specimen but not to distort it, and if not too strong will preserve the colour. The vapour technique is useful for anopheline mosquito eggs. Dried-out specimens may be soaked in 10 per cent. glycerin until suitably swollen. Methylated spirit (impure ethyl alcohol) should be avoided. 5. The removal of harmful interior parts. Often the specimen may deteriorate if its viscera are left intact. The soft body parts of larger specimens may be removed through a lateral slit between two abdominal segments. Moths may be emptied by slitting the ventral surface of the abdomen longitudinally. The cavity should then be filled with cottonwool soaked in preservative. 6. Mounting on slides for microscopic examination. Sclerotised specimens may first require maceration by boiling or steeping in 10 per cent. caustic potash solution before processing. All specimens must be dehydrated by passing through 70 per cent., 90 per cent. and absolute alcohol, cleared in xylene or cedar-wood oil, and mounted on the slide in resin such as DPX or Canada balsam. 7. Embedding in plastic. For demonstration purposes this is an excellent method. Several processes are available, and once the difficulties of "silvering" and the formation of air-bubbles have been overcome, durable and very presentable results may be achieved. 8. The Enemies of preserved specimens come under three groups: i) Pests: these include mites, moth larvae, beetles and their larvae, and psocids, which will feed on collections or on moulds formed on the specimens. These pests are discouraged by the use of paradichlorobenzene crystals which may be placed in cells or linen bags, or be mixed with molten paraffin wax evenly poured over the cork lining of store boxes. Dibutylphthalate repellent liquid may be squirted from a pipette along the joints of store boxes. ii) Moulds and fungi: species of Penicillium and Aspergillus will form on collections which are at all damp. Moulds may be inhibited or killed by the vapour of medicinal creosote applied to cottonwool pads. Only a small amount isrequired, otherwise celluloid stages etc. may become buckled. iii) Verdigris: where nickel plated brass pins are used, the silvery coating is frequently eaten into by certain dilute acids in the specimen: this allows the moisture in the atmosphere to attack the copper of the pin, forming a basic copper compound, verdigris. This appears as long green strands where the pin passes through the specimen. Verdigris will not form where stainless steel pins are used instead of brass. Damaged specimens may be cleaned by immersion in dilute ether. 9. Some actually used improvisations were shown: a petrol can cut up, soldered, and made into collecting boxes during the 1939-45 war; several methods for posting pinned mosquitoes, e.g., where the cork pinning sheet was fixed to the cork of a 3 in. x 1 in. glass tube and enclosed in it; and where a roll of celluloid plugged with corks in which specimens were pinned made a very strong postal package. A slide mountant was also shown of transparent plastic scrapings dissolved in chloroform.
L O N D O N S C H O O L OF H Y G I E N E A N D T R O P I C A L M E D I C I N E DEPARTMENT OF PARASITOLOGY
Professor P. C. C. G a r n h a m and Mr. R. Killiek-Kendrick: Plasmodium pinottii, a m a l a r i a parasite of the B r a z i l i a n toucan. P. pinottii is maintained in pigeons, which die of the infection in about a week.
If quinine is administered, the life of the bird is prolonged, and exoerythrocytic schizonts develop in the capillaries of the brain from the 21st to the 28th day of th~ disease. The mature schizonts occur in two forms, producing macro- and micro-merozoites respectively, with no intermediate stages. Such a phenomenon was thought to occur also in P. gaUinaceum, until Huff (1954, Exp. Parasit., 3, 433) questioned the theory by showing that there is a complete series connecting the two forms; now, the situation will have to be re-examined.
P r o f e s s o r P. C. C. G a r n h a m : D i s c o v e r y o f Besnoitia in the basilisk l i z a r d . Besnoitia spp. have been reported from cattle, rodents and other mammals. This is the first demonstration of the presence of these parasites in cold blooded animals. They were found in basilisk lizards in British Honduras. Free "spores" were seen in blood films and spleen smears, and typical cysts in the spleen, intestine, heart, skeletal muscle, lung and liver. Small collections of organisms were found in macrophage cells in the lung, which Dr. Jacobs suggests might be comparable to the proliferative ("pseudocyst") stage of Toxoplasma.