British Museum (Natural History). Instructions for Collectors: No. 12.—WORMS. Lettre “ oe, BU a \ 32756 s/ LONDON: 1915. PRICE THREEPENCE. (All rights reserved.) Specimens intended for the Natural History Branch of the British Museum should be sent to THE BRITISH MUSEUM (NATURAL HISTORY), — CROMWELL ROAD, LONDON, 8.W. All communications should be addressed to the DIRECTOR. NOTE.—Boxes of specimens so addressed are not opened by the Customs Officers at the Docks, but are forwarded to the Museum under seal. PRESENTED BY The Trustees or BRITISH MUSEUM. ——a | C Re ® ™ Instructions for Collecting Worms. INTRODUCTORY REMARKS. On COLLECTING. Tue term “ Worms,” as here used, embraces a great many animals which bear very little resemblance to the popular conception of a worm. Some little idea of their variety and of the situations in which they are most likely to be found may be gathered from the lists given below. It will be seen that the parasitic groups are A 2 and 3, C, D, E, and those mentioned on pp. 10-11. The majority are internal parasites of vertebrate animals; but their position in their hosts varies very greatly. Many live in the alimentary canal, but many occur in other cavities and organs of the body, eg. the heart, liver, lungs, kidneys, bladder, body-cavity, nasal cavities, or even in the blood-vessels, bronchial tubes, and among the muscles and connective tissue. Some of the Trematodes and Leeches are external parasites of fishes and other aquatic or amphibious animals, and may be found attached to their skin, especially on the fins, or on the gills and in the cavity of the mouth. Hence one must not be content merely to open the intestines of an animal and make a hasty examination there, but all parts of the animal ought to be inspected. Even the connective tissue between the skin and flesh often contains parasites. Filarice (Nematodes), for instance, often choose this situation, where they lie coiled up in capsules or “cysts’’; and the larval forms of various other parasites may be discovered in similar positions. When found in the connective tissue or in the peritoneum covering the internal organs and body-cavity, or in the walls of the stomach, etc., it is advisable to cut out and preserve the cyst with a small portion of the surrounding tissue. It is worth while to collect all parasites seen, since it is often only the most careful microscopic investigation that can determine & species, Some small Nematodes parasitic in the intestines, more especially of birds, are so fine that they are difficult to see unless some of the contents of the intestine or ceca are shaken up in salt solution or water, when the little hair-like creatures can be picked up with a needle, to which they adhere readily. For many 2 Nematodes it is a good plan to shake or stir up the intestinal matter in a vessel of weak salt solution, and allow it to stand, when the worms will sink to the bottom, and the dirty fluid can be poured off. Clean fluid may then be added, and the process repeated until the worms are clean and free from débris. In collecting Tape-worms the intestine of the host should be slit up with a pair of scissors (preferably blunt-ended) and the contents scraped out gently with the back of a knife or scalpel. The scraping ought to be so managed as to secure the heads of the worms, which may be attached more or less firmly to the lining of the intestine. If any are very firmly attached a small portion of the intestine may be cut out round them, and placed with the a b Cc d FIGURE 1. Portions of an adult Tapeworm, Taenia serrata, natural size. a, the head (scolex) and some of the young segments ; d, the most fully developed (gravid) segments, near the hinder end of the worm: 3%, e, portions from intermediate regions. [CESTODA.] worms in a dish of weak salt solution, when by gentle persuasion, by means of scalpel or needles, the worms may be made to loosen their hold. In general, however, it is best to place the mass of worms and ingested matter in a tall vessel of salt solution, any floating lumps being broken up, and the worms then washed by gentle “shaking, as directed on p. 22. On LABELLING. It is extremely important to label all specimens fully. The label bearing data as to the host in which a _ parasite occurred, its position in the host, the locality, date, and any other Siseamiions made at the time of collecting, is almost as valuable as the specimen itself; a specimen without its label is practically useless. These remarks, of course, apply to free-living forms as well as to parasites—notes as to the position in which they were found should always be given. The method of preservation used should also be stated. In addition to the labels giving such information, it is well to number the specimens, and to keep a note-book with full notes of each specimen or set of specimens opposite its number. In the case of parasites, the species of the host (if known) should always be given. If unknown, an accurate description of it, and if possible a drawing, will help greatly in the labour of determination. In some cases (e.g. small Fishes, Reptiles, Mammals, &c.) FIGURE 2. it would be well to preserve the ip pra \ ey eo br a host as well as_ the parasite, ing the hooks and two of the giving it a corresponding label and ‘Suckers. From a Ee ea number.* Labels should» be written clearly with a soft lead pencil on unsized paper, and placed in the preserving fluid with the specimens. Ordinary imk will not do for this purpose, as it will wash out. Certain kinds of waterproof Indian ink, however, are good, if allowed to dry thoroughly before being placed in the liquid. In some cases an additional label in ink may be placed outside the bottle. * Fishes, Amphibians, Reptiles, &c., are best preserved in spirit; in the case of Birds and Mammals the skins must be preserved, and the skulls of Mammals should be dried and labelled to correspond with the skins. (See “« Instructions for Collectors,’”’ Nos. 3, 2, 1.) 4 SCIENTIFIC AND POPULAR NAMES OF THE CHIEF GROUPS OF “ WORMS.” A. PLATYHELMINTHES (Flat-worms). Including :— 1. TuRBELLARIA (Planarians). 2. TREMATODA (F'lukes). 3. Crsropa (Tape-worms). B. NEMERTINEA (Long unsegmented worms, mostly marine). C. NEMATODA (Thread-worms or Round-worms). D. NEMATOMORPHA or GOR- DIACEA (Hair-worms). E. ACANTHOCEPHALA (Includ- ing Echinorhynchus). F. CHATOPODA (Bristle-worms). Including :— 1. OuicocHmtTa (Earth- worms and their allies). 2. PotycH@Ta (Marine bristle-worms). G. GEPHYRBEA. H. HIRUDINEA (Leeches). I. PHORONIDEA (Phoronis). J ENTEROPNEUSTA (Balano- glossus). Some of the distinguishing features of the groups mentioned above are outlined in the following : : BRIEF SYNOPSIS, OF 2s IGURE 5. ‘ Fasciola hepatica, the Liver-fluke CHIBF GROUPS OF WORMS. toler mad onlargal spouse 2> A. PLAT YHEUMIN DEES diameters. The veneuntnee ‘Ooimals of flattened shape, their behind the mouth. In some yiscera not contained in a “ body- related species it ag amnat? cavity.” The alimentary canal, which is either simple or branched, has no anus or vent. Mostly aquatic or parasitic ; exceptionally lving on land in moist situations. These include :— (1) TurBEeLLaRiA (see Fig. 14). Usually with soft, oval or elongate, unsegmented body, covered with minute cilia, or vibratile “hairs.” Mouth often in centre of ventral surface, or even further 5 back, with protrusible tubular pharynx. Alimentary canal simple or much branched. Chiefly aquatic (marine and fresh-water) ; some terrestrial, living in damp places, generally under logs or stones, (2) Tremaropa (e.g. Fasciola hepatica, the well-known Liver- fluke of the Sheep.—See Fig. 3). Internal or external parasites ; usually of oval shape. Unsegmented. With suckers and some- times horny hooks for attachment to host. Alimentary canal bifurcate, sometimes branched. (3) Cestopa (eg. Tenia of various species, parasitic in man, dog, etc.—See Figs. 1 and 2). HKlongate parasitic creatures, of ribbon-like form, with a rounded “ head”’ bearing suckers or hooks and suckers, followed by a series of segments Increasing in size posteriorly. The hinder segments are generally full of eggs. The larval or young forms (see Fig. 4) of these worms are found as_ bladder-like ‘cysts’ of various sizes embedded in the organs (e.g. the liver) of some animal which is liable to be eaten by their final host. Alimentary canal absent in all stages. { Note—In collecting Tapeworms great care must be faieen to find the head (Figs. 1 and 2), which is very minute and easily broken off, as it may be attached firmly to the host’s tissues. (See p. 2.) | B. NEMERTINEA (see Fig. 15). (Such as Linews of the sea- shore.) Generally very elongate, unsegmented, FIGURE 4. more or less flattened worms, capable A “Bladder - worm,” Cysticerevs of oreat extension and contraction. tenuicollis, enlarged about 12 : 5 ' : diameters. This is the larval Sometimes several yards in length Teworas of ees and te when extended. Possessing a remark- found in the lining of the body- able protrusible proboscis, in a sheath eavities of Sheep, Cattle, etc. [CESTODA.] lying above the alimentary canal. , Mostly marine, rarely on land or ip fresh water, living under stones, etc. . C. NEMATODA (see Fig. 5). (Such as Ascaris lumbricoides, the largest round-worm found in man. Mostly parasitic worms of elongated form, with flexible but not extensible cylindrical, unsegmented body. (There are many free- living Nematodes, but these are mostly very minute, living in 6 water or in the soil; some are also para- sites of plants.) Tail of male nearly always curved downwards, and proyided either with a membranous expansion, or with curved horny spicules, or both. D. NEMATOMORPHA (Gordiacea) (see Fig. 13). Extremely long, thin, wiry-looking worms, traditionally compared to ‘animated horse-hairs.” Parasitic at first in the body-cavity of Insects; after- wards emerging from them to lead a free life, usually in fresh water. EK. ACAN- THOCEKPHALA (Hchinorhynchus, etc.—See Fig. 6). Unsegmented, cylindrical worms, more or less resembling Nematodes, but having a_ pyro- bosecis armed with many hooks (see Fig. 7), with which theyattach themselves to the tissues of the host—usually to FIGURE 6. ies : Gigantorhynchus gigas, natural the lining of the © size,of the Pig. This isa male Pye ees Z specimen, the female being stomach or intes- considerably larger. [ACAN- tines. Larvee in THOCEPHALA. ] cysts, usually in aquatic insects or crustacea, sometimes in fishes, preyed upon by the final host. Alimentary canal absent. F. CHAATOPODA. Body divided into a number of “rings” or The large Nematode, Segments,each of which Ascaris megalocephala, 3.4 aces a sea Fateral/e dine OF the 218. DrOvided syriun certain intestines of the Horse. groups or rows of This specimen shows the } istles curvature of the tail, UFIStLeS. characteristic of the male (1) OLIGOCHETA sex of many Nematodes. ah ‘ i [NEMATODA.] (Karthworms, é&c.). FIGURE 5. fi Chiefly living below the surface of the soil (often crawling on the surface after rain, &¢c.), or among rotting timber or other vegetation. Others, of small size, in fresh water, or in mud at the bottom of ponds and streams. (2) Potycuamra (see Figs. 8, 9 and 16). Each segment bears generally a fin-like expansion on each side, from which bristles project fanwise. These expansions may be wanting, or confined to the front end of the body, in forms which inhabit tubes. Marive. Some live under stones, or buried in sand at low-water mark ; many in tubes of their own construction, at- tached to _ solid objects under water. The tubes are of very -yarlous appearance, some being hard and chalky, others covered with sand- grains, bits of shells, FIGURE 7. The “head” end of a typical Echinorhynchus, greatly magnified, showing the eylindrical proboscis covered with hooks. [ACANTHOCEPHALA. |] seaweed, &c., others quite transparent. FIGURE 8. An example of a free-living Polychete worm, Lepidasthenia elegans, slightly en- larged, showing the characteristic paired appendages. {CH ®- TOPODA.] Many of the tube-living worms (see Fig. 9) have a flower-like crown of feathery “ gills ”’ on the head, which can be protruded from the opening of the tube. A few Polychetes lead an entirely free- swimming life at the surface. Certain forms, rather unlike the ordinary conception of a ‘‘ worm,” are included in this group (Polycheta)—such as the Poly- noids, some of which are of woodlouse- like shape and appearance; and Aphrodite (“ Sea-mouse ”) and its relatives, which are thick-set animals covered with strong bristles and beautifully iridescent hairs (see Fig. 16). G. GEPHYREA (see Fig. 10). Marine forms, usually with a simple distended sac-like body, which shows no external traces of seg- mentation. Sometimes a crown of short ten- tacles may be seen expanded at oneend. The viscera are enclosed in a spacious body-cavity. 8 H. HIRUDINEA. (Leeches.) Worm-like animals, narrower at the head end than at the tail, and having a rounded sucker at each end. Ex- ternal, temporary parasites mostly found on vertebrates, sometimes entering the mouth or nose; living in water (either fresh or salt) or on land in moist places, sometimes climbing on to low bushes, &e. I. PHORONIDEA (see Fig. 11). Unsegmented worms living in tubes, commonly found associated in large numbers. Tube membranous, some- times covered with mud and other foreign FIGURE 9. substances. One of the tube-forming Poly- cheetes, Sabella bombyx, slightly The mouth enlarged. The specimen has is in the been removed trom its tube, and shuws the crown of tentacles. middle of a [CHATOPODA.] terminal crown of tentacles, arranged on a more or less obvious horseshoe-like plan. Alimentary canal U-shaped, the intestine running up by the side of the cso- phagus and opening near the mouth. The blood is red, with conspicuous.red corpuscles. Marine. May be found attached to solid objects in shallow water, or inhabiting tunnels bored in calcareous rocks, shells, &e. J. ENTEROPNEUSTA (see Fig. 17). Soft-bodied worms which may have a strong odour of iodoform. The front end has the form of a conical or cylindrical ‘proboscis,’ which 1s followed by a short region termed the “collar,” encircling the mouth. The rest of the animal, unsegmented and often of considerable length, is composed of several distinct regions, the first of which is often produced into large Jateral flaps. A double series of pores FIGURE 10. occurs on the back or at the sides of Echiurus vulgaris, the “ Spoon- this * br: shial’’ regi worm,” slightly enlarged. 118 IvVaNneN1a region. (GEPHYREA.] wv) Marine; to be looked for on sandy shores at extreme low tide-mark. [ Note.—The body is so easily injured that great care is required to obtain complete specimens ; but they are worth preserving if the front half can be obtained in good condition. | FIGURE 1l. A portion of a crowded colony of Phoronis, seen from above and slightly enlarged. The tentacles of the individual worms are not fully expanded. [PHORONIDEA.] 10 OTHER PARASITES WHICH ARE LIABLE TO BE CONFUSED WITH WORMS. When searching, by dissection or otherwise, for parasitic worms, it is not improbable that various other parasites of more or less worm-like appearance will be found. The importance of collecting everything of this kind, especially if there is any question as to its identity, can- not be too strongly emphasised. Hence a brief reference is made here to some of the parasitic creatures which bear some re- semblance to worms. Certain parasitic Arthropods, in which the limbs are degraded or entirely lost, re- semble worms’ in general appearance and are commonly confused with them. Among these may be mentioned the insect larve or MAGGOTs, which make their way into the mouth, nostrils or stomach, or under the skin of Mammals, FIGURE 12. including Man. These A large Pentastomid, Pentastoma polyzonum, found in ar , o ¥ the Puff-adder ; natural size. [PENTASTOMIDA.] Bre the lary al stages of certain Khes, and most of them attack Ungulates—(Horses, Cattle, &c.). Some, like the ‘“‘Warble’”’ flies, raise tumours of considerable size under the skin, in which the larve live and feed, and from which they eventually emerge. The so-called “Screw-worm”’ similarly attacks Man. In other cases, like the Gastrophilus of the Horse, the eggs swallowed by the animal hatch in its stomach, and the larve thereupon attach themselves as true internal parasites to the stomach-wall. They are true maggots and have the usual shape of such creatures, tapering in front and broadened behind, and armed with various rings of spines encircling the body. 11 The Pentastomipa (Pentastoma or Linguatula, &c.) are curious worm-like creatures (see Fig. 12), of doubtful affinities, usually considered to belong to the Arachnid group, 7.e. to be allied to Spiders and Mites. But in general appearance they resemble worms, having a comparatively soft elongated body, without limbs, and the head being armed with two pairs of sharp, horny hooks. In their immature stages they may occur enclosed in cysts among the tissues of almost any part of the body, and in almost any Mammal or Bird, or living free in its various cavities, more especially the general body-cavity. The adult forms usually occur in Snakes or other carnivorous Reptiles, and mostly take up a position in the lungs of their host. Parasitic CrusTAcEA of various sorts may also occur. They are found mostly on Fishes and Whales, or on other Crustacea, on Mollusca and other aquatic invertebrates; they may assume a very different appearance from a typical Crustacean. The body may be reduced to a mere soft sac, without appendages, or with only vestiges of them. Sometimes there is a pair of elongated bags of eggs attached to the body, and looking almost like a pair of legs, or like a pair of long strings. These parasites are very commonly found, for example, clinging to the gills of Fishes. Any of the above-mentioned creatures may be safely treated by ordinary methods of killing, such as corrosive sublimate, and preserved in alcohol. Insect-larvee may be killed by immersion for a few seconds only in boiling water, and then placed in weak spirit, in which they are to remain till hardened (two or three weeks). They can finally be transferred to stronger spirit. HABITAT OF WORMS. The following is intended as a rough indication of what may be looked for in a particular kind of locality, though it is impossible to give a complete guide :— (i) Inland localities. (a) Parasitic worms.—Any kind of mammal, bird, reptile, amphibian, or fresh water fish may be found to harbour parasites of many kinds. NrmaropEes, TREMATODES, CESTODES, and ACANTHOCEPHALA occur in all these groups of animals, either in their larval or adult stages. As a rule, dissection is the only method of obtaining them, a special search being made in the alimentary canal. But some of the TREmMATopEs attach themselves to the skin of Fishes. LEECHES are sometimes found adhering to animals (e.g. Frogs) which have recently been in or near water. Reptiles, especially Snakes, are some- times found to harbour Pentastoma (or Porocephalus, see 12 Fig. 12), a wormlike creature of doubtful affinities, which usually takes up a position in the lungs of its host. Molluses frequently contain the immature stages of TREMATODES. Insects, such as beetles, grasshoppers and flies, often harbour the immature stages of ‘“‘ Harr-worms ”’ (Gordius and a Nematode worm of somewhat similar appearance, Mermis). These live coiled up in the body-cavity of the host. It is to be desired that the adult stages of these worms should be obtained, and if possible some knowledge acquired as to their eggs and larvee, and the mode of infection of new hosts by the larve. It is recommended, therefore, that when these worms are seen emerging from insects, or found crawling about, an attempt should be made to keep them alive for a time in damp earth, water, etc., in the hope of learning more of their life-history, as the mature stage is usually only reached after leaving the insect host. Some small Nemaropss are also parasitic in insects, but usually require the microscope for their detection. (b) Fresh-water worms. TURBELLARIA are commonly found in any standing water, crawling on water-weeds or on the bottom, or even upside down on the surface-film. Since many of them are carnivorous, and have a liking for decaying matter, they may sometimes be conveniently caught by sinking a pot of dead earthworms in a pond or ditch, drawing it up after a day or two and collecting the Planarians that have been attracted by it. Fresh-water fishes,* amphibians, etc., as mentioned above, should be examined for parasites. Some of the Insects and Crustacea also harbour NematopEs and NEMATOMORPHA. NEMERTINEA are rare in fresh water, but may be found occasionally. Many Nematopa occur in fresh water, in the mud at the bottom, or among plants, but these are chiefly of microscopic size. NematomorpHa habitually pass their adult stage of existence in fresh water, sometimes in standing water, sometimes in quite rapid streams. They may often be * Fishes frequently harbour parasitic Crustacea, especially on their gills. These parasites should, if possible, be preserved, with a note of the position in which they were found, and information about the ‘‘ host.” 13 found tangled together (see Fig. 13) in great numbers, and twisted round weeds, ete., on which their eggs are laid. The fresh-water CHa#ropops are practically all OuigocH#TA, chiefly small forms living in the mud round the roots of aquatic plants, or plants growing near the edge of the water. Samples of mud scraped up from the bottom of any pond, ditch or stream may contain many of these worms. PotycHmTA are exceptionally found in fresh water, and these are most likely to be obtained by dredging in large lakes. In brackish water, on the other hand, PoLtycHaTES are numerous, living in the mud, under stones, ete., as do those of the sea-shore. LercuHesare often abundant in ponds, ditches and streams, where they swim with an un- dulating movement, or attach themselves to solid objects, and walk like “looper” caterpillars. (c) Land forms. A number of PLANARIANS (see Fig. 14) crawl about in moist places, or may be found among wet vegetation, such as mosses, or under stones and decaying logs. Some of them suggest slugs, othersare more elongated, but they are to be recognised by their extremely soft and slimy bodies, having no tough skin like molluses or other worms. FIGURE 13. Land Planarians are chiefly, A tangled mass of Gordius aquaticus, but not exclusively, tropical. natural size. [NEMATOMORPBA. } NEMERTINES are rare on land, but one or two have been recorded. They are likely to be found in wet places, under stones, «ce. NemMATopDEs of minute size are abundant in soil and among roots of grasses and other plants. Some are parasitic on plants, and form various kinds of galls and other diseased growths on grasses, &c. Mermis, as already mentioned, is at first parasitic in Insects, but after emergence from them is often found crawling about on the soil or on plants, sometimes in large numbers, especially after rain. It resembles Gordiws in its very wiry appearance. 14 Gorpius and its relatives are also sometimes found crawling on the ground, among dead leaves, &c., especially — [EERE FIGURE l4. A land Planarian, Geoplana fryi, natural size. [TURBEL- LARIA. J in wet forest districts. OxicocHm®Ta (Harthworms and their allies) are abundant every- where in damp soil, under stones, or among rotting vegetation, some- times even in the accumulated water and vegetable débris in hollows in the branches of trees. Many species are very small and inconspicuous. ‘The earthworms may be obtained by digging, but many of them emerge from, their burrows after rain, and may then be collected, while some species habitually lie with part of their bodies out of their holes at night, except in frosty weather or bright moonlight. They may be caught with forceps when found in this position, but are very sensitive to light and vibration, and dart back into their burrows with astonishing speed if disturbed. Land-leeches occur chiefly in the tropics, lying in wait for their vertebrate prey, either on the ground or on forest undergrowth, always provided that the situation is moist. They often attack man and domestic animals passing through such places. (ii) Marine localities.* (a) Parasites. As on land and in fresh water, all kinds of mammals, birds, reptiles, and fishes may be examined for PLATYHELMINTHES, NemaropA, ACANTHOCEPHALA, and HirupINnEA (see Synopsis). The last-named are not always attached to their hosts (usually fishes),. but may be found free in the water, or on stones and weeds. *Much valuable information about the collection and preservation of Marine organisms will be found in a most useful book, ‘‘ Science of the Sea,” issued by the Challenger Society. London: John Murray. 6s. (0) 15 It is also worth while to examine the gills and body- cavities of crustacea (crabs, &c.), for larval NemaropEs and TREMATODES, and the various cavities of shell-fish for larval TremMatopEs, and their empty shells for NEMERTINES, &c. Free-living forms in the water or on shore. PLANARIANS and NEMER- TINES (see Fig. 15) are numerous, being generally found under stones between tide-marks or dredged in shallow water. The NemEr- TINES are often in tangled masses. Hmpty shells should be examined for them, and also sea-weeds. Some Nemertines & make gelatinous tubes, or even burrows in the mud. Pelagic Nemertines, swimming freely at the surface, are very rare. Free-living NEMATODES are known in the sea, but many more no doubt remain to be discovered. A single NrmatomorrH, Nectonema, has sometimes been caught by the tow-net swim- ming at the surface. PotycHmTa (see Figs. 8, 9, and 16, and p. 7) are very abundant. Unless dredging operations can be carried out, they are best obtained by shore- collecting at low tide. The burrowing kinds often betray their presence by leaving es “casts”? of sand which has FIGURE Li. passed through the alimentary Nemertine, Hunemertes gracilis, canal on the surface of the ily twisted position. [NEMER- ground, and may be obtained Cian! by digging in such spots. GEPHYREA (see Fig. 10) will, as a rule, only be obtained by dredging, but a few may be found between tide-marks, burying themselves in mud, or forming tubes, or tubular excavations in soft rock. Some live as ‘‘ com- mensals ” with corals or sea-urchins, or in empty shells. 16 OLIGOCHETA are rare on the shore, but a few forms have been found among seaweed above high-water mark, and some small species are partly marine. For LrencHes, cf. i (a), p. 14. For PHORONIDEA, see p. 8. IJNTEROPNEUSTA (see Fig. 17) may be looked for on sandy shores at extreme low-tide mark. They FIGURE 16. Hermione hystrix, one of the spiny Polychetes. reluted to the “sea- mouse”; slightly enlarged. [CH#- TOPODA. | form large sand-casts, resembling those of Polychetes, and they may be found by digging in the neighbourhood: of these casts. They must be handled with great care (see p. 9). LIST OF USEFUL CHEMICAL REAGENTS. Acetic ACID, GLACIAL (see “ Herries Sublimate’”’). Only a very small quantity of this will be required. Its use is to counteract the shrinkage of tissues, caused by solutions containing corrosive sublimate. A few drops added to the solutions are sufficient Anconon. The best is “pure spirit,’ which can often be obtained more easily abroad than in England, where the heavy duty makes its price almost prohibitive. For export only it may be obtained from English firms at about 2s, 6d. per gallon, or 10s. for a 10-gallon drum (retur nable). The strength of the spirit as purchased should be about 90 per cent. For most purposes a strength of 70 per cent. is sufficient for use; this is obtained by adding 70 volumes of the 90 per cent. spirit to 20 volumes of water. Similarly, a 50 per cent. solution is obtained by diluting 50 volumes of 90 per cent. spit with 40 volumes of water Ordinary “‘ methylated” spirit can be used for some animals, but cannot be diluted, as a white precipitate is formed on addition of water. For delicate marine animals it is quite useless. What is known as Industrial methylated spirit is, however, suitable for preserving specimens. In. England it can only be obtained by special permit from the Customs authorities, and only in considerable quantities. Sudden changes in the strength of this reagent should be avoided. Thus if it be desired to transfer specimens from corrosive sublimate to alcohol, they should be placed, after washing with water, in a weak solution of alcohol before being transferred to stronger spirit. Cutorat Hyprarr (Crystals), used for ee es narecotizing certain kinds of worms (e.4. auisseeg ape eatl Nemertines) before killing them, so as to diameters. [ENTERO- cause them to die expanded. A few of the . crystals may be added to the sea-water, or the animals may be placed in a 1 per cent. solution of the substance made up in sea water. Further remarks about narcotizing are made on page 21. 18 Corrosive SuBpuimate (HgCl,). A saturated solution of this exceedingly poisonous substance in fresh water (or sea-water for marine animals) is a very useful killing and preserving fluid. Its action is extremely rapid, and is made even more so by heating. The solution is best made by putting more of the crystals in a vessel of water than will dissolve; but as solution takes place slowly the mixture should be made some hours before it is likely to be wanted. As the solution is used more water can be added at intervals. So long as some of the crystals remain undissolved the solution will remain “saturated.” For delicate specimens which are not calcareous it is useful to add a few drops of glacial acetic acid to the solution. Steel instruments should not be brought into contact with solutions of corrosive sublimate. Objects hardened with corrosive sublimate should be soaked for some hours in, water or, better, in iodised alcohol (alcohol 70 per cent. 100 parts; tincture of iodine 2°5 parts) before being placed in alcohol for keeping. Otherwise the sublimate may crystallise out in the tissues, and spoil the specimens, when subsequently mounted for the microscope. N.B.—The saturated solution of sublimate looks exactly like plain water, and the bottle containing it should be labelled ‘“« Poison.” FormMAuiIn. The commercial formalin as bought is a solution of the gaseous compound formaldehyde in water. Its strength is about 40 per cent. For use it may be diluted with several times its bulk of water; hence it is very convenient for certain purposes, owing to its portability. It is also much cheaper than spirit. The strength most convenient for preserving animals is a 10 per cent. solution of formalin (or, which is the same thing, a 4 per cent. solution of formaldehyde). This is made by diluting one volume of commercial formalin with 9 volumes of water. After haying been preserved in this solution specimens may be kept in a weaker one—say 5 per cent. (5 parts commercial formalin to 95 of water). Note.—As formalin does not give good results with many kinds of animals, some caution must be used in employing it. It is advisable not to preserve all the specimens of a kind in it, but to keep some in alcohol. Further, formalin contains a certain amount of acid, which is injurious to animals with calcareous structures, such as worms with calcareous tubes, or jaws in which the hardening matter is calcareous. Hence Polycheetes should generally be preserved in alcohol rather than in formalin. For marine animals the formalin may be diluted with sea-water instead of with fresh water—an advantage which this reagent has over alcohol. N.B.—Formalin should not be used for preserving Nematodes, as it is liable to cause the specimens to burst. ae GLYCcERINE. This may be used, with special precautions, to preserve the natural colour of an animal. But as this reagent produces strong shrinkage in the tissues, it is essential to transfer the specimen into it gradually. This can be done by using successively stronger solutions of glycerine. The specimen may first be placed in a 10 per cent. solution of formalin containing a smal/ quantity of glycerine. After a suitable interval (perhaps half an hour) the specimen, now hardened by the formalin, may be placed in a stronger solution of glycerine in water; and this process must be repeated each time with an increasing strength of glycerine. If the colour is not too fugitive, the specimen may be hardened in alcohol for a short time, transferred to a dilute solution of glycerine in alcohol, and then left in an open dish for the glycerine to concentrate itself by evaporation. In a hot, dry climate a formalin solution of glycerine might be allowed to concentrate itself in the same way. Whatever method is adopted, the specimen should be brought into glycerine of full strength before the colour is lost. TopINE, TINCTURE OF. A small quantity of this will be useful (see “ Corrosive Sublimate ”’). Soptum CHLORIDE (common salt). A weak solution of this substance should always be kept ready to hand for the preliminary washing of parasitic worms. 1 gramme of salt dissolved in 99 cubic centimetres of water will give the required strength (or about 1 oz. in 5 pints). APPARATUS. A few dissecting instruments will be required by those who intend to look for parasites. They need not be many or costly, but the following would be indispensable :— Forceps.—Two or three pairs of various sizes, including one large and strong pair, and one with slender points. Scissors.—A large strong pair for cutting open animals (some- times better than a knife). One or two pairs of smaller dissecting scissors. Scalpels.—Three or four of different sizes and shapes. Needles.—A few of different sizes mounted in wooden handles. Camel's hair brushes.—One or two. Glass vessels.—Specimen tubes, with corks, of various sizes, will be required, and can be obtained cheaply from dealers in natural history requisites. 20 For larger specimens any kind of wide-mouthed bottles with sound corks or stoppers will do, but perhaps the best are those with screwed-on metal lids and rubber rings. Tins, closed by soldering, may be used for specimens in spirit. They should under no circumstances be employed for specimens in formalin, which corrodes the metal. Glass ptpettes—Fitted with rubber caps, are useful for picking up and transferring small specimens. ‘“ Dipping tubes,” or straight pieces of glass tubing, with their cut edges rounded by placing for a few seconds in a flame, are useful for similar purposes, especially for capturing small creatures contained in deep vessels of water. (The method of using a dipping tube is as follows: close one end with a finger, and bring the other end down into the water until close to the specimen. Then remove the finger, when water will rush up the tube, carrying the specimen with it. Replace the finger over the upper end of the tube, and withdraw the tube from the vessel.) Test-tubes.—A few of these will be useful in cleaning speci- mens, as described below. Dishes.—Shallow glass or porcelain dishes may be used for containing the fluids in which specimens are to be killed or washed, but an ordinary saucer, plate, or other such vessel will serve most purposes equally well. Hnamelled iron vessels are particularly useful when travelling—e.g. for heating the alcohol with which Nematodes are to be preserved. For this purpose they need only be wide enough to allow the specimens to be straightened out, and should not be too shallow, in order to lessen the possibility of the spirit being set on fire. SPECIAL METHODS FOR THE TREATMENT OF SPECIMENS. CLEANING, Having collected the specimens, the first thing to do is to clean them from foreign matter, which may be difficult to remove after they have been killed. All parasitic worms may be washed by shaking in the 1 per cent. salt solution. Nematodes and Trematodes can be shaken up vigorously in a test-tube without damage, but care must be taken with Tapeworms, which are very fragile. In some cases, such as Cheetopods, a soft camel’s hair brush may be used to remove particles of sand, ete., which cling to their bodies, especially if they 21 are very bristly. Water alone may be used for the washing if salt solution cannot be had, but specimens, especially Nematodes, must not be left long in it. Kinurne. After washing, the specimens must be killed with some reagent which leaves them in a suitable condition for subsequent examination—7.e. which “fixes” their tissues in a condition as nearly as possible approaching that of life. The+ most generally useful reagent is Corrosive Sublimate (see list of reagents). The effect is more instantaneous if the solution be heated, and the animals then plunged into it; but it may also very well be used cold. . An hour or two in this solution will be long enough for the largest specimens; for smaller ones a few minutes will suffice. For necessary treatment after corrosive sublimate, see this heading in the list of reagents. Formalin (10 per cent. solution) is also good for most parasites, except for Nematodes, but it cannot be recommended for the majority of marine worms; specimens left in this reagent for 24 hours can then be transferred to the weaker solution, or to alcohol. For killing parasitic worms, see also other methods given below. Narcotizine. As it is desirable to get the animals to die in an expanded condition, it is useful in some cases to anzsthetize them before killing. This is especially the case with Nemertines, and worms which live in tubes. They may be placed for from 6 to 12 hours in a solution of chloral hydrate in sea water (1 per cent.), or a few of the crystals may be simply added to the sea water in which they lie. When fully extended and showing no sign of sensation, they may be preserved with formalin or alcohol. Cocaine may be used with advantage instead of chloral hydrate. Another method is to place the worms in a shallow dish of sea water, and gently pour about ,, part of strong alcohol on the surface. This will slowly mix with the water and anesthetize the animals gradually. They can then be transferred to alcohol to harden, and preserved in it. Some of the free-living Polycheta must be treated in this way, or they will break themselves into a number of little pieces when a killing reagent is added. Note.—In tropical countries narcotizing is sometimes found difficult, owing to the fact that decomposition sets -in before the aneesthetic has had time to take effect. In such cases the alcohol method will be found to give better results than chloral hydrate. 22 SpeciAL Metuops For Parasitic Worms. Nematodes. After washing thoroughly as above directed,* kill by plunging each separately into a quantity of hot 70 per cent. alcohol. A temperature of 50°-60° C. is high enough. N.B.—Care must be used, as the vapour given off is highly inflammable.) The advantage of this method is that the worms should die in a straight position. After being killed in this way the worms are to be stored in fresh 70% alcohol (never in formalin). Trematodes. After the first shaking up in salt solution the dirty liquid is poured off, and a small quantity of fresh salt solution introduced. The worms are shaken vigorously in this, and an equal quantity of saturated solution of corrosive sublimate added quickly, the shaking being then continued for several minutes. This should kill and fix the worms in an extended position. They may be left in the fluid for several days, or transferred at once to water, in which, in any case, they should be washed for about twelve hours. After this they may be stored in 70% alcohol as usual. [Formalin may be used instead of corrosive sublimate, in a solution of 10% strength, followed by a weaker solution (about 3%) - as a storing fluid. ] Cestodes. Wash gently in salt solution ;f then fix in a solution containing equal parts of saturated corrosive sublimate and 70% alcohol, to which add a few drops of glacial acetic acid—the whole heated to about 50°C. The Tapeworms should remain in the fixing fluid till it is cold; they are then washed gently in running water for twelve hours, and stored in 70% alcohol. PACKING OF SPECIMENS. The separate small bottles containing worms should be filled as full as possible with the preserving fluid (alcohol or formalin), and damage to the specimens by shaking prevented as far as possible by a light packing of cotton-wool or tissue-paper. Care mau, “however, be taken to insert the wool in such a way that the * In the case of Nematodes with a mouth-capsule, a thorough shaking-up is necessary to remove parts of the tissues of the host, or other débris, from it. + When the Tape-worms are long and tend to become much tangled they should be washed in a shallow dish, and picked out one by one on a splinter of wood (not a metal instrument) and transferred to the fixing fluid. 23 small specimens cannot get entangled init. The liquid must on no account be allowed to dry up. When they are to be forwarded all the small bottles should be placed in a large, wide-mouthed bottle with a good sound cork or a glass stopper, or better still, a screwed-on lid with rubber ring. In this the small bottles should be packed lightly with wool, horse-hair, or soft paper screwed up into loose balls, and the whole filled up with the fluid. It is a good plan, if the large bottle or jar is well stoppered and protected against breakage, to leave the small bottles without corks, simply plugging their necks with a pellet of cotton-wool or a stopper of pith—e.g. elder-pith—and then stand them upside-down in the large jar. There is then much less risk of the fluid in the small bottles getting dried up, so long as there is plenty in the big jar. Air-bubbles in the small bottles should be avoided as far as possible. N.B.—Small bottles packed in this way, immersed in fluid, must have their labels imside, and not gummed on the outside. These ‘“ Instructions” have been drawn up by Mr. H. A. Baylis, Assistant, Department of Zoology. Srpney F. Harmer, Keeper of Zoology. Br:tisH Musrum (NaturAL History), CROMWELL Roap, Lonpon, 8.W. February, 1915. LONDON : PRINTED BY WILLIAM CLOWES AND SONS, LIMITED, DUKE STREET, STAMFORD STREET, S.E., AND GREAT WINDMILL STREET, W.