Text-Book of Embryology 2-11 (1919)
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Kerr JG. Text-Book of Embryology II (1919) MacMillan and Co., London.
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Chapter XI Hints Regarding the Practical Study of the Embryology of the Various Types of Lower Vertebrates
Amphioxus
The interest and importance of Amphioxus to the student of Vertebrate morphology are due to the fact of its position near the base of the Vertebrate phylum. It is true that in its adult structure Amphvlomus is intensely specialized in correlation with its burrowing habit. Further, it is necessary to recognize that a burrowing like a pelagic mode of life, in which the environmental conditions are comparatively uniform, is likely to lead to a kind of fixing of the organization which will be fatal to its adaptability to new sets of conditions and consequently to its capacity for evolving along new lines. We must therefore regard it as improbable that the Vertebrata passed through an ancestral condition of specialization for a burrowing habit and the specialized features of the later stages of the life history of Amphioxus cease on that account to have a phylogenetic interest. The main interest to the Vertebrate morphologist lies therefore in the earlier stages before the specialization of the adult has developed——in such features as segmentation, gastrulation and the origin of the main systems of organs. And the interest of these stages is heightened by the fact that food yolk-— that potent disturbing factor—is present to a far smaller extent in the egg of Ampiaxioxus than in that of any other of the lower Vertebrates.
Unfortunately the known localities in which fresh ernbryological material of Amphioxus can be obtained in abundance are still few, and in most laboratories recourse must be had to preserved material purchased from supply stations such as the Naples aquarium.
The best locality so far known for obtaining developmental stages of Amphiomus is the pantano or shallow lagoon at Faro near Messina. Here the spawning takes place each evening, when conditions are favourable, during the summer months from April to July. The eggs pass to the exterior through the atriopore. If in a dish on board a boat the eggs are liable by its movements. to become distributed through the water and they are then apt to become drawn by the inspiratory current in amongst the buccal cirri. When the
558 CH. xx PRACTICAL HINTS 559
Amphioams becomes inconvenienced by such entangled eggs amongst the cirri it is able suddenly to reverse the respiratory current so as to clear them away, and in this Way there is produced a misleading appearance as if the eggs were being laid through the mouth. The first meiotic division has been completed before oviposition while the second is in the spindle stage at this period. Fertilization probably takes place immediately, spermatozoa being disseminated through the water.
It is best (Cerfontaine, 1906-7) to bring the adults into the laboratory and wait until they spawn which operation may be considerably delayed. To a dish of pure ‘sea-water is added a little sea-water containing sperm then the eggs, collected with a pipette as soon as extruded, are added.
Batches of eggs are fixed periodically, preferably in strong Flemming’s solution or Hermann’s solution. After dehydration they are placed in a mixture of 2 parts clove oil and 1 part collodion in which they may be kept indefinitely. For examination whole the egg or embryo is placed on a slide or|coverslip in a drop of the clove-oil-collodion. After the specimen has been arranged in .the desired position by means of needles a drop of chloroform is applied in order to cause the collodion to solidify. The whole is then cleared with cedar oil and mounted in canada balsam. For the preparation of sections the procedure is similar, only in this case the slide or coverslip should be coated with paraffin as a preliminary to allow the collodion block to become detached, and the latter should be embedded in paraffin. ,
PETROMYZON.-The various species of Lamprey make their way up streams to suitable gravelly spots for spawning in the spring or early summer (April, May, in the northern hemisphere). Material
for emhryological study is best got by “stripping ” the ripe males and females 7}.e. by passing the hand back along the body with gentle
pressure so as to force out the eggs or sperm. The gametes from the male and female are collected separately in two small dishes: they are then mixed together, stirred gently with a feather, and water added. This “ dry ” method gives a smaller proportion of unfertilized eggs than when the eggs are received from the fish directly into water (Herfort, 1901). As fixing agent the ordinary corrosive sublimate and acetic acid is quite satisfactory.
MYxINOIDs.——-The only Myxinoid eggs that have been obtained in any numbers are those of Bdellnstoma which are dredged near Monterey, California, on shelly and gravelly bottom at a mean depth of about 12 fathoms (Bashford Dean, 1899). Much still remains to be done in working out the details of their development but it is clear that this is of a highly peculiar and specialized type.
ELASMOBRANCHI1.-—The eggs are fertilized in the upper part of the oviduct. They may traverse the oviduct comparatively rapidly and be laid as in Birds at an early stage of development [0’h'£mae'ra, Scylliidae, Castration, Rain] or they may remain in the oviduct for a prolonged 560 EMBRYOLOGY or THE LOWER VERTEBRATES
FIG. 247.—-Blastoderm of Torpedo with meulnllary folds x 18). (After Ziegler, 1892.)
A, stage four (b'c.'unmon, 1911); B, stage six; 0, stage ten. The rounded projection near the anterior edge of the blastoder-m is the bulging I‘00f of the so-glm-IlL:1l.inlI (::|\'il.,\-'. In (I the bl()0«‘l-islamls form :1. row of (‘HlI.\‘pl(.'llIJll.*~‘. I-h-\'.'tt-inns of the :«'11rl':-we of the l)l:lh'lA)ll(‘I‘lll p:u':alls-I to its 4*Il;_{4-,
- r 14
- :
CH.
period and the young born in an
advanced stage [Notidam/us, Mus—'
talus, Galeus, Uarcharias, Zygaena, Lamna, Alopias, Uetorlzxinus, Acumtlmlas, Scymnus, Squatina, Torpedo, Trygonidae,Myliobatidae]. Amongst the viviparous Elasmobranchs preserved developmental stages of Torpedo (Fig. 247) may be obtained from Naples, and of Acanthias from various marine laboratories.
Amongst the oviparous forms certain species of Skate (Rania) are used as food-fishes and their eggs can frequently be obtained in quantity at trawling centres. In such cases arrangements can be made with local fish-dealers to send on by post the “ skate-purses ” taken from the oviducts when the fish are cut up.‘ The eggs of the different species differ in size and in the characters of the shellshape, colour, degree of translucency (Williamson, 1913). Of the European species B. batvls is the most convenient species to use; the normal period of spawning is from December to April but the retarding effect of the low temperature i.s so great that December eggs are practically overtaken in their development by the April eggs. The complete period of development is roughly 20 months, most of the eggs hatching about August.
The eggs should be posted in damp seaweed. On arrival the soft sticky marginal zone of the shell, which separates off except at
one end and serves to anchor the.
egg to the sea-bottom, is removed, and the date is marked in ink with a wooden style upon the flat portion of shell between the two horns.
- 3 —u-: t ‘
1 Jamieson observed out of many thousa.°nds of eggs only one case of the inclusion
of two eggs within a common shell. XI PRACTICAL HINTS—ELASMOBRANCHII 561
For hatching boxes it is convenient to take ordinary fish boxes freely perforated with anger holes, provided with a cross partition in the centre, and pitched inside and out to discourage the growth of seaweeds. The hatching boxes are moored afloat in pure sea-water within a breakwater or other shelter. About 20 eggs are placed in each compartment.
On alternate days the boxes are drawn a few times backwards and forwards through the water to dislodge any sediment that may have accumulated. Once a week they are hauled’out of the water and each egg-shell tested by rubbing the finger over its surface. If a slippery mucus-like layer has developed on its surface the egg is useless and should be got rid of.
When the egg has reached the desired period of development it is removed from the Water, placed in a horizontal position with the more strongly convex side below and opened by carefully removing the greater part of the less convex side of the shell. The isolated piece of shell must be lifted off very carefully as the albumen is very adhesive and the vitelline membrane extremely delicate.
In the early stages the embryo is almost invisible in the fresh state so the egg, still held carefully in a horizontal position, is gently submerged in fixing fluid. The blastoderm then comes into view and after a short time may be excised and floated into a watch-glass to complete fixation and the subsequent processes.
In later stages (Fig. 248) where the body of the embryo is constricted off from the yolk—sac, it is narcotized by submersion in sea-water containing alcohol and then the yolk-stalk is ligatured with thread and the embryo excised for further treatment.
Embryological material of the Sharks is to be preferred to that of the Skates or Rays on account of their less specialized character but unfortunately it is more diflicult to obtain in quantity. Small sharks of the genus Scyllelum and allied genera occur commonly round the shores of the various continents and their eggs may be found attached to seaweed at extreme low tides.
' On the British coasts a well-known spawning ground for Sag/llium canicula exists at Careg Dion about 2% miles from Beaumaris on the Anglesea side of the Menai Straits in between 3 and 4 fathoms of water and in spots not exposed to strong tidal currents} The eggs are deposited usually in the morning, the shorter stouter pair of filaments which issue first from the cloacal opening being trailed about /amongst tufts of the seaweed Halidrys siliquosa until they become entangled when the fish swims round so as to wind the elastic filaments firmly amongst the seaweed. The eggs can only be obtained at very low and specially favourable spring tides and as White finds at one time embryos of all stages of development it would appear that oviposition is not limited to any definite season. ~ Sag/llwlum not infrequently deposits its eggs in aquaria and at the
1 For the details in regard to this locality I‘ have to thank Professor Philip J. White of Bangor.
\roi.. 11 2 o 562 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.
Berlin Aquarium it has been observed that pairs of eggs were deposited at intervals of about ten days. The methods of technique mentioned in connexion with the Skate are also applicable to the eggs of Scyllium.
It should not be forgotten that, as mentioned earlier in this
FIG. 248.—Raia batis, embryos.
at, atrial portion of heart; E, eye; c, conus ; f.g, foregut; H, heart; l, lens; Zi, liver; ot, otocyst; pin, pineal organ ; rh, thin roof of fourth ventricle; v.c.I, etc., visceral clefts; y.s, yolk-stalk; V, VII, VIII, cranial nerves.
volume, one of the greatest desiderata in Vertebrate embryology is an oviparous shark with eggs of small size. ‘
TELEOSTOMI.-—-The most archaic and therefore the morphologically most important surviving member of this group is.Polypterus and strenuous efforts have been made to obtain ‘developmental material. Harrington lost his life on an expedition to the Nile with this object. Budgett made two expeditions to the Gambia, one to. Nigeria and XI PRACTICAL I-IINTS—-FISHES 563
the Nile, and a fourth to the Niger Delta. with the same object in view. The three first expeditions were fruitless but on the fourth he was fortunate enough to obtain ripe males and females and to accomplish fertilization of a number of eggs. Unhappily Budgett did not live to work out this precious material, falling a victim to blackwater fever soon after his return to England. The Budgett material has been investigated (Graham Kerr, 1907) but further material is urgently needed to work out much of the detail.
On the Gambia and on the Upper Nile Budgett found females with eggs in the oviducts during July and August; in the Niger Delta during August and September. During these periods he found that at any one time only a small proportion of males had active motile spermatozoa in their urinogenital sinuses so that it looks as if the actual breeding season of each individual male were very short. The fertilizations which were successful were effected with teasedup testis, the tubules being much distended and the sperm clear instead of opaque as it frequently is. In some cases Budgett found that eggs from the splanchnocoele gave a larger percentage of successes than those from the oviduct. .
The fertilized eggs adhered strongly to the bottom of the dish and this supports the statements made by the natives that in nature the eggs are attached to sticks and stems ol' plants under the water.
Nothing is known regarding the development of the other surviving Orossopterygian-—Oalamichthys.
Of the Actinopterygian ganoids, whose haunts are more accessible
and less unhealthy than those of I’ol_2/pterus, the development has’
been worked out more or less completely in the case of each of the main types———the Sturgeon (Acipenser), the Garpike (Lepidosteus), and the Bowfin or Dogfish (Amia). .
At the large fishery stations such as those on the Elbe or Delaware Rivers ripe Sturgeons are caught during a brief season on their way into the river to spawn. The eggs and spermatozoa may be obtained by “ stripping ” the fish 73.6. by firm pressure passed backwards along the sides of the body, or by opening the fish. The eggs are immediately placed in a dish and a little of the sperm mixed with a small volume of Water is poured over the eggs, the whole being stirred gently for about ten minutes. They are then distributed in a single layer over the bottom of a submerged shallow tray made with coarse mosquito netting to which the eggs adhere firmly within twenty minutes. ‘The trays are then placed in wooden hatching boxes with gauze ends and moored in the river so that they are traversed by a constant current. The dark-coloured somewhat tadpole-like larvae hatch out in from three to six days.
Lepidosteus (Dean, 1895) breeds at Black Lake, N .Y., normally between the middle of May and the middle of June, the eggs being fertilized at the moment of spawning and being distributed over the bottom in shallow water, adhering firmly to stones and other solid 564 EMBRYOLOGY OF THE LOWER VERTEBRATES C1--I.
objects. For laboratory purposes it is best to employ artificial fertilization as in the caseof the Sturgeon. T Amia (Dean, 1896) spawns at Black Lake during the latter half of April or May. The eggs are deposited on a compact site over which the vegetation is pressed aside so as to form a clear space with about a foot of water over it. The eggs, fertilized at the moment of laying, adhere to roots or other portions of the water—plants. The rate of development as in other cases varies greatly with the
F10. 249.—Stages in the development of Symbmnchus. (After Taylor-, 1914.) our, optic rudiment; .I’.F, pectoral nu rudiment.
temperature and from four days to fourteen have been observed to elapse between the deposition of the eggs and their hatching.‘
Of Teleostei (Figs. 249 and 250) by far the most convenient for systematic laboratory work are the Salmon (Salmo salar) and the Trout (S. famlo), eggs of which can be obtained in quantity from the various hatcheries. The eggs obtained by “stripping” are fertilized artificially and may then be sent by post packed in damp moss. Small hatching boxes suitable for laboratory use can also be purchased?
The eggs and larvae of marine Teleosts are often obtained in great
1 Excellent developmental material of Lepidosteus and Anita may be obtained from the Woods Hole Laboratory or from Mr. J. C. Stephenson, Washington University,
St. Louis. 9 E.g. from the Snlway Fisheries Co., Dumfries, Scotland. but these are not so con XI PRACTICAL HINTS——-—I<‘ISHES 565
numbers in the tow-net
venient for investigation on account of their reduced size. As there is little doubt that the 'l‘eleostei have been evolved out of ancestral forms with large eggs investigations are particularly desirable on those teleosts, mostly freshwater forms inhabiting warm climates, in which the large size of the egg has been retained. There is an important field for investigation in the embryology of tropical freshwater fishes. Of individual families the Siluridae, Characinidae and Gymnotidae call especially for investigation.
DIPNOI.-— The Lungfishes form a group of much importance to the Vertebrate morphologist on account of, on the one hand, their great antiquity and the retention of many archaic features in their organization and, on the other hand, of the -fact that they present to us foreshadowings of various features which become prominent characteristics in the tetrapoda or terrestrial animals. A knowledge of their embryology consequently became one of the great desiderata of Vertebrate Embryology. The first
- Fm. 250.——-Bla.stmlei‘1ns anal mubryos of Trout dlscovered of the three (Salmo fa/riu). (After Kopscll, 1898.)
surviving representatives of the gI‘0llp—-L6}9?«d0- y, oxpo.~ae(l su1'fa.ee of yolk.
Iv), eye; at, otocyst; p.f, pect.0r:ll Mn; -rh, rhombeneephalou; 566 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.
s7?rem—remained unknown so far as its development was concerned until 1896 when Graham Kerr succeeded in obtaining abundant embryological material in the Gran Chaco of South America.
The developmental stages of Protopterus, the next representative of the group to become known to science, were first obtained on the Gambia River by Budgett who had taken part in the Lepidosiren expedition a few years earlier. Ceratodus, the last of the surviving genera to become known in the adult condition, was the first to be made known embryologically by Caldwell and Semon as already mentioned (p. 435).
The Lung-fishes like other animals living under similar conditions breed at the commencement of the rainy season (Protoptems, Gambia, August; Lepidosiren, Ohaco, November but incidence of rainy season irregular and may be de1ayed—till e.g. J une-—or omitted altogether; Oeratodus, September to December). In the case of Oemtodus the eggs are scattered loosely about amongst the water plants, while in Protoptems and Lepidosiren they are deposited in a special burrow at the bottom of the swamp where they are guarded by the male parent.
¢ Dipnoans live well in captivity and there is little doubt that it will be found easy to induce them to breed by using similar methods to those described under the heading Amphibia. It is particularly desirable that this should be done in the case of Lepidosiren on account of the large size of its histological elements which make it a peculiarly suitable type for the investigation of various problems of histogenesis.
The eggs of Dipnoi, especially of Lepidosiren, are of large size and this makes it especially advisable to use celloidin in addition to paraffin methods of embedding. When paraflin is used it is necessary to remove the egg envelope by slitting it up with fine scissors, care being taken to keep the point of the scissors close to the envelope so as to avoid injury to the surface of the egg.
Corrosive sublimate and acetic acid is a good stock fixing agent. For stages before hatching 107 formalin is convenient.
AMPHIBIA.—-The most easi y obtained embryological material is that of the common Frogs of the genus Roma the masses of spawn of which are familiar objects in pools during the early weeks of spring in temperate climates. The exact time differs with climate and also with species, some species such as R. esculenta in Europe and R. catesbiana in North America lagging several weeks behind the others. The spawn, fertilized as deposited in the early morning, may conveniently be kept during its development in earthenware pans. The water should be left stagnant and unchanged during the period prior to hatching as under these circumstances the spawn is less liable to be attacked by fungus but the hatched larvae should be at once transferred to clean water.
Investigations are greatly needed on the embryology of Anura outside the genus Rana (of. Figs. 251, 252, 253 and 254). The different genera and species differ greatly in the size of the egg XI DIPNOI, AMPHIBIA 567
and its richness in yolk and there is no group of Vertebrates which ofi"ers anything like the same facilities for studying the influence of yolk upon the course of development. Further it will be only after greatly extended studies on different species that we shall be in a position to have a really com- prehensive idea of typical Anuran development.
Many tropical species of Frogs and Toads fire to be Flu. ._')l.—-String of eggs of unknown Frog from the Gambia. Obtalned ahve from Individual variations in the rate of developnu.-nt are indicated animal dealers and hy the varying size of the yolk-plug.
in these it may be taken as a general rule that breeding takes place at-the commencement of the rainy season, or in other words when environmental conditions become favourable after a prolonged period during which they have been unfavourable. By bear ing this principle in mind such tropical T i amphibians may usually be induced to breed in captivity. Bles in his excellent account of the life-history of Xenopus (1905) describes a method which will be found to be of general use. The pair of animals were kept in a Budgett tropical aquarium consisting of a glass bell-jar 20 inches in diameter dipping into a galvanized iron water-tank heated by a small Bunsen burner and oxygenated by plants of Vallisnemla. During summer the temperature of the water in the belljar was kept at about 25° C. The water was not changed. The frogs were fed daily with small earthworms or thin strips of raw calf’s liver until they would eat no more. In December the temperature was allowed to fall to 15°-16° during 1*‘1G-252--EI§ibI_'y0 of 1’hz/llmIwd'u~sja the day and as low as 5"-8° during the "”P"”f;::l”“l’8 "““‘°”°d °“t 1" night. As the temperature rose With the °;lfiI1:ucca'1wity_M bmmpm_ onset of spring the frogs became more N’ mes, n1c.s'odcrnlsnigincnts. 4, actives Wilkmg “P out of the lethargic condition induced by the winter's cold. Breeding was induced by simulating the natural conditions of the rainy season. The temperature was raised to about 22° 0. Each morning and evening about two gallons of the water was drawn off, allowed to cool for twelve hours and then returned to the aquarium in the form of a fountain of spray from the upturned 568 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.
end of a glass siphon drawn out to a fine point so as to produce the effect of a shower of rain. Within a week or two breeding took place.
The chief difiiculty in the way of cutting sections of _Frog’s eggs is due to the presence of the jelly-like envelope. This may be got rid of by prolonged soaking, six months or more, in -5% formalin (Ogushi, 1908), or by fixing in Zenker’s fluid and leaving the efgs 111 this fluid renewing it after 2 to 3 days and continuing the treatment
FIG. 253.-—Stages in the development of ]’l:.y/llunwdusa /1.3/po¢:h¢mal7"£alz's. E, eye; e.g, external gill; op,opercu1um; oz, otocyst.
for 8 to 14 days or longer, shaking gently so as to remove the envelopes (Kallius, 1908). '
For cutting sections paraflin is commonly used but it should be supplemented by celloidin e.g. the clove-oil method mentioned under Ampimloxus.
In the Urodeles the eggs are commonly laid singly in water and attached to water plants (Triton) or other solid objects such as logs or stones (Proteus, Necturus). In Oryptabranchus and Amphiuma they form a beaded string, adjacent envelopes being connected together by a narrow isthmus.
Fertilization is rarely external (0'ryptob'ranchus——Smith, 1912). In the Newts the female takes up a spermatophore into the cloaca. xx ' PRACTICAL HINTS-——AMPHIBIA 569
Such internal fertilization leads up to the condition in the Salamanders where fertilization takes place in the upper part of the oviduct and the developing embryo is retained for a less or more prolonged period within the body of the parent. In Salamcmdm mooculosa larvae about an inch in length are born in May resulting from fertilization during the preceding summer. '
As in the Anura wide differences exist in the richness of yolk and consequent size of the egg—the latter varying from under 2 mm. in the Newts to 6 mm. (Necturus) or 7 mm. in diameter (Org/ptobranchus japom'cus): so that here again though not to the same extent as in
F10. 254.-—Tadpo1e of unknown Frog from Tropical Africa.
A, side view; B, ventral view. inc, huccal cavity; c.o, ('(‘.lllf‘.I|l.r organ ; rz, anus; E, eye; e.g, external gill; u/,/', olfactory organ; up, operculum.
the Anura there is an excellent field for investiga c tion into the influence of yolk upon developmental processes. The eggs of Urodeles are commonly collected under natural conditions and kept in earthenware dishes. Or the adults just about to breed may be brought into the laboratory and allowed to deposit their eggs in a suitable aquarium;
The Urodela form one of the relatively primitive groups of Vertebrates and their embryology‘ deserves much greater attention than it has hitherto received. Most of the older literature deals with special details i.n the development of the Newts but comprehensive monographs, including “normal plates” on the development of such genera as Proteus, Siren and Amphiuma are much wanted. A general account of the development of the American species of Uryptobranohus has been given by Smith (1912), while the Japanese species has been dealt with by Ishikawa (1918), De Bussy (1915) and Dan. de Lange, Jr. (1916). Of Necturus normal plates with accompanying tables have been worked out by Eycleshymer and Wilson (1910).
The Gymnophiona—-—though an aberrant group of Amphibians highly specialized for a burrowing existence—are of much embryological interest and have provided the material for work of great morphological importance, such as that of Brauer upon the excretory organs. A general account of the development of Icltthyophis 570 EMBRYOLOGY or THE LOWER VERTEBRATES on.
will be found in Sarasin (1887-90) and of Hypogeophis in Brauer (1897).
The eggs, fertilized internally, are normally deposited in the soil and the embryologist has, as a rule, to depend upon such scanty material as can be obtained by digging in the damp soil of localities where Grymnophiona are abundant. Ty/phlonectes in South America and _De7~mophz's in West Africa are viviparous.
Of the group in general it may be said that a comprehensive monograph on the development of each genus beyond Ichthyoplmls and Hypogeoph/is is a great desidcratum.
As standard fixing agents for Amphibia corrosive sublimate and acetic acid, and for the later larval stages strong F lemming’s solution, may be used. For the early stages (segmentation and gastrulation) quite good results are obtainable from eggs that have been preserved alive in 10,°/O formalin: in this case it is well to treat the egg before dehydration for an hour or two with corrosive sublimate solution as without this precaution the formalin-preserved eggs are diflicult to stain well. When any other fixing agent than formalin is used it is necessary, as a preliminary, to remove the egg envelopes. In the case of the larger eggs of the Urodela and Gymnophiona this can be accomplished with the aid of fine scissors and forceps.
REPTILIA. — For gaining practical knowledge of Reptilian development the student will find the group Chelonia most convenient as it is possible to obtain 1 excellently preserved series of developmental stages of Terrapins (Ohrysemg/s) and Snapping Turtles (0helg/dm). In particular localities especially in warm climates he may have opportunities of obtaining the eggs of Lizards, Snakes or Crocodilians. In all cases the same technique may be used as in the
case of the Fowl. ' AVEs.—The Birds, although showing conspicuous differences in
external appearance and in minute details of structure, form a very compact evolutionary group and there is little likelihood of important differences in principle existing in their development. Interesting differences in detail however are to be found—such as the presence or absence of neurenteric canals. Groups which there is any reason to suspect of being particularly archaic——such as Divers, Grrebes, Penguins-—-are worthy of careful scrutiny for possible persistence of Reptilian features.
LITERATURE
B103. Trans. Roy. Soc. Edin., xli, 1905.
Brauer. Zool. Jahrbiicher (Anat.), x, 1897. do Bussy (do Lange), L. P. Eerste ontwikkelingsstadién van Megalobatrachcpos
Mamimus, Schlegel. Amsterdam, 1905.
Cerfontaine. Arch. de Biologie, xxii, 1906.
Dean, Bashford. Journ. Morph., xi, 1895.
1 E.g. from Mr. J. C. Stephenson,‘Washington University, St. Louis,'or The-Marine Biological Laboratory, Wood's Hole. XI - LITERATURE 571
Dean, Baahford. Quart. Journ. Micr. Sci., xxxviii, 1896.
Dean, Bashford. Kupifers Festschrift. J ena, 1899.
Eerfort. Arch. mikr. Anat., lvii, 1901.
Iahikawa. Mitt. Deutsch. Gesell. Natur- und Viilkerkunde Ostasiens, xi, 2, 1908. Kalliua. Anat. Anz., xxxiii, 1908.
Kerr, Graham. The Work of John Samuel Budgott. Cambridge, 1907.
Kopsch. Arch. mikr. Ana.t., Ii, 1898.
do Lange, Dan., Jr. Onderzoek. Z061. Lab. Groningon, iv, 1916.
Ogushi. Anat. Anz., xxxiji, 1908.
Sarasin, P. and H. Ergebnisse nnturwissenschaftlicher Forschungen auf Ceylon, ii.
Wiesbaden, 1887-90.
Soammon. Keibels Normentafeln, xii. Jena, 1911.
Smith. Journ. Morph., xxiii, 1912.
Taylor. Quart. Journ. Micr. Sci., lix,\1914.
Williamson. Fisheries, Scotland, Sci. Imwst., 1912, i. 1913.
Ziegler, H. E. and F. Arch. mikr. Ana.t., xxxix, 1892.
APPENDIX
THE GENERAL METHODS OF EMBRYOLOGICAL RESEARCH
EMBRYOLOGY is one of the youngest of the sciences and it offers a wide field for fascinating and important research. Regarded as a branch of morphology its main object is to gain information concerning the lines along which the structure of existing groups of animals has evolved. In the phylum Vertebrata there is an immense amount of work still to be done and it is important that the would-be researcher should be guided by certain general principles as to the technique of the subject, otherwise he is apt to achieve no more than the addition of relatively unimportant details to the vast accumulation of details which during the past few decades has tended to hide away general principles and incidentally to smother interest in the subject.
The incompetent or inexperienced investigator frequently betrays-himself by his choice of subject: he chooses a problem of relatively minor interest when there lie ready at his hand others which are of real importance, or he chooses a subject really important but of such difficulty that the probabilities are heavily against the feasibility of its solution under existing conditions. The beginner then should see that he has the aid of some competent adviser before he decides upon his line of research.
Having chosen his particular problem he has next to decide regarding the particular animals upon which his research is to be carried out. The earlier workers were guided mainly by the accessibility of the material. Fowls and Rabbits -—-of which embryos were easily obtained and easily investigated—--provided the material for the great pioneers of vertebrate embryology and the embryology of to-day suffers much from the difficulty of getting rid of general ideas founded on such narrow bases. N ow that embryology has taken its place as a branch of evolutionary science we recognize the importance of basing our general ideas upon the phenomena of development as displayed by the more primitive existing groups. In attempting any important problem of vertebrate morphology, evidence must be got from Elasmobranchs, Crossopterygians, Lung-fishes, Urodeles, before we can feel completely confident as to general principles: in other words we must go to groups which are admittedly archaic. Apart from directly adaptive features an animal which is archaic in its adult structure may be expected to show primitive features in its development. Naturally we should not look for this in cases where development takes place under peculiar conditions, for these necessarily involve adaptive modification. A pitfall into which investigators frequently stumble is that, starting from
573 574 EMBRYOLOGY- OF THE LOWER VERTEBRATES' APP.
some particular group—-say Ampluloxus, or the Mammalia——with whose structure they'happen to be thoroughly familiar, they assume its general organization to be primitive. As a matter of fact it may be assumed with considerable probability that every existing vertebrate is to a certain extent a mixture of primitive features and specialized. It is only by careful comparative study that it can be decided which features are probably primitive and it is quite certain that these will not be found all within one group. Consequently speculations based upon the intensive study of one particular group are to be distrusted, though there is always less ground for distrust if the group is one which is recognized for reasons other than embryological, as being on the whole archaic. ‘ When minute histological details are concerned another qualification
which should be possessed by the animal chosen for investigation is large size of its cell units.
The material should be abundant. Not only should there be a continuous series of stages but there should be numerous specimens of each stage. There is no such thing as an absolutely normal individual: the conception “normal” is an abstraction based upon the observation of numerous individuals. Only by observing numerous individuals can we therefore arrive at a knowledge of normal development. Work carried out on a? few specimens may of course provide isolated observations of much interest and value but it is inadequate to serve as a basis for general conclusions.
In all descriptive embryology it is necessary to have some method of specifying the stage of development of individual embryos. Unfortunately there has been a great lack of uniformity as to the particular method of doing this. One of the most frequently used is that of specifying the period of time during which development has been going on as for example a “chick embryo of 40 hours’ incubation.” This method is quite unsatisfactory, owing to the fact that the actual stage of development of any individual embryo is a function of other factors in addition to mere time, such as temperature and individual idiosyncrasy. Thus in many tropical freshwater animals a statement of the age of the embryo is practically worthless unless accompanied by a record of the temperature, and even then there remains the unknown element of individual peculiarity such as is for example illustrated by Fig. 251 where a number of sister eggs of a Frog are seen to have “lost step” with one another to a marked extent even at a comparatively early stage of development. In other words eggs or embryos of the same age are liable to vary greatly in their degree of development, and a statement of their age is not adequate as a precise indication of the stage of development. The want of precision varies in different cases: it is less for example in a Eutherian mammal where development takes place at a fairly definite temperature than it is in a Fish or Amphibian inhabiting a tropical pool or swamp where the temperature is liable to great variation. ,
It is necessary then in referring to particular stages of development to define them by structural features. Here however a new difficulty presents itself in the fact that the relative rate of development of different organsystems is not the same in different individuals. It follows that if a. number of individuals be grouped together as being at the same stage of development as judged by a particular organ A it Wlll be found that other APP. METHODS OF EMBRYOLOGICAL RESEARCH 575
organs B, C, etc. are not exactly at the same stage of development—some are less developed some more in the various individuals. Still for practical purposes this is a useful way of indicating roughly the stage of development. For example early stages in the development of Vertebrates may be defined by giving the number of mesoderm segments which have developed——these being fairly conspicuous structures and definable by a number. A much better system, however, is to use numbered stages defined by the general external form——the first structural feature met with in the examination of an embryo. Keibel has published “normal plates” of the development of various Vertebrate types in which standard stages in development are defined by accurate figures. Unfortunately some of the normal plates are incomplete as regards the earlier stages during segmentation and gastrulation, but wherever the plates extend over the whole period of development they should be made use of by the working embryologist as his standard stages. Where no normal plates exist the embryologist should m_ake_it his first business to construct one by carefully working over the external features of development and defining by careful drawing and description a series of stages which he judges to be roughly equidistant. The embryology of any animal is an account of the observable changes which take place in its structure from the zygote stage up to the adult. Logically the investigation of its embryology should proceed similarly from zygote to adult but in actual practice it is better to work in the opposite direction—-—to commence by getting a clear idea of the adult organization and then to work back from the known to the unknown of earlier stages. An embryological investigation should commence with a careful study of the entire embryos or larvae at the various stages. Each stage should be examined first alive by transmitted and reflected light, careful note being taken of any movements due to muscular contraction, ciliary action etc. Particular attention should be paid to the arrangement of the blood-vessels, the time of commencement of heart movements, of circulation of the blood and of the appearance of haemoglobin in the corpuscles. The appearance of chromatophores should be noted: the seat of their first appearance and their reactions-——whether by changes of form, movement of pigment granules in their protoplasm, or by actual migration-—in
response to changes in direction or intensity of light. During this phase_
of the work constant use should be made of the binocular microscope and rough sketches should be made.
Embryos of each stage should be submitted to the action of various fixing agents and it is important to watch the embryo during the process of fixing, for the fluid as it gradually penetrates the tissues often makes special structures stand out distinctly for a short space of time——to disappear again with further penetration. The fully fixed embryo should be subjected to further careful scrutiny by reflected light under the Greenough binocular. To detect small inequalities of the surface it will be found necessary to arrange the lighting carefully. The light from an mcandescent gas-mantle may be concentrated by a large condenser and caused to illuminate the embryonic surface in a tangential direction. It is often well to cover the specimen with a little house of opaque cardboard or metal resting on the stage of the microscope and possessing two apertures one in its roof through which the observation is made and one at the side through which light is admitted. The embryo must of course be 576 EMBRYOLOGY OF THE LOWER VERTEBRATES APP.
completely submerged in fluid and is preferably contained in a round glass dish with a layer of pitch or black wax on the bottom in which, if necessary, small excavations can be made in which the embryo can rest securely in the desired position. The glass vessel should be rotated slowly during the observations so as to allow of the incidence of the light from different directions. It is important to observe a number, preferably a considerable number, of embryos of the same stage, as owing to individual variation particular features may be much more distinct in some than in others.
A number of thoroughly typical specimens of each stage should be picked out for further investigation and these should be carefully drawn under the camera lucida, a piece of millimeter scale being placed by the side of the embryo and drawn at the same time so as to form a reliable record as to dimensions.
' At this stage the normal plates should be constructed if not already in existence and the embryos classified in accordance with them,
For the study of internal structure the great method is that of cutting the embryo into serial sections‘ but a much older method, that of dissection, should by no means be ignored. Careful dissections made under the Greenough binocular are often extraordinarily instructive. It is advisable to experiment with embryos fixed according to various methods as diffcrrent methods give difl‘erent degrees of consistency, opacity etc. Van Beneden and N eyt’s fluid will be found in many cases to give very good results.
In section - cutting a fetish to beware of is excessive thinness of sections. The expert section cutter is liable to become so interested in his feats in accomplishing the preparation of sections of an extraordinary degree of thinness that he is apt to forget that the criterion of good sections is not simply their degree of tenuity but the relation which their thickness bears to the size of the cell-elements of the particular embryo. Thus while in some cases it is of advantage to have sections so thin as 1 [L2 or even '5 ii, in other cases, such as segmentation and gastrulation stages of some of the large heavily-yolked holoblastic eggs, the sections should reach as much as 80 p. or 100 ,u in thickness.
Before an embryo is cut into sections its soft protoplasm has to be supported by infiltration with some suitable embedding mass. For this purpose the two substances used at the present time are parafiin of high melting-point and celloidin. Of these the first is used frequently alone but the student should realize from the beginning that if he is to obtain reliable results, especially yvhere yolk is present in the embryonic tissues, he must use both methods and control and check the results obtained from one by those obtained from the other.
The process of infiltrating the embryo with paraflin is usually carried out in a hot-water oven heated by oil, gas or electricity and kept at a temperature just above the melting-point of the paraffin by a thermostat. The melted paraflin may be contained in small copper pans preferably plated inside with silver or nickel. An essential preliminary is a very thorough dehydration followed by a very thorough soaking in the clearing agent. To get the best results it is well to take the embryo through
1 A useful guide for beginners is.Sect7}on- Cutting by P. Jemieson in preparation. For those who already possess an elementary knowledge of the subject an .xcellent work of reference is Bolles Lee's Miicrotomicfs V ads-nwcum. .
9 1 p.=n1n millimeter. APP. METHODS or EMBRYOLOGICAL RESEARCH‘ 577
three changes each of 90% alcohol, absolute alcohol, and xylol or other clearing fluid. The actual process of infiltration with paraffin should last for the minimum time (which will have to be determined by experiment 1) and be carried out at the minimum temperature.
It may be remembered that the complicated and bulky water-bath with its thermostat is in no way necessary for the embedding process. A very simple apparatus which is perfectly eflicient consists of a small metal trough (copper, or tinplate) resting upon a metal table kept heated at one end by a small flame. By sliding the trough lengthwise along the table a position can be found such that the entire thickness of paraflin is fluid at the end next the flame and solid towards the other end. Between these two points stretches an inclined plane of solid paraffin upon the surface of which the embryo rests without any risk of the temperature rising appreciably above melting-point. A simple embedding trough of the kind indicated is of great use in the field as there is no method of storing and transporting embryos so free from danger of accident or of histological deterioration as having them embedded in solid paraffin.
'I‘o get a block of parafiin in good condition for section~cutting the embryo should be transferred to a bath of fresh paraffiu as soon as it is infiltrated. With certain clearing agents, e.g. cedar oil, it is well to give two or three changes of paraffin. 'l‘he vessel containing the embryo in"a considerable volume of paraflin should now he floated on cold water so as to give a homogeneoustranslucent block of solid paraffin. On no account should the vessel be actually submerged in the cold water for in this event the contraction of the inner paraffin as it cools within the already rigid outer layers will lead to the formation of cavities into which the water penetrates.
For the actual process of section-cutting it is necessary to use a mechanical microtome. The Cambridge Rocking microtome is one of the most convenient for ordinary enibryological work while the ReinholdGiltay ‘microtome is a most excellent instrument both as regards accuracy and rapidity of working. '
The paraflin block containing the embryo is trimmed down so as to be rectangular in section and is then fixed by, the interposition of a hot spatula to the parafiined surface of the microtoine carrier in such a position as may be necessary to give the required direction of sections.
Where the object is a “diflicult” one, e.g. containing much yolk, it is advisable to have it surrounded by a paraffin block of considerable size. A considerable mass of paraffin above the specimen makes it out better, while a considerable mass to the side causes successive sections, with their long edges, to adhere better together and form a continuous ribbon. The embryo should be near one of the lower corners of the block to facilitate exact orientation.
For thorough investigation of the structure of embryos it is advisable to have specimens cut into sections in the three sets of planes-—transverse, psagittal or longitudinal vertical, and coronal or longitudinal horizontal. To obtain these it is tiecessary to have the embryo orientated exactly on the microtome. In most cases this can be accomplished with a sufficieutly close approximation to accuracy when fixing the paraflin block on to the
‘ E.g. for a Chick at about the middle of the second day about 20 minutes will be found to be suflicient. VOL. II 2 1» ' 578 EMBRYOLOGY OF THE LOWER VERTEBRATES APP.
carrier, especially if care has been taken to trim the surfaces of the block parallel to the three chief planes of the embryo.
Where greater accuracy is needed, as in the case of very small embryos, they should be arranged in position in the melted paraffin with warm needles under the prism binocular microscope. This may be done by placing the watch-glass or other vessel on the top of a small flat copper cistern full of water, provided with inlet and outflow tubes, and heated up by contact with the top of the water-bath or hot stage. In the bottom of the embedding vessel is placed a small plate of glass on the upper surface of which are engraved parallel lines intersecting one another at right angles. When the embryos have been accurately orientated with regard to the engraved lines a stream of cold water is allowed to run through the cistern and this causes the paraliin rapidly to solidify. When the block is quite llard the glass plate is picked off and the ridges formed by its engraved lines serve as accurate guides to the position of the embryo.
Still greater accuracy is obtainable by arranging that the melted paraffin in which the embryo is being orientated is already in its definitive position on the holder of the microtome, the paraffin being kept melted as long as necessary by an electric current passing through a loop of high resistance wire.‘
' For the actual cutting care must be taken that the razor (solid ground) or other knife has a very fine edge which does not show irregularities when examined under the low power of the microscope. The blade should be thoroughly cleaned with pure spirit before commencing work. If very thin sections, e.g. of l [L in thickness, are required it is well to commence with sections of 5 pt, then without stopping to change to 4 p., then to 3 p., then to 2 p, then to l p.——cutting a continuous ribbon throughout and going ahead rapidly when the 1 /1. sections are cutting properly.
The celloidin method should be constantly used as a check on the paraffin method. Where yolk.y eggs or embryos are being cut the celloidin method gives the only trustworthy sections as by it the yolk granules are held in position and prevented from sticking on the edge of the knife, ploughing through the tissues and destroying much of the fine
detail, as is always liable to happen if paraffin alone is used under such ‘
circumstances.
In cases where there is no need for specially thin sections (say under 25 pi.) a convenient method is that in which the celloidin block is hardened
by exposure to: chloroform vapour and then cleared by immersion in cedar-wood oil.
The block of celloidin is usually fixed to a block of wood which is
gripped by the holder of the microtome. Care should be taken that such wooden blocks are baked for several days so as to ensure their being
absolutely dry. Otherwise moisture will diffuse out and produce a milky opacity in the celloidin which ought to be absolutely clear and transparent. Sometimes it will be found that the block becomes too hard and will
not cut properly, its edges frilling or breaking. This is sometimes due to the presence of a trace of chloroform in the cedar oil used for clearing.
When this is the case the cut surface of the block should have perfectly pure cedar oil applied to it with a brush just before each section is ‘cut.
‘ A special apparatus for this purpose is made by the Cambridge Scientific Instrument Company. APP. METHODS OF EMBRYOLOGICAL RESEARCH 579
To obtain thinner sections it is necessary to embed the celloidin block containing the object in parafiin. This may be done simply by transferring the block saturated with cedar oil to melted paraffin. A better method is to use a solution of celloidin in clove oil of about the consistency of treacle. The object, thoroughly permeated by this and surrounded by a small quantity of the celloidin, is hardened and cleared in chloroform. The block is then carefully trinnned with one face accurately parallel to the plane of the required sections. It is now immersed in melted paraiiin for a minimum time (ten minutes suilices for a small object). After cutting and mounting the sections the slide is immersed in xylol ,until the parafiin is dissolved out, then in absolute alcohol, then in a mixture of equal parts of absolute alcohol and ether until the celloidin is removed. The slide is now taken down through the series of alcohols and the sections stained and mounted in the ordinary way.
The arriving at a clear idea of the structure of an embryo from the study of a series of sections involves fitting the successive sections together into a continuous whole. To a great extent this reconstruction of the whole from the successive sections can be done mentally but where complicated structures are being investigated, some aid- is either absolutely necessary or at least desirable for the sake of accuracy. The preseht writer finds the most reliable as well as the most convenient of such aids in the method of reconstruction by means of glass plates.‘ Successive sections are drawn with a hard (9 H) lead pencil by means of a camera lucida upon finely ground sheets of glass such as is used for photographic focusing screens and then the successive drawings are fitted together, a fluid of as nearly as possible the refractive index of the glass being interposed between them so that the ground surfaces disappear and the heap of plates appears as a clear block with the structures drawn running through it and appearing as a kind of solid model.
The following details may be noted. Sections are cut to a standard thickness of 10 ,u (z'.e. T55 mm.): the glass plates are 1 mm. thick: the drawings are made at a magnification of 100 diameters. But it will be found in practice that much use can be made of the method even if these three dimensions are not so exactly correlated. The outlines made with pencil of the particular organ that is being studied are filled in with water colour. Vermilion is the most generally useful colour for it retains its opacity and light-reflecting properties to an unusually high degree when submerged in fluid of high refractive index. When the plates are dry N o. 1 is laid, ground side up, on a flat surface——_preferably a glass stage with a_ mirror beneath so that light may be refleeted up through it—a few drops of the fluid used, e.g. clove oil or cedar oil or a mixture of fennel oil (two parts) and cedar oil (one part) as recommended by Budgett 2 are placed by a pipette on the centre of the ground surface and then plate N o. 2 is lowered gently into position and fitted into its place over plate N 0. 1. The outlines of the drawings should be made to coincide exactly, and the two plates should be pressed firmly into contact care being taken to avoid interposed air bubbles which act as elastic cushions and prevent the upper plate from settling down into contact with the other. Successive
1 Quart. Jowm. Micr. Sea, xlv, 1902. 2 Trans. Zool. Soc. Landon, xvi, Pt. 7. 1902. 580 EMBRYOLOGY OF THE LOWER VERTEBRATES APP.
plates are fitted on in a similar manner until the particular organ stands out like a solid model in the mass of plates. _
The same set of drawings may be used for different organs : the clove oil is removed by treating with strong spirit, and the water colour by holding under the tap, and then, after drying, a new organ can be coloured in. By colouring merely the cavity of an organ the relations of the cavity can be displayed as by an injection. When finally done with the drawings are removed by scrubbing with “ Monkey brand” soap.
By this method, after a little practice, reconstructions can he made with great rapidity and accuracy.
Though less accurate and much more tedious the older method of reconstructing with plates of wax is useful for building up a permanent model. Its use is also indicated where only a single specimen is available. Instead of wax plasticine may be used 1 which allows of a kind of dissection being made, in as much as particular parts of the model may be bent out of the way to display structures which would otherwise be hidden.
1n investigating the development of the skeleton the cartilage is often found to pass by imperceptible gradations into unmodified mesenchyme. The absence of sharply defined surfaces in such cases makes the reconstruction method unreliable and it is advisable to supplement it by subjecting the embryo to treatment with a specific stain which picks out the cartilage while leaving the other tissues uncoloured so that the cleared and transparent specimen may be studied as a whole under the binocular microscope.
An excellent stain for this purpose is v. Wijhe’s Methylene Blue.” The embryo is fixed preferably in '5% watery solution of corrosive sublimate, with 10% formalin added just before use, and preserved in alcohol. When about to be stained it should be treated for a day or two with alcohol containing :1-% hydrochloric a.cid——care being taken_ to renew this so long as it develops any yellowness due to traces of iodine. The stain consists of a solution of 1% methylene blue in 70% alcohol to which 1% hydrochloric acid has been added some time before use. The embryo is stained for a week and is then treated with 70% alcohol containing {/0 hydrochloric acid and renewed several times the first day and thereafter once daily until no more colour comes away. The embryo is now dehydrated, cleared gradually in xylol, passed through stronger and stronger olutions of canada balsam in xylol, and preserved eventually in balsam so thick as to be solid at ordinary temperatures though liquid at 60° C.
An excellent method of cleaning small cartilaginous skeletons is to
place. them amongst Frog tadpoles which remove the muscle etc. from the surface of the cartilage by means of their oral combs.
In regard to the general principles of embryological research it need hardly be said that, as in other branches of science, accuracy of observation occupies the first place. And yet, curiously, accuracy may become a fault. In those branches of science which are more effectively under the control of mathematics it is well recognized that in any type of investigation there is a limit of- probable error of observation-—due to instrumental or sensory imperfections or to disturbing factors of one kind or another— ‘ Harmer, Pterobranclyia of Seiboga Expedition, 1905. 9 Proceedings Akad. Wetensch. Amsterdam, J une 1902. APP. METHODS ‘OF EMBRYOLOGICAL RESEARCH 581
beyond which it is mere waste of time to push observation. In all biological observation the limit of probable error is particularly high yet this fact is peculiarly apt to be ignored and it is no unusual thing to find dimensions or other numerical data stated to three or four places of decimals when anything beyond the first place is worthless for the reason indicated. _
To secure accuracy of observation not merely training and experience in the art of observing is needed but also a proper psychological outlook: the observer must be able to take a completely detached point of view and must ever be on the watch to guard against some particular hypothesis or preconceived idea causing actual error instead of fulfilling its proper function of keeping the powers of observation tuned up to the highest pitch of alertness. '
The whole spirit and aim of scientific investigation is directed toward
‘the seriation of facts and the devising of general expressions or formulae _ which unite them together. In this it contrasts with the more primitive
state of mental development which observes isolated phenomena, noting the differences between them but blind to the common features which link them together. In embryology as in other departments of knowledge the able investigator sees the general principles which run through and organize the masses of detail: he interests himself in discovering the likeness which is hidden under superficial difference; he is constructive not destructive.
In this volume embryology is treated as a branch of morphology but it must be borne in mind that morphology and physiology are inseparably intertwined. The living body whether of an embryo or an adult is above all a piece of exquisite mechanism fitted to live and move and have its being, and to ignore this is to make morphology as sterile and as misleading as would he the study of machinery apart from the movements and functions of its various parts. More particularly in attempting to delineate the evolutionary past of .an organ, or set of organs, speculation must always be rigidly controlled by the reflexion that at each phase in evolution it nmst have been able to function.
When at length the stage is reached of putting results into form for publication the first thing to aim at is absolute clearness of expression. It must be remembered that clearness of language and clearness of thought are closely interdependent. Sloppy obscure language means sloppy obscure thought. The greatest care should be taken in the correct and precise use of technical terms. Argumentation in regard to scientific and other matters is, when the disputants are equally well informed, due as a rule to some word or expression being used in slightly different senses. Elegant literary style, however desirable, must always be subordinate to clarity and precision of language. Indeed actual harm is sometimes done to scientific progress by the writer whose literary skill carries away not merely himself but others of uncritical and impressionable mind. Scientific problems are eventually settled not by skill in dialectic but by increase of knowledge.
As a rule the proper presentment of an embryological thesis involves pictorial illustration. In this the elaborate coloured lithographs of former days may conveniently be replaced to a great extent by simple line or half-tone drawings in India ink ‘or process black which can be reproduced photographically and inserted in the text in contiguity with the passage which they illustrate. Their function is to render more clear the statements of the author: they represent as accurately as possible phenomena as observed by the skilled and trained eye with a brain behind it. Actual photographs, which repr'(:sent merely details lying in one particular plane and as seen by the untrained photographic lens, should be avoided. Apart from the imperfections indicated they are so blurred by the ordinary processes of reproduction as to be liable to misinterpretation and in these days of skilful manipulation they are of course useless as guarantees of truth.
