Publicly accessible
URL: http://nzetc.victoria.ac.nz/collections.html
copyright 2006, by Victoria University of Wellington
All unambiguous end-of-line hyphens have been removed and the trailing part of a word has been joined to the preceding line, except in the case of those words that break over a page. Every effort has been made to preserve the Māori macron using unicode.
Some keywords in the header are a local Electronic Text Center scheme to aid in establishing analytical groupings.
Journal of the Biological Society
Victoria University College
Wellington New Zealand
Few people there must be who have not at some time or other come lace to face with one of those most peculiar of insects popularly known as ‘Stick Insects’. These grotesque animals, which at close quarters wear an almost human quizzical expression, are normally denizens of tropical lands, but strangely enough they are very common throughout the length and breadth of New Zealand.
Tropical forms of stick insects are generally winged, but all the New Zealand forms, some 19 species in all, are apterous and occur from North Auckland to Stewart Island, on the lowlands and on the highlands, up to heights of 4,000 to 5,000 feet. Some dwell on scrub, others in the forest, while high-mountain forms are usually found on tussock, sedges or Dracophyllums. Of cryptic colouration and concealing habit, they are seldom seen except when moving, and are obtained by collectors usually through the beating or sweeping of foliage. When disturbed stick insects are generally cataleptic, and will release their hold on foliage and fall or slide down the stems of a plant to the ground, or some other solid resting place, where they will remain motionless for hours with the fore legs held outstretched and parallel beyond the head, and with the middle and hind legs directed posteriorly and pressed against the sides of the body. When at rest amongst foliage, these insects either hang below branches and twigs or arrange themselves in such a way that their elongate bodies merge with the surrounding branch system so as to resemble a twig; hence the popular name of stick insect. All New Zealand species are protectively coloured so that their colour and colour pattern blends with their surroundings. This is particularly so with one species of Clitarchus which normally rests with its body pressed flat against the trunk or branches of manuka and kanuka trees. Except to the trained eye this species would pass unnoticed on the bark until it moved, so beautiful is its camouflage.
All stick insects are foliage feeders, but except for odd occasions they do little visible damage to shrubs or trees, and cannot be classed as economic pests. Occasionally, however, large numbers are found defoliating a single tree, sometimes in a private garden. Outbreaks of this nature arise from a culmination of factors such as the continued presence of a gravid female insect in that locality during the preceding season, favourable and mild climatic conditions, and luscious growth of the plant. Usually they last for one season only, the insect numbers being rapidly reduced with the onset of early winter frosts and cold weather.
The male stick insect is easily recognised, having a body shorter than and very much thinner than that of the female; some males are mere
Acanthoxyla; all these species exist as females only, reproducing parthenogenetically. That one exception is A. senta found only on Great Island of the Three Kings group, North Auckland. To the genus Acanthoxyla belong all the common medium-sized green and brown spiny stick insects of New Zealand which range over the lowlands of the North, South and Stewart Islands. The very large brown spiny stick insects found in forest trees in many parts of New Zealand, and measuring up to 14 in. in length, belong to the genus Argosarchus.
Returning to the breeding habits of these insects, in those New Zealand species in which males do occur both fertilized and unfertilized eggs are laid by the female, and both hatch. The fertilized eggs produce both males and females but the unfertilized eggs produce females only. The common smooth-bodied New Zealand stick insects belonging to the genus Clitarchus behave in this way, and will reproduce either parthenogenetically or sexually, though males of the species are almost as common as females. Clitarchus hookeri can produce two broods during the spring-summer season by parthenogenesis, and from a single original female kept in captivity I have continued to breed successive parthenogenetic generations for five years.
When mating occurs the male stands upon the back of the female and passes its abdomen down and round one side of the female's abdomen to reach the genitalia below. In this position the male is carried around by the female, often for several days or even one to two weeks at a time, each insect feeding independently as the female moves from place to place. The coitus is periodically interrupted while some eggs are laid, but during this egg-laying period the male does not necessarily leave the back of the female.
With insects kept in captivity it is not unusual to find males of one species attempting to mate with females of another species, or even another genus.
Although foliage feeders, each New Zealand species of stick insect has very definite food requirements. All species will eat manuka, but only Clitarchus hookeri can be successfully brought through its entire life history on a diet of manuka alone. Species of Acanthoxyla or Argosarchus confined on manuka will thrive for a few weeks, but then slowly die unless fed some of their specific food plant. In the case of species of Acanthoxyla rata or pohutukawa leaves are essential, or possibly also rimu and totara. With Argosarchus, Myrtus bullata, the ramarama is the essential food plant.
Newly-hatched stick insects, unless given their appropriate food plant within 24-48 hours of hatching, will always later die. even though they may live for some considerable time and grow, on the wrong foliage diet.
Acanthoxyla sp. A colony of New Zealand Acanthoxyla prasina was introduced into England accidentally in 1910, near Paignton, and is still thriving there on Japanese cedar. Both A. prasina and. Clitarchus hookeri have found their way from New Zealand to the Scilly Isles, where they are established in the gardens of Tresco Abbey.
Stick insects will feed during the daytime, but for the most part they are nocturnal, remaining concealed in foliage during the day. They walk fairly rapidly with a peculiar ambulatory or swaying motion, and individuals will travel considerable distances, often over open country, for no apparent reason other than an urge to migrate.
One of their most fascinating habits is the performance of the ‘dance’, a peculiar motion in which the body is swayed from side to side by a flexing motion of the legs at the trochanteral-femoral and femoral-tibial joints. This may continue for half an hour or more without stopping. It starts and stops for no apparent reason, and has no apparent purpose. I have seen more than a hundred young stick insects standing on the glass of a breeding cage, all performing this dance together and in unison — an extraordinary sight. I have also seen lone individuals in the field going through the same antics, usually at a little before sunset.
Most stick insects in New Zealand occur in two forms, a green form and a brown form. They always hatch green coloured, but may change to brown at the second moult. The adult insects cannot change colour, though green ones do tend to go a brownish green a week or so before dying. All species undergo four ecdyses during growth to maturity, and some can live for periods up to two years.
Stick insects are sometimes found with one or more legs shorter than the others. This arises from the accidental loss of a leg which is later regenerated. A regenerated leg is always shorter than the normal leg, its actual length depending on the instar in which it was lost. For instance, if it is lost in the first instar it will be only slightly shorter than normal when the insect is full grown; if it is lost in the second or third instars it will be correspondingly so much shorter; a leg lost after the last moult cannot be regenerated, and remains as a stump. Legs lost are usually broken off at the coxal-trochanteral joint which covers over with a small black cap, underneath which the new leg gradually grows. As growth proceeds, the cap extends until a small loop or hook-like structure results, within which can be seen the perfect leg in miniature. This persists in this state until the next moult, and when the insect emerges from its old skin the loop has been replaced by a perfect though smaller leg.
It is not unusual for a leg to be lost during moulting, and in fact stick insects suffer a severe mortality rate during moulting as they sometimes fail to emerge properly from the old skin and become strangled, as it were, by it.
Stick insects belong to the Orthoptera, Family Phasmidae. They are absolutely harmless and are quite amenable to handling, though easily injured by crushing.
The Samoans are still ‘navigating’ people and have remained so ever since the time they were named ‘Navigators’ by the early discoverers of the Islands. This navigating instinct, strictly speaking, refers to two practices: firstly, crossing to and from neighbouring islands, and secondly deep-sea fishing — catching bonito fish with a rod and line; catching sharks with a certain rope-lasso.
Shark-fishing is, to the Samoans, just as old as their island myths and legends, but it is still exciting and customarily honoured at present as though the pre-civilized era were still existing in full. Why? The seasonality of the pastime creates immense pre-fishing felicitous anticipation; the excitement in performing the art of catching sharks and economically the high utility of the meat to the consumers, to say nothing to some extent of the fact that the pastime is still a periodical custom and tradition.
While the seasonality of shark-fishing still remains the same, it is interesting to note now that as far as the seashore fringing villages are concerned, the practice has degraded from general to regional. By regional it is meant that only those villages which have no lagoons or have poorly fish-infested ones are still engaged in shark-fishing, e.g. Falailua side, Falealupo, Miafu, Saleqa, Falefa, Fatufia, Tofua (all in Savaii), A'leipata, etc., in Upolu. Likewise the capability, the interest and the bravery naturally required to carry out the game, if it is the from father-to-son knowledge of deep-sea navigation, also shows degradation.
Rowing boats of any length are now generally used to go out fishing shark, although bonito-canoes as in the past are occasionally seen
According to the village Fatufia, there are two noted seasons in the year for shark-fishing, from April to May, and November to December. The first season is more prominent owing to the fact that at that time the sharks are exceptionally ferocious. And this makes them dash nearer and daringly to the boat. How the Samoans know this very critical time is when a certain ‘long-leaf’ grass (called Sefa) bears blossoms. The second season, of course, comes on after the ‘Palolo’ time.
Shark-fishing as a game has certain ‘play-grounds’ — it is not done anywhere in the deep sea. There are certain shallow locations in mid-ocean; these are known to the fishermen as ‘Aau’ or Reef. Some locations are closer to land — about twelve miles, but some are so well out in the open that land is totally invisible. Apart from the ‘reefs’ there are other fishing points right in the fathomless ocean. But these places are marked, by experience, by the fact that the ocean currents converge to and assume in those spots a boiling or whirling appearance.
The fishermen, apart from taking enough provisions for themselves, take bait — eels, canned meat (putrified), a killed dog, guts of a pig, or flying foxes.
For fishing implements they take three to six rope lassos, knives, specially-made sticks called ‘Taova'a’ and ‘Paletua’ respectively, Tui-ipo or string of shells, the string and floater or ‘Fau-ma-le-uto’, and, of course, the important signalling shell-horn.
As soon as the boat reaches the fishing location selected by the leader, one bait tied to the stern and another to the bow are hung down into the water. If it is night-time the string and floater will then be used because it is the only means the men have of knowing the presence of sharks. Note that the floater is a flat piece of board about 3 in. × 8 in. × 14 in. On its top flat surface there are baits fastened to it with wires or strong strings, and one of its ends is fastened to a string which is held by one of the men on board. The floater-and-bait is really a tamer and a teaser at the same time to the sharks.
It is said that the sharks find great difficulty in biting off the baits on the floater, and this makes them more ferocious and madder. At the same time the holder of the floater-string slowly pulls the floater and bait closer to the hull of the boat where the leader with the rope-lasso is in readiness. The lasso is half submerged, and as soon as the shark's head goes through the loop the leader pulls on the rope, having the loop locked around the gill-area or thereabouts. With patience and might the operator holds on fast to the rope, and at the same time tries to raise the shark's head upwards. At the same time one of the men with the ‘Taova'a’ shoots the latter as far as possible into the shark's mouth and throat, while another man with the ‘Paletua’ or wooden hammer strikes the shark's head with heavy blows. Within an instant the ‘catch’ is in the boat kicking about.
While this excitement goes on, one of the crew will keep his head steady and keep on throwing down bits of bait to tame the shark's darting about.
At day-time fishing the ‘bait floater’ will not be used, for the men will plainly see the sharks.
In case there may be one or two sharks or nothing at all in a certain location, then the men will use the ‘Tui-ipu’ or string of coconut shells (about six to ten shells strung through a 4ft. stick). The splashing and the crackling noise of the shells on shaking in the water imitates the splashing and the darting about of a school of big fish preying on a shoal
By custom, the leader is the first to catch a shark, and then everybody's turn follows. Every shark caught means a blow of the horn.
Some names relating to shark-fishing:—
Va'alepa: Va'a = boat; lepa = flood; Va'alepa means the floating boat. ‘Va'alepa’ refers strictly to any rowing-boat used at the time for shark-fishing.
Va'aeva: Vaa = boat; eva = sail for joy. ‘Va'aeva’ is another name which refers strictly to a rowing-boat used at the time of shark-fishing. A synonym for Va'alepa.
Aata: Refers to a school of sharks.
Ua manu le ata: ‘Ua manu’ means plenty sharks, and are surfacing. The saying refers to a school of sharks surrounding the boat and showing up their fins in the air.
Formalin, now the most abundantly used preservative for bulk zoological material, was introduced following the simultaneous discovery of its value for this purpose by Hermann and by Blum independently in 1893. It is difficult to-day to appreciate that its use was much debated and in question even after 1900. The solution of the pungent formaldehyde in water was variously called formalin, formol, and formalose, each being a commercial name. Its efficiency, cleanliness, general stability, economy in use, availability and freedom from custom control have combined to favour formalin over alcohol as a bulk preservative; but its effect on the skin of a person handling formalin-preserved material has always offset its many other advantages.
There is a wide range in this reaction to formalin, from those persons who seem essentially immune from its effects in low concentrations, such as after preserved material has been subjected to prolonged washing, to those unfortunates who are highly sensitive and develop a rash even on areas of skin, such as the face, not actually in contact with formalin-solution. These are extremely rare; but commonly it is found that sensitivity to formalin increases with time. The reaction shows on the hands. The skin becomes dry, grained, it splits and the quick round the nails splits, the nails become hard, and ultimately a slight general red rash may appear.
Such effects from formalin were previously countered by prolonged washing of specimens before use, repeated washing of the hands, and by palliative treatment with glyerine and rosewater, and lanoline. Too frequently, even in spite of such care, it was necessary to stop work on formalin-preserved material. The sensitivity would remain high, even ten years later. Recently the washing of specimens in solutions of urea or hypochlorite was practised, but while helpful, such washings are no complete safeguard.
The subject of reactive dermatitis has been important in industry and much researched into. During the early war years, a form of protective hand-cream was discovered. The hands are washed clean, carefully and thoroughly smeared with a thin coat of the protective cream. This rapidly dries to an invisible layer which, properly applied, acts for three or four hours as a complete protection against a wide range of harmful substances. They are termed ‘barrier’ creams, and fortunately one such cream gives full protection to the hands against the effects of formalin. This cream, a so-called ‘wet’ cream, was introduced into general use in our zoology laboratories this year with so far complete success for a large number of students. After much dissection-work on washed material, the skin of the hands remains soft, smooth, and without sign of splitting or of cracking. There is no indication of tingling or other sensations. One person with a long-standing acquired hypersensitivity has been able to handle formalin-preserved material without discomfort for the first time in ten years.
It appears that a suitable barrier-cream now removes one of the major disadvantages of formalin as a preservative.
In the last century when microscopy was a popular hobby, as in the days when Kirk, Maskell and their associates formed a Microscopical Section of the Wellington Philosophical Institute, the ciliates were much studied and admired for their size, varied and often beautiful form, and activity. None are really small. The ordinary powers of the microscope are adequate for the study of their behaviour, of the often strikingly attractive detail of their structure, and of the cilia and ciliary mechanisms responsible for locomotion and feeding in most species. Observations are best made on the live animal, or at best with simple staining techniques, so that an expensive laboratory is not needed except for the most advanced work. They were, and are, readily obtainable and can be easily cultured. Hence their former popularity, which it is hoped this survey will assist to restore, since it presents not only adequate data for the recognition of many common species, but in adding eleven species (marked with an asterisk in the list) to the 123 species previously known from this country, it shows that the study of these animals is still scientifically profitable in this country.
The species included in the present account have been taken from a variety of habitats, permanent ponds and pools, bush tarns, slow-running streams, aquarium tanks, etc. Ciliates have quite marked habitat preferences, and it is accordingly necessary to examine a range of habitats if any number of species are sought. In fact, the preference is so strong in many, that species found close to the surface may be different from those
Vorticella and Paramecium, which are so freely used for laboratory studies; but in addition other, and often equally useful, animals such as Spirostomum and Stylonychia will be found.
Bary (1950) has reviewed the knowledge of our freshwater ciliates, and in this came to the conclusion that the species found here are essentially cosmopolitan. Thus the New Zealand microscopist has the advantage of a world-wide literature, and is not faced with the study of a group of animals peculiar to this country and difficult through their systematic novelty. The study of Protozoa is now well organised, and the microscopist who commences work in this group will find that the literature listed in this paper will provide an adequate guidance through most systematic problems. In fact, this and many other aspects of the ciliates make them most suitable for researches in the home or in small or poorly-equipped laboratories.
Culturing is simple, a convenience and an advantage, since by culturing species can be kept alive in the laboratory over long periods and when wild stock is cultured different species ‘bloom’ at different times. If a new culture (subculture) is started when a species ‘blooms’ and is dominant in the original culture, it is not difficult to establish a pure culture of that one species which can be then repeatedly cultured, and so kept alive for intimate detailed study of its structure and life-history. Kirby (1950) gives an extensive account of the many different techniques which can be used in the study of ciliates and other Protozoa; but the following will prove adequate for most free-living freshwater ciliates.
All ciliates are best studied as live material which will yield data adequate for the diagnosis of nearly every species ordinarily encountered. This can be done with the ordinary wet-mount preparation on a 3 in. × 1 in. slide with a coverslip, but a ‘hanging-drop’ mount will often keep a single specimen alive for one or more days and so allow prolonged study. A glass ring ¼ in. or less in thickness is cut from ½ in. internal diameter glass tubing and ground on a carborundum stone to give parallel sides. This ring is sealed with vaseline to a microscope slide, and the upper surface lightly smeared with vaseline. A small drop of water with a specimen in it is placed on a coverslip. The slide is inverted, the edge of the ring touched lightly on the coverslip with the drop centered in the ring so that the coverslip adheres to the ring, and the whole gently but quickly turned over so that the drop of water now hangs from the centre of the coverslip. This preparation is examined in the usual way. The drop must be as small as practical, or the specimen cannot be kept in focus under high power.
If a preparation is made with very dilute Indian ink in water, the action of cilia, the manner of feeding and the formation of food vacuoles can be very clearly observed. While it is not essential to slow most species for observation, and many will slow down on a slide after some minutes, the following methods can be used when necessary.
Gelatine Solution: Make a 3% solution of gelatine with distilled water. One drop of this stock solution is placed on a slide and warmed slightly. Add to this one drop of culture fluid. This gives a gelatine concentration of approximately 1.5% which slows most ciliates successfully. Some of the more delicate species may survive only for a short period.
Copper Sulphate: One drop of 1% copper sulphate in one drop of culture, giving a solution of 0.5%. Species were slowly killed with little change in form until death, when autolysis occurred. An 0.2% solution gives a longer period of immobility before death.
Urethane: This crystalline narcotic was found useful in immobilising species such as Coleps and Stylonychia. Place a crystal of urethane to one side of the coverslip in contact with the culture medium. Distortion occurs if too high a concentration of the narcotic is used, but if in sufficient dilution, movement is retarded.
Isopropyl Alcohol: Used as the vapour (Bary's modification of Bills' [1922] method). Expose a drop of culture fluid on a slide to the fumes of a 2 to 5% solution of the alcohol for several minutes. Slows down individuals for a short period before they explode. This is recommended by Bary and was most useful for species of Paramecium and Halteria grandinella.
Cooling: Place slide on block of ice for several minutes. A drop of acetic acid on the coverslip eliminates condensation.
Nuclei are well stained with dilute methyl green (methyl green 1.0 grm., glacial acetic acid 5 ml., aq. dist. 95 ml.). Permanent preparations are not easy since it is difficult to fasten specimens to a slide, but if a smear is made and allowed to nearly dry and is then gently immersed in Schaudinn's fixative, it can be stained with Heidenhain's or Ehrlich's hæmatoxylin and mounted, after dehydration, in Canada Balsam (see Tuatara Vol. V [1], p. 14).
Cultures can be held in 1 in. test-tubes, 4 in. × 1 in. vials, or 100 cc. conical flasks. These are plugged with cotton wool. The object in preparing a culture is to supply an adequate stock of bacteria as food, and an environment in which the oxygen content of the water can be controlled. If a conical flask is nearly filled, the surface area is smaller than in a partly-filled flask, and as a result the former has a slower oxygen uptake than the latter and so is suitable for many mud-inhabiting species, while the latter can be used for surface-dwelling species whose oxygen requirement is higher. Since the nutrient requirements of the bacteria are
Paramecium and other species needing a low oxygen content, a culture can be maintained for one or two years simply by adding small amounts of hay-stalks, lettuce or grain to the old culture as necessary, or by pouring off three-quarters of the old medium and replacing with fresh. Hay infusion was found a most useful general medium in this study, and lettuce, wheat-grain and flour-hay infusions were satisfactory.
Culture Media, etc.: (Quantities specified below for culture media — after Kirby 1950).
Hay Infusion: Boil 2 grm. of hay in water and add water (distilled or rain-water) to make 1.000 cc. The infusion may be made up in a more concentrated form (10 grm. hay to 1,000 cc), autoclaved and stored in sterile containers for future use. Dilute to the former concentration before use. Timothy hay is usually stated as the best medium. In the present study the dried flower stalks of cocksfoot (Dactylis glomerata) or similar grass was found very satisfactory.
Lettuce Infusion: Dry lettuce leaves in oven until crisp or brown (do not burn). Powder dried leaves. Boil 1.5 grm. lettuce powder in 1,000 cc. distilled water for 5 minutes. For use, add 1 part distilled water to 2 parts of stock lettuce infusion. (Note: Lettuce powder may be stored.)
Wheat Infusion: Boil wheat grains in a small amount of water for 2 or 3 minutes. Add the boiled wheat grains to the culture water. The number of grains used varies with the type of ciliate to be cultured, e.g. Paramecium 60-70 grains to a litre, Vorticella and Stentor 20 grains to a litre, and hypotrichs (in general) 40 to a litre.
Flour-hay Infusion: 0.1 grm. chopped hay and 0.13 grm. white flour. Boil for 10 minutes in 100 cc. water. Stand for 24 hours and dilute with an equal amount of water for use.
Media can be kept for some time in sterile tubes (place tube in boiling water for 15 minutes, then plug with cotton-wool and cool).
Body form constant, barrel-shaped; 13 to 15 rows of regularly arranged, smooth indurated ectoplasmic quadrangular platelets; narrow intervening furrows soft, possessing cilia; cytostome. anterior surrounded by slightly larger cilia than those of the body; three posterior spinous projections; single terminal contractile vacuole, clearly visible in life; macronucleus, rounded with micronucleus lying close to it. Length: 0.038 to 0.075 mm.
Commonly found in fish ponds, etc., and usually recognisable by its light brown colour and constantly rapid revolving motion with frequent changes of
Bary (1950) described C. hirtus Muller from a Wellington locality. This species differs from C. elongatus in being slightly smaller in size, 0.04 to 0.065 mm. as against 0.038 to 0.075 for C. elongatus, and in having a greater number of rows of ectoplasmic plates, viz. 15 to 20 rows. Both species possess 3 caudal spines.
References: Kudo, 1946; Bhatia, 1936.
P. caudatum is the most widely distributed species of Paramecium and occurs in freshwater ponds, streams, etc., particularly those with a high bacterial content. The species is described and figured adequately in all elementary zoological texts. Length 0.18 to 0.30 mm. Two species of paramecium that may be confused with P. caudatum are P. multimicronuleatum and P. aurelia. The former is similar in shape to P. caudatum but is distinguished by having 3 to 7 contractile vacuoles and at least 4 vesicular micronuclei. P. aurelia is also similar in shape but smaller than P. caudatum, but differs from the latter in having 2 small vesicular micronuclei and a more rounded posterior end. P. caudatum cultures, well in hay infusion, fixes well with warm Schaudinn's and stains readily with Heidenhain's or Erhlich's hasmatoxylin. Reference: Kudo, 1946.
Body, foot-shaped, somewhat compressed and ellipsoidal in cross-section; body ciliation uniform except at posterior end where cilia are slightly longer; peristome very broad; macronucleus large, micronucleus compact; 2 contractile vacuoles; symbiotic zoochlorellæ often present. Length, 0.10 to 0.15mm., width 0.05 to 0.06 mm. Freshwater ponds, etc. Cyclosis is well marked, but locomotion is slow (not as active as P. caudatum). This species may be confused with P. putrinum which is similar in size and shape but has one contractile vacuole, an elongate macronucleus and no zoochlorellæ. Fix and stain as for P. caudatum. Reference: Kudo, 1946.
Body, oblong and somewhat compressed; peristome, long and conspicuous; macronucleus, kidney-shaped; micronucleus single and compact; 2 contractile vacuoles. Length, 0.06 to 0.12 mm. Common in stagnant waters, cultures readily in lettuce and wheat infusions. Reference: Kudo, 1946.
Body form constant, oval; ciliary rows run obliquely from right to left across the body anterior to the cytostome and longitudinally behind this region; an obvious junction marks the meeting point of the oblique and longitudinal ciliary rows; 4 posterior elongate cilia; cytostome roughly triangular and terminating in a food vacuole; macronucleus oval; contractile vacuole posterior. Length. 0.09 to 0.15 mm.
Fresh and salt water. Cultures easily in lettuce infusion. Found here in Waiwhetu Stream and Judgeford Stream. References: Kudo, 1946; Bary, 1950.
Body, elongate, ovoidal; cilia long, fine, rigid, arranged in longitudinal rows but anterior truncated extremity devoid of cilia; caudal seta long and posteriorly directed: peristome with large hood-like membrane along the edge; macronucleus spherical and central; contractile vacuole terminal in position. Length, 0.02 to 0.03 mm.
Cosmopolitan, in swamps and ponds and found here in the Karori Reservoir, Wellington. Locomotion jerky. Cultures well in lettuce infusion, not so successfully in hay infusion. References: Ward and Whipple, 1945; Kudo, 1931; Bary, 1950.
Ctedoctema acanthocrypta (Stokes)*. (Fig. 20.)
Body ovoid, slightly truncate anteriorly; caudal cirri present; pellicle refractile: peristome at right midline with a membrane on right peristomial ridge; macronucleus anterior ovoid and associated with small micronucleus; contractile vacuole posterior in position. Length, 0.02 to 0.03 mm.
Common amongst decaying vegetation in beech forest tarns, Butterfly Creek, Wellington. Size range of the present specimens smaller than those given by Kudo, but otherwise the characters are those of C. acanthocrypta. Reference: Kudo, 1946.
Body form variable, when extended oblong to fusiform; body ciliation uniform and longitudinal, some specimens having larger cilia forming a spiral at the end; peristome conspicuous, slightly spiralled, beginning at the anterior end and extending to the middle of the body; when contracted, peristome distinctly spirally coiled; cytopharynx short; macronucleus ovoidal to elongate; conspicuous terminal contractile vacuole. Length, 0.12 to 0.20 mm. Obtained from tropical aquarium tank. Cultures well in lettuce infusion. Reference: Kudo, 1946.
Body form variable, narrow to widely oval (according to habitat) but always easily recognised; anterior end rounded or slightly pointed, posterior end broadly rounded and possessing many rather elongate cilia; body ciliation relatively uniform, in longitudinal rows except for a zone of 5 to 7 rows of cilia which lie anterior to, and parallel with, the peristomial groove, and spiral diagonally in the same manner as the groove; peristome a conspicuous groove, spirally coiled, commencing anteriorly and passing diagonally across the body to terminate posteriorly in an undulating membrane; macronucleus spherical, situated half-way along the body; contractile vacuole large and terminal; a prominent group of large granules are found in the anterior region of the body; colour variable — yellowish to brownish violet. Length, 0.075 to 0.50 mm. Swims with a flexible rotating motion. Obtained from a tropical fish tank. Reference: Kahl, 1932.
Body elongated, cylindrical, length to breadth ratio 10: 1; caudal cilia thigmotactic and secrete mucous threads; ectoplasm with well-developed myonemes (which are arranged lengthwise independent of the ciliary rows), hence the body is highly contractile; peristome extending two-thirds down the body and closely lined with a short membranelle; macronucleus moniliform and twisted, beads rounded to oval or elongated and tapering at both ends, numerous micronuclei rarely visible and less in number than the beads of the macronucleus; excretory vacuole large and terminal, with canal; colour yellowish to brown. Length, 0.8 to 1.0 mm. Cosmopolitan species found in ponds with fair amount of vegetation, and found here in an aquarium tank; easily seen with the naked eye. Locomotion rapid, spiralling. Quick contraction makes for rather marked distortion of shape on fixing with Schaudinn's. References: Bhatia, 1936; Kudo, 1946; Bary, 1950.
Elongate, cylindrical, slightly depressed, broadening in the peristomial region; adoral zone of cilia slightly longer than the body cilia ectoplasm with highly contractile myonemes arranged lengthwise and independent of body cilia; peristome situated about half-way along the body, short, and lined with short membranelle; macronucleus moniliform extending almost the full length of the body; excretory vacuole large, terminal with a long dorsal canal extending almost to the anterior end; colour, yellowish-brown. Length, 0.28 to 0.31 mm., width (average) 0.020 to 0.030 mm.; length-breadth ratio 10:1.
Freshwater, found in an aging Paramecium culture of material obtained locally. The body length of the present specimens is shorter than the 0.40 to 0.60 mm. recorded by Kahl but all other characters for the species are in accordance with those given by Kahl. References: Kahl, 1932; Kudo, 1946.
Body elongate and with groups of long cilia here and there; highly contractile; anterior end, trumpet-shaped; posterior end drawn out and terminating in a flattened attachment disc; longitudinal and horizontal striations visible in the anterior trumpet-shaped region; anterior disc curves and sinks towards the cytostome and bears long cilia which create a feeding current; cytostome leading to ciliated cytopharynx clearly visible in feeding individual; macronucleus long, and band-like; contractile vacuole large and distinct, situated anteriorly on the left of the cytopharynx and with a collecting canal extending for two-thirds the body length; yellowish-white to greyish-white in colour with many food vacuoles. Length: 0.5 to 1.0 mm. Collected from freshwater aquarium. Reference: Kudo, 1946.
Body urn-shaped, setiform cirri arising in threes from obliquely placed equatorial indentations; peristome small with vibratile membrane on one side
Body spherical or broadly fusiform; 15 large cilia form a clockwise adoral zone; equatorial region of body bears long bristles but is not ciliated otherwise; macronucleus oval and micronucleus also present. Length, 0.04 to 0.05 mm.
Common in pond water and found here. Distinguished from the variety cirritera by the presence of zoochlorellæ and more slender body-cirri. Locomotion is by rotation about the oral-aboral axis of the body accompanied by sudden leaps. References: Kudo, 1946; Ward and Whipple, 1918.
Horseshoe-shaped, with the anterior end slightly pointed towards ventral surface; posterior end irregularly truncate; dorsal surface more convex; right side of body with one dorsal and one ventral ciliary row in the posterior region; usually three conical tooth-like structures without spines on the left side and four on the right side in the anal region; comb-like structures lie posterior to the oral aperture; two large oval macronuclei situated close together in a dorsal position; large posterior ventral contractile vacuole. Length, 0.038 to 0.045 mm.; width, 0.027 to 0.030 mm.
Lives in sludge and found in tropical fish tank. Locomotion by irregular, slow jerky movements. Good permanent slides can be made from Schaudinn-fixed, iron hæmatoxylin stained material. References: Kahl, 1932; Kudo, 1947.
Body form constant, ovoid to reniform, ventral surface flat, dorsal convex; eight frontal, five ventral, five anal and three caudal cirri, and with a continuous border of marginal cirri; peristome half as wide as the body; two macronuclei, oval or elongate; contractile vacuole single, spherical, situated near the posterior angle of the peristome. Length, 0.125 to 0.150 mm. Cosmopolitan species, common in ponds and found here in fish-ponds. Locomotion rapid, both by swimming and creeping. Cultures readily in lettuce infusion. References: Kudo, 1946; Bhatia, 1936; Bary, 1950.
Body ovoid, left side straight; ‘giant’ and ‘dwarf’ forms may occur along with normal individuals; 8 frontal cirri (typical of the genus) are usually present and 5 ventral cirri, but variable, sometimes with 7 ventrals; peristome with a protruding upper lip and an undulating membrane; 2 large macronuclei each with a closely associated compact micronucleus; contractile vacuole on left side posterior to peristome. Length, 0.10 to 0.30 mm.
Abundant among decaying vegetation in Waiwhetu stream. Feeds very actively on algæ and diatoms. Reference: Kahl, 1932.
Body form fairly constant, but proportions of length to breadth variable (2: 1 up to 2.5: 1); posterior end tail-like, anterior end flattened, left side straight, right side convex, making the body asymmetrical and bluntly rounded anteriorly; marginal cirri long, with three barely differentiated caudal cirri; peristome roof-like and has a ventral ectoplasmic fold or inner lip; large undulating membrane present between outer and inner ectoplasmic lip and ends in the same position as the outer lip; endoplasm yellow, thickly but evenly granulated. Length, 0.08 to 0.13 mm. Reference: Kahl, 1932.
O. Peritricha. S.O. Sessilia. Tribe Aloricata. F. Epistylidæ.
Pyxidium cothurnoides Saville Kent*. (Fig. 8.)
Body vasiform, widest centrally; frontal ciliary disc small and oblique and has two circles of fine cilia; stalk simple (not branched) and very short; body surface smooth, no striations; peristome not constricted from the body proper; macronucleus elongate bean-shaped; single contractile vacuole. Length, 0.09 to 0.10 mm.
Attached to freshwater animals (Kudo) and found here in ‘silage’ heap vegetation. References: Kudo, 1946; Ward and Whipple, 1918.
Body form bell-like, slightly ovate with a narrow ciliated peristomial disc surrounded by a thin ring-like margin; pellicle annulated; contractile stalk present and contractile fibres often visible towards the base of the body; peristome short but with long œsophageal fibres; macronucleus large and band-like, micronucleus present; large, contractile vacuole; colour, yellowish. Length, 0.065 to 0.083 mm.; width, 0.022 to 0.050 mm.. Stalk size: Length, 0.020 to 0.385 mm.; width, 0.0015 to 0.0040 mm.
Solitary, attached to submerged objects. Obtained from a slow-moving stream at Butterfly Creek, Wellington. The species readily encysts. At the commencement of encystment the stalk contracts and coils spirally; at the same time the body encysts, the peristomial disc being contracted and the body folding up and over the peristomial region. Encystment is rapid under artificial conditions. The species has the ability to live in bacteria-rich cultures. Telotroch formation takes place under conditions of low oxygen tension. This was observed on more than one occasion in the present study. The sessile animal develops a posterior ring of strong cilia, breaks away from its stalk proximally and leads for a time a free swimming existence. The time occupied from the appearance of the cilia (Fig. 10B) until the animal becomes free varies from 5 to 10 minutes. During this time the cilia grow in length and their beat becomes progressively stronger until suddenly the body of the animal is freed from its stalk. Fig. 10 shows the formation of the telotroch.
References: Kudo, 1946; Bary, 1950.
Body of an inverted bell-shape with slight but constant changes of form; pellicle smooth; stalk highly contractile; contractile fibres often visible towards the base of the body; peristomial disc broad and ciliated with a lip and vestibule leading into the cytostome; macronucleus horseshoe-shaped, micronucleus? (not seen in any of the material in the present study); contractile vacuole small and towards the distal end. Length, 0.053 to 0.067 mm.
Usually attached to submerged water plants (often Nitella). Gregarious. forming large masses easily visible to the naked eye. Reference: Bary, 1950.
The publication of this issue of Tuatara ends the longest period between issues since the journal was first published, and we hope you will accept our apologies for the delay. An unusual combination of circumstances brought this about and we hope that such circumstances will not recur, and that it will be possible to provide you with future numbers regularly. We appreciate the interest in Tuatara that is shown by the number of enquiries we have received about the progress of this issue. Our subscription cards are fully up to date, and you will be notified when new subscriptions become due. The journal is at present in a quite sound financial position.
Your attention is drawn to the advertising section, and we hope that you will support our advertisers (mentioning the journal), for without their support it would not be possible to publish Tuatara.
At our request Dr.
Sixty years ago the chemist's contribution to botany and agriculture was limited to the analysis of soils and manures. He advised farmers concerning the fertility of their soils and what manures should be applied to obtain the highest crop production. In these early studies the main emphasis was placed on nitrogen, phosphates, potash, and perhaps lime. The other so-called minor elements such as boron, magnesium, manganese, zinc, copper, etc., that are now known to be essential for plant growth were not then considered. This work led naturally to the analysis of plants, particularly of the mineral contents that remained after plant material had been carefully burnt. Thus attempts were made to correlate the uptake of minerals by crops with the amounts of those minerals in the soil.
But nitrogen requirements of plants had intrigued chemists from the start, first as to whether plants could draw on atmospheric nitrogen; and later when it was recognised that most plants drew their nitrogen from the soil, the problem of explaining the stimulus to plant growth that resulted from additions of small quantities of nitrates (20 Ib. per acre) to soils that already contained several thousand pounds of nitrogen per acre. It soon became apparent that the total amounts of nutrients in the soil, whether minerals or nitrogen, were not available to plants but only a portion that was soluble in the soil water. Nitrogen was of course driven off during the combustion of plant material, and could not be isolated from the ash. In the early days it was measured as total nitrogen by techniques in which it was removed from its chemical compounds as ammonia. The compounds in which it occurred in the plant were not identified, but were hypothesised as ‘proteins’ and ‘non-proteins’ (amino acids, etc.).
Though some attempt had been made to study physiologic processes such as photosynthesis prior to 1920, the major consideration of plant chemistry during the 1920's was directed towards an evaluation of the nutritive value of plant products in terms of carbohydrates, proteins, fibre, and later minerals. The techniques often ignored the actual identity of the proteins and sugars concerned and aimed only at estimating the total food value under general headings such as protein, fat, fibre, ash, and by subtraction, carbohydrates. A natural corollary of this work resulted in studies of the utilization of foodstuffs by animals, their digestibility and
By such means of trial and error parts of the photosynthetic and of the respiratory processes were located, but their significance in the physiology of the plant or animal was not fully established until we had means of labelling individual atoms and molecules, and tracing their movement through various chemical compounds that constitute the organic body. For such studies the chromatographic techniques are ideal in that they require very little labour and permit analysis of minute quantities of material, and the separation and identification of individual compounds even though these may be represented by only a few molecules of each. Once the compounds have been separated those that are labelled with radio-active elements can be readily recognised. As the process proceeds for longer periods more and more compounds acquire radio-active elements and so the sequence in which compounds are formed can be determined. If radio-active carbon which has an atomic weight of 14 and is usually represented by C14 is brought into contact as C14O2 with the actively photosynthesising plant, the first stable product in which the radio-active carbon accumulates is phosphoglyceric acid (probably 2-phospho-d-glyceric acid), a compound with three carbon atoms in the molecule. In this the radio-active carbon is located in the carboxyl (C14 OOH) group of the acid. In photosynthesis the radio-active carbon appears early in phosphopyruvates and pyruvic acid besides other compounds, and shortly afterwards in glucose. The position of the C14 atoms in the first formed glucose suggests that this substance is derived by a chemical fusion of two molecules of glyceric acid in such a way that two carboxyl (C14 OOH) groups condense and the two carbon atoms become the two central atoms of the six carbon atom chain which forms the backbone of the glucose molecule.
Such information could scarcely have been obtained by the older methods of chemical analysis — and the outline given above is but a very small part of what has been achieved by paper chromatographic techniques in the study of photosynthesis.
Chromatography has been freely used in many fields of physiologic research, both plant and animal, and can be adapted to study any compounds that occur in plants or animals provided the molecular structure permits their movement with the solvent. The method has been adapted also to the isolation and identification of inorganic compounds.
In New Zealand the techniques have been used for the past fourteen or fifteen years — notably at the Plant Chemistry Laboratory at Palmerston North, the Medical Research Council Laboratories, the Wallaceville and Ruakura Animal Research Stations, the Dominion Laboratory, the Fats Research Laboratory, and other laboratories.
In other fields it is adapted to the study of vitamins, hormones, growth substances (auxins), nucleic acids, respiratory processes, physiologic differences between resistant and susceptible plants, the study of physiologic differences of closely-related genotypes, hydrolytic products, enzymes and their products, bacterial physiology and many other types of the research. The methods of developing and identifying the compounds which are isolated are just as intriguing. Radio-active isotopes are autophotographic and may be identified by the fogging they produce on a photographic plate. Other substances are fluorescent or may be made so, while the commonest method is probably to develop the paper by treatment with a reagent that brings about a colour change of the compound. This may require heat to bring out the colour. Vitamins may be identified by biological assay — i.e. the adding of a culture medium which lacks a given vitamin or other food material to the paper, and inoculation with a specific organism (bacterium or fungus) that will grow only when the vitamin or other substance being studied is present. Thus when the chromatogram is incubated, growth occurs only on the spots in which the vitamin is located. Occasionally two compounds may move on the paper as one, but require different reagents for their development. The scope and possibilities of the techniques are numerous and offer a wide scope for ingenuity and imagination.
Last year Dr. Harvey prepared the following schedules for the Botany course as a practical introduction to the techniques. The procedure was simplified so that initial separations could be made with ordinary laboratory equipment, e.g. corked test-tubes, filter paper and about four or six solvents and reagents. After the first separation of known compounds in prepared solutions had been made, the class found considerable interest in identifying the amino-acids and sugars in several fruits and vegetables. The technique is easily mastered and is adaptable to many projects that could be carried out in post-primary school laboratories such as the changes that take place in the ripening of different fruits, seasonal changes in the amino acid and carbohydrate compounds of growing and/or dormant plants, the changes in seeds during germination, or during their formation, and many other projects. The advantage of the method is that only one drop of liquid extract is necessary for most analyses. The main precautions are; (1) the drop applied to the paper must be allowed to dry before the paper is placed in the solvent to be run; (2) the drop must not be so near the
The technique of paper chromatography, although of comparatively recent origin, has assumed such importance in the last few years that it is now no longer a novelty and every worker in the fields of chemistry and the biological sciences should have some knowledge of the scope and limitations of the method. The aim of this article is to give an introduction to the subject, sufficiently detailed to enable the non-specialist to make use of the technique in at least its simpler forms.
When Consden, Gordon and Martin, in 1944, published their experiments with paper chromatograms, several branches of pure and applied chemistry were almost revolutionised overnight. Problems which up to that time had seemed virtually incapable of solution became amenable to attack and such was the simplicity of the equipment required and the techniques involved that interest in the method spread rapidly and its use is now commonplace in laboratories throughout the world.
Paper chromatography is essentially an analytical tool for separating chemical compounds, and in this respect is comparable with distillation, crystallisation, precipitation, etc. The latter methods all depend on differences in volatility or solubility of the substances involved, whereas partition chromatography in general, and paper chromatography in particular, rely on differences in another physical property — namely the partition coefficient. If a pure substance is shaken up with two immiscible solvents the ratio of the concentrations of the substance in the two liquid layers is known as the partition coefficient. For a given solute and solvents at constant temperature the partition coefficient is a constant, and, in particular, is independant of the actual concentration of solute in the liquid phases. Partition coefficients vary from 0 to 1 and it is the small differences in the partition coefficients of various substances which are utilised for their separation.
Paper chromatographic methods possess several advantages compared with the more conventional methods of separation. Perhaps the most important of these is the facility with which substances which are extremely similar chemically can be separated. The classical example of this kind is the separation of the amino-acids and of the carbohydrates. So great are the difficulties associated with the separation of members of either of these important groups of substances by methods other than chromatographic ones, that the analysis by conventional methods of even comparatively
Another major asset is the small amount of material required. Qualitative analyses can be carried out on amounts which may be as small as a few micrograms of each component, and only slightly larger quantities are often sufficient for approximate quantitative results. Few other analytical procedures can be adapted to this minute scale, yet in the case of material of biological origin, amounts greater than a few milligrams may be almost unobtainable.
The wide versatility of paper chromatographic methods is illustrated by the fact that they may be successfully employed for separations in such diverse series as the amino-acids, sugars, plant pigments, phenolic constituents of wood, etc., volatile fatty acids, sugar phosphates, and related compounds, nucleic acid degradation products, compounds such as adenosine triphosphate (A.T.P.) and adenosine diphosphate (A.D.P.), metallic ions, etc.
In view of this wide range of application and the fact that the equipment required is simple and the technical skill required not great, it is not surprising that the methods of paper chromatography have assumed such importance in a short space of time.
Partition of a solute or solutes between two immiscible liquids was utilised by Craig in his elegant counter current distribution technique which is essentially a method of carring out a series of extractions of one liquid phase with another in a definite order. However counter current distribution studies often require considerable amounts of material, may require expensive precision equipment and are frequently time-consuming. Martin and Synge conceived the idea of keeping one of the liquid phases stationary and allowing the other liquid to pass continuously over it, thus essentially having an extremely large number of extremely small-scale extractions. If one of the liquid phases is water it may be held stationary by absorbing it on the surface of a material such as silica gel, diatomaceous earth, starch or cellulose. This idea led to the development of partition chromatography using columns of materials such as these saturated with water, with solvents such as chloroform or butanol flowing slowly down the column. This technique is of great importance and is used extensively for the separation of substances in amounts of the order of 5-10 mg. upwards.
For smaller quantities Consden, Gordon and Martin showed that a sheet of filter paper serves admirably to hold the stationary phase (water) and the moving phase may be made to travel slowly up or down the paper by capillary action and/or gravity: thus paper chromatography came into being.
In practice the mixture to be separated is applied in the form of a small spot a few millimetres in diameter near one end of a strip of filter paper which is then hung in an atmosphere saturated with both water and the
The ratio F value, is a constant which, for a given solvent at a given temperature, is characteristic for the substance, and thus ‘unknown’ substances may be identified with some degree of certainty by measuring their RF values preferably in several different solvents. RF values should be reproducible, but sometimes careful control of conditions is necessary for this to be the case, and it is normal practice to run standard spots simultaneously alongside the ‘unknowns’.
A number of factors influence the ease with which paper chromatograms can be successfully prepared, and of these brief mention may be made of the design of the equipment, the type of paper, the nature of the solvent, and the method of detecting the spots.
The original apparatus used by Consden, Gordon and Martin consisted of a drain pipe closed at the lower end by immersion in a dish containing the aqueous phase and at the upper end by a sheet of glass. A small glass trough resting on the shoulder near the top of the pipe contained the solvent and the paper, held in the trough by means of a glass rod or sheet, passed over a glass rod and hung down inside the pipe. If the paper was hung directly over the rim of the trough difficulty was experienced with solvent syphoning out between the paper and the trough. It is convenient to be able to follow the progress of the solvent down the paper visually and a tall glass jar is a more satisfactory container. The trough must then be supported on some type of stand preferably of glass (to resist the attack of solvents). Metal troughs have the great advantage of not being fragile but are often unsatisfactory with strongly acidic or basic solvents. It is an advantage, for reasons which will appear later, if the solvent can be run into the trough through a small hole in the top cover so as to avoid opening the jar completely to the atmosphere.
The ascending technique requires less complicated apparatus and any container in which the paper can be hung so that it dips into a pool of solvent at the bottom may be employed. Again a glass container is
In general the descending method is preferred for accurate work, although the ascending technique is somewhat simpler and requires less complicated apparatus. The former method has the advantage that the solvent may be allowed to run off the bottom of the paper and, although RF values can not then be determined, substances with very low RF values can be separated more completely. As mentioned above, standard spots are usually run alongside the ‘unknown’ so that the fact that RF values are not obtainable is not of major importance. The ascending method, on the other hand, has the advantage that the solvent cannot ‘run off’ the paper and thus the chromatogram needs less attention.
A combination of the two methods in which the paper dips into a trough, runs up over a glass rod and then hangs down is useful especially when handling large sheets of paper as it obviates the use of a large tall glass container and vessels such as glass aquarium tanks may be employed Whatever the type of equipment used it is most desirable that it can be sealed as completely as possible.
A large variety of types of paper have been tried, some of which possess advantages for specialised work. Whatman No. 1 filter paper is most widely used and will give satisfactory results in the majority of cases. Thicker papers such as blotting paper are not usually suitable.
The choice of solvent depends on the type of substances to be separated, the most common ones being alcohols such as butanol with or without the addition of acids such as acetic acid or bases such as ammonia or amines, phenol, hydrocarbon solvents, collidine, etc. The solvent phase is saturated with water except in certain cases where the solvent is actually miscible with water. (Although it would appear at first sight that such a solvent would not give any separation this is, for reasons which will not be discussed here, not always the case.) It is perhaps unfortunate that the more common solvents are often unpleasant to handle and toxic, and care should be taken to avoid unduly prolonged exposure to their vapours.
The lower limit to the quantities of substances which can be separated usually depends on the sensitivity of the method used for detecting the resulting spots. Detection is usually carried out by spraying the dried
As a general rule the spots applied to the paper should be small, preferably not more than a few millimetres in diameter and the concentration of the individual substances should not be too high or ‘tailing’ and excessive spreading of the spots will take place. In most cases a single small drop — say 5 microlitres — of a 1% solution is about the amount of substance required. For quantitative work an accurately known amount must be applied to the paper and for this a micro-pipette is required, but this elaboration is unnecessary for qualitative studies.
It is often desirable though not essential that the paper after the application of the material be allowed to stand in an atmosphere saturated with respect to both the aqueous and mobile phases before the chromatography is commenced. For this reason it is desirable that the solvent can be added to the trough or, in the case of ascending chromatograms, the paper may be lowered into the solvent, with the least possible dismantling of the apparatus.
The paper must almost always be dried before spraying and this is best accomplished either in a hot air oven or in a current of warm air such as that provided by a hair drier. Because of the toxic nature of the solvents drying should be carried out in a fume cupboard, or failing that in a well-ventilated place. Overheating of the papers should be avoided.
The spray should be applied lightly so that the paper becomes merely damp and not saturated in order to avoid undue migration and diffusion of the spots. For the same reason most spraying reagents are made up in non-aqueous solution.
With complex mixtures it is often impossible to obtain complete resolution with any single solvent and in such cases two-dimensional chromatograms may be resorted to. This may lead to a very high degree of resolution of complex mixtures but it is not possible to run standard substances simultaneously.
Quantitative paper chromatography may involve measurement of spot sizes, intensities of coloured compounds developed by sprays or elution of
The following practical directions should enable the beginner in the field to become familiar with the general methods and the suggested materials for investigation should provide a guide to those interested in further studies.
Whatman No. 1 filter paper is cut into strips which will fit easily into an ordinary test-tube and the strips are creased lengthwise to form a V-shaped trough. A line is drawn lightly across the strip about 2 cm. from the end, and by means of a fine glass capillary tube or a match-stick, a small spot of the sample (about 2 mm. in diameter) is placed on the line in the centre of each half of the V. The spots are allowed to dry then the paper strip is placed, spotted end down, in a test-tube containing 1-2 cc. of solvent in such a way that the strip touches the side of the test-tube only at the outside top corners. It is essential that the starting line marked on the strip be above the solvent surface, and the paper strips will stay in position readily if the tube is placed in a slightly slanting position. The test-tube is corked and set aside undisturbed until the solvent front rises almost to the top of the strip. This may take up to about one hour, depending on the temperature and the nature of the solvent. The strip is removed, the position of the solvent is marked, and the solvent is evaporated either by allowing the strip to stand in the air or, preferably, in a hot air oven or in a current of warm air. The chromatogram is sprayed lightly with the detecting reagent and heated if necessary, the position of the spots is marked, and their RF values determined. For this purpose the ‘distance moved by the solvent front’ is the distance between the starting line and the final position of the solvent front. The ‘distance moved by the substance’ is usually taken as the distance from the starting line to the ‘centre of gravity’ of the spot.
Wide-necked bottles such as milk-bottles are most satisfactory and the paper can be suspended from a glass or wire rod passed through a cork or rubber stopper. If the support can be moved vertically it is possible to allow the paper strip to hang in the atmosphere inside the bottle for up to 24 hours before it is lowered into the solvent, and if this is done more regular spots may be obtained and there may be less tailing. The additional width of the paper strip allows several samples to be run side by side. If this is done the individual spots should be spaced at intervals of 1.5-2 cm. along the starting line.
When a large number of samples must be run side by side or for two-dimensional chromatograms with sheets of paper up to 40-50 cms.
The spots are placed on a line 6-8 cm. from the end of the paper strip which should be about 40-50 cm. long. The paper is arranged in the trough so that the starting line is well clear of the edge of the trough, a dish of water saturated with solvent is placed in the bottom of the container, the solvent is added to the trough and the container is closed. Once again, if possible, it may be desirable to allow the paper to equilibrate in an atmosphere saturated with both liquid phases before the solvent is added to the trough. The chromatogram may be allowed to develop as long as desired and if necessary the solvent may be allowed to run off the lower edge of the paper. If this is to be done the lower edge of the paper should be serrated to facilitate an even flow of the solvent. When development is complete the paper is removed, dried and sprayed in the usual way.
When complex mixtures have to be analysed two-dimensional chromatography is a valuable modification because of the large increase in resolving power. A single spot of the mixture is placed near one corner of a sheet of paper 30-50 cm. square and the chromatogram is run in the usual way using either the descending or the ascending technique. At this stage the mixture will have partially separated into a series of spots distributed along a line near one edge of the sheet. The paper is removed and dried, then run in the direction at right angles to the original with a different solvent, then dried and sprayed in the usual way. The resulting chromatograms will show a series of spots distributed over the paper, and although standard substances cannot be run simultaneously, the identification of a spot is simplified by the fact that its position depends on a combination of two RF values. ‘Maps’ showing the positions which substances will appear in, with various solvent combinations have been published or can easily be constructed from tables of RF values. For two-dimensional chromatograms the amount of material placed on the original spot should be rather greater than for single dimensional ones, and the first solvent should be removed as completely as possible before running in the second direction. It is frequently found that there is a preferred order in which the two solvent combinations should be used.
For demonstration purposes amino-acids are the most satisfactory although some sugars can also be separated reasonably well in a comparatively short time.
The amino-acids can be applied as 0.2-1% solutions and mixtures are most readily obtained by applying the individual amino-acids successively to the same spot, the paper being allowed to dry between each application. For small-scale chromatograms in test-tubes or bottles, 80% phenol water (W/W) is a suitable solvent (this is rather cheaper to use than phenol saturated with water) and combinations of amino acids such as valine, glycine and aspartic acid or leucine, threonine and glutamic acid separate readily.
0.2% Ninhydrin (triketohydrindene hydrate) in water-saturated butanol is the spraying reagent, and the sprayed papers must be heated briefly at about 110° to develop the (usually blue) spots.
Sugars and related substances may be separated using a solvent mixture containing 5 volumes of acetic acid, 25 volumes of water and 110 volumes of butanol, although RF values are low and complete resolution of mixtures is difficult on a small scale. However, mixtures of monosaccharides and disaccharides such as glucose and lactose separate comparatively readily. The solvent mixture given above approximates in composition the organic (upper) layer obtained by shaking together butanol, acetic acid and water in the volume ratios 4: 1: 5 — a solvent which is very widely used in chromatographic work. This solvent mixture slowly changes in composition due to esterification and should not be kept indefinitely.
As spraying reagent, ammoniacal silver nitrate or 2% aniline hydrogen phthalate (made from 0.93 g. of aniline and 1.60 g. of phthalic acid in 100 ml. of water-saturated butanol) may be used. With either spray the paper must be heated to 105-110° to develop the spots.
Aiken, Miriam A., Living Fossils of the Plant Kingdom. 4. (2): 47-53.
Allan, H. H., A Note on Lichens with a Key to the commoner New Zealand genera. 1. (3): 20-35.
A Note on the Crustaceous Lichens of New Zealand (and a Key to the commoner families and genera). 2. (1): 15-21.
New Zealand Lichens (Key to Umbilicariaceae). 4. (2): 59-62.
Allen, K. R., The New Zealand Grayling — A Vanishing Species. 2. (1): 22-27.
Allen, K. R., and
Allison, K. W., Some Notes on Mosses with Key to commoner New Zealand genera. 2. (3): 131-147.
Correction to Moss Key. 3. (1): 42.
Bary, Brian McK., Sea-Water Discolouration. 4. (2): 41-46.
Batham, Elizabeth J., Life at Plymouth Marine Station To-day. 1. (1): 24-26.
Butler, N. J., A Guide to the Collection of Botanical Specimens. 3. (2): 67-77.
Cairns, D., New Zealand Freshwater Eels. 3. (2): 43-52.
Fishing Industry in China. 1. (2): 13-18.
Callaghan, F. R. C., The Scope of the Biologist in Plant Research Institutions in New Zealand. I. (1): 5-10.
Campbell, J. T., Some Basic Ideas in Statistical Method. 2. (1): 9-14.
Cassie, R. M., and
Class Project, The Frog, Hyla aurea, as a Source of Animal Parasites. 5. (1): 12-21.
Crawford, D. Alleyne, Phytoplankton. 1. (1): 15-20.
Cunningham, Ashley, National Forest Survey. 5. (2): 39-48.
Davis, John H., Evidences of Trans-Oceanic Dispersal of Plants to New Zealand. 3. (3): 87-97.
Dawbin, W. H., Editorial. 1. (1): 3-5.
Editorial. 1. (2): 1.
Biological Interests at a Whaling Station. 1. (3): 14-20.
The Tuatara. 2. (2): 91-96.
A Guide to the Holothurians of New Zealand. 3. (1): 33-41.
Dawson, J. W., A Key to the New Zealand Lycopods. 5. (1): 6-11.
Dell, R. K., Key to the Common Chitons of New Zealand. 4. (1): 4-12. The New Zealand Cephalopoda. 4. (3): 91-102.
Dellow, U. V., and
Falla, R. A., Biology in New Zealand Museums. 1. (3): 4-5.
Identification of New Zealand Mainland Shags. 2. (3): 116-120.
Fell, H. Barraclough, A Key to the Littoral Asteroids of New Zealand.
1. (1): 20-23.
A Key to the Sea Urchins of New Zealand. 1. (3): 6-13.
New Zealand Littoral Ophiuroids. 2. (3): 121-129.
A Key to the Sea Urchins of New Zealand. Additional Species. 3. (1): 42. New Zealand Crinoids. 3. (2): 78-85.
Filmer, J. F., Biological Aspects of Animal Research in New Zealand. 1. (2): 3-6.
Fleming, C. A., The Geological History of New Zealand. 2. (2): 72-90.
Forster, R. R., A Key to the Common Spiders of the Wellington District. 1. (2): 22-27.
Garrick, J. A. F., and
Godley, E. J., Cytology and Genetics and their Application to New Zealand Plants. 2. (3): 109-115.
Harris, W. F., Climatic Relations of Fossil and Recent Floras. 3. (2): 53-66.
Harvey, W. E., Paper Chromatography. 5. (3): 100-110.
Hatch, E. D., Checklist of New Zealand Orchids. 4. (1): 28-40.
Hay, J. A., and
Healy, A. J., The Place of the Botanist in Soil Conservation. 1. (2): 19-22.
Hodgson, E. Amy, The Classification of New Zealand Hepaticae. 3. (1): 20-32.
Classification of New Zealand Hepaticae (Addenda). 3. (2): 86.
Hurley, D. E., New Zealand Terrestrial Isopods. 3, (3): 115-127.
Knox, G. A., A Guide to the Families and Genera of New Zealand Polychaetes. 4. (2): 63-85.
Krefft, S., and Richardson, L. R.,
Laird, M., Mosquito-Borne Disease and the War in the Pacific. 1. (1): 10-14.
Lee, K. E., Role of Earthworms in New Zealand Soil. 4. (1): 22-27.
Leed, Heather M., Kapiti Island. 1. (2): 28-30.
Levy, Bruce E., The Conversion of Rain Forest to Grassland in New Zealand. 2. (1): 37-51.
Manter, Harold W., Collection of Animal Parasites. 4. (2): 56-58.
Mare, F. A. de la, In Memoriam —
Marples, B. J., Vertebrate Palaeontology in New Zealand. 2. (3): 103-108.
Miller, D., Shakespearean Entomology. 1. (2): 7-12.
Morton, J. E., Collecting and Preserving Zoological Specimens. 3. (3): 104-114.
Oliver, W. R. B., The Fossil Flora of New Zealand. 3. (1): 1-11.
Ralph, Patricia M., A Guide to the Athecate Hydroids and Medusae of New Zealand. 5. (2): 59-75.
Richardson, L. R., Inflation of the Abdomen in Cephaloscyllium. 1. (3): 39.
A Guide to the Brachyrhynchous Crabs. 2. (1): 29-36.
A Guide to the Oxyrhyncha, Oxystoma and Lesser Crabs. 2. (2): 58-69.
Corrections and Additions for the Guides to the Brachyura. 2. (3): 130.
The Generic Status of the New Zealand Lancelet. 3. (2): 86.
Design and Maintenance of Marine Aquaria. 4. (3): 87-90.
Principles of the Balanced Freshwater Aquarium. 5. (1): 1-5.
Use of Barrier Cream in the Dissection Laboratory. 5. (3): 85-86.
Richardson, L. R., and
Richardson, L. R., and
Rigg, Sir Theodore, The Cawthron Institute. 2. (1): 2-8.
Rollo, L. J., Type Terminology. 4. (2): 54-55.
Salmon, J. T., Vegetable Caterpillars. 4. (1): 1-3. Stick Insects. 5. (3): 77-81.
Schmidt, K. P., To a Tuatara Alive in my Hand. 2. (2): 90.
Smart, Cynthia M., Plant Virus Research. 5. (2): 52-58.
Spiller, D., The Biology and Control of Beetles Attacking Seasoned Timber. 3. (1): 12-19.
Stout, J. D., Protozoa and the Soil. 4. (3): 103-107.
Tuioti, Siaosi E., Shark Fishing in Western Samoa. 5. (3): 82-85.
Wenzel, R.,
Woodward, T. E., Collection and Preservation of Insects. 4. (1): 13-21. See also corrections, 4. (2): 53.
Wright, A. C. S., Some Recent Researches on the Soil Organic Cycle. 3. (3): 100-103.
Zotov, V. D., Rata the Killer. 1. (3): 36-39.
Animal Research in New Zealand, Biological Aspects of, by
Annelida — See Keys to groups.
Aquaria, Design and Maintenance of Marine, by
Aquarium, Principles of Balanced Freshwater, by
Arachnida — Key to the Common Spiders of the Wellington District, by
Asteroids — Key to the N.Z. Littoral, by H. B. Fell. 1. (1): 20-23.
Barrier Cream, Use of, in Dissection Laboratory, by
Batoidei, Key to, in A Guide to Lesser Chordates and Cartilaginous Fishes, by
Beetles Attacking Seasoned Timber, Biology and Control of, by
Beech, Request for Observations on Flowering of, by
Birds — N.Z. Mailand Shags, Identification of, by
Biological Society Notes. 1. (1): 26-29.
Botanical Specimens — A Guide to the Collection of, by
Botanist in Soil Conservation, The Place of the, by A.
Brachyura, Gymnopleura: Lyreidus australiensis Ward from Cook Strait, by
Brachyura — A Guide to the Brachyrhynchous Crabs, by
Brachyura, Corrections and Additions for the Guides to, by
Brittle Stars — New Zealand Littoral Ophiuroids, by H. B. Fell. 2. (3): 121-129.
Bryophyta:
Mosses, Some Notes on, with Key to the Commoner N.Z. Genera, by
Mosses, Correction to Key, by
Liverworts — Classification of N.Z. Hepaticae, by E. Amy Hodgson. 3. (1): 20-32.
Liverworts — Classification of New Zealand Hepaticae, Correction to, by E. Amy Hodgson. 3. (2): 86.
Cawthron Institute, by Sir
Cephalopoda, New Zealand, by
Cephaloscyllium, Inflation of the Abdomen in, by
Cestoidea — Gyrocotyle, A Peculiar Parasite of the Elephant Fish in New Zealand, by
China, Fishing Industry in, by
Chitons — A Key to the Common Chitons of N.Z., by
Chordates, Lesser — Key to, by
Chromatography, Paper, by
Ciliates, Freshwater, from the Wellington Area including Eleven Species Recorded from N.Z. for the First Time, Report from a V.U.C. Zoology Class Project. 5. (3): 87-99.
Class Project — The Frog, Hyla aurea, as a Source of Animal Parasites, by a V.U.C. Zoology Class. 5. (1): 12-21.
Climate Relations of Fossil and Recent Floras, by W. F. Harris. 3. (2): 53-56.
Coelenterata — See Various Groups.
Collection:
Botanical Specimens, A Guide to the Collection of, by
Zoological Specimens:
Animal Parasites, by
Insects, Collection and Preservation of, by
Marine Invertebrates, by
Marine Micro-organisms — Sea Water Discolouration, by
Conifers, New Zealand, by
Conservation, Soil, The Place of the Botanist in, by A.
Crabs — See Brachyura and Crustacea.
Crinoids, N.Z., by H. B. Fell. 3. (2): 78-85.
Crustacea:
A Guide to the Brachyrhynchous Crabs, by
A Guide to the Oxyrhyncha, Oxystoma and Lesser Crabs, by
Lyreidus australiensis Ward (Brachyura, Gymnopleura) from Cook Strait, by
Corrections and Additions for the Guides to the Brachyura, by
Isopods, N.Z. Terrestrial, by
Cytology and Genetics and their Application to N.Z. Plants, by E.
Disease, -Mosquito Borne, and the War in the Pacific, by
Dispersal of Plants to New Zealand, Evidences of Trans-Oceanic, by
Earthworms, Role of, in N.Z. Soil, by
Echinoderms:
Asteroids of N.Z., Key to the Littoral, by H. B. Fell. 1. (1): 20-23.
Echinoids — Key to the Sea Urchins of N.Z., by H. B. Fell. 1. (3): 6-13.
Ophiuroids, N.Z. Littoral, by H. B. Fell. 2. (3): 121-129.
Holothurians of N.Z., A Guide to, by
Echinoids — Key to Sea Urchins to New Zealand, Additional Species, by H. B. Fell. 3. (1): 42.
Crinoids, N.Z., by H. B. Fell. 3. (2): 78-85.
Ecology — Role of Earthworms in N.Z. Soil, by
Protozoa and the Soil, by J. D. Sout. 4. (3): 103-107.
National Forest Survey, by
Editorial, by
By
Eels, N.Z. Freshwater, by
Epiphytes — Evidences of Trans-Oceanic Dispersal of Plants to N.Z., by
Entomology, Shakespearean, by
Feather-Stars — N.Z. Crinoids, by H. B. Fell. 3. (2): 78-85.
Fish:
Eels, N.Z. Freshwater, by
Grayling, The N.Z., A Vanishing Species, by K. Radway Allen. 2. (1): 22-27.
Fisheries, Problems of Marine and Freshwater Fisheries Biology in N.Z., by
Fishes, Major Groups of Cartilaginous, Key to, by
Fishing, Shark, in Western Samoa, by Siaosi E. Tuioti. 5. (3): 82-85.
Fishing Industry in China, by
Flora:
Fossil Flora of N.Z., by
Climate Relation of Fossil and Recent Floras, by W. F. Harris. 3. (2): 53-66.
Evidences of Trans-Oceanic Dispersal of Plants to N.Z., by
New Zealand Conifers, by
Forest — Survey, National, by
Frog — The Frog, Hyla aurea, as a Source of Animal Parasites, by a V.U.C. Zoology Class. 5. (1): 12-21.
Fungi — Vegetable Caterpillars, by
Galeoidea, Key to Species — see Selachii.
Geology (see also Palaeontology):
Geological History of N.Z., by
Climate Relation of Fossil and Recent Floras, by W. F. Harris. 3. (2): 53-66.
Genetics and Cytology, Application to N.Z. Plants, by E.
Genetics, Discussion Group, by A.
Grasslands in N.Z., The Conversion of Rain Forest to, by E. Bruce Levy. 2. (1): 37-51.
Gyrocotyle, A Peculiar Parasite of the Elephant Fish in New Zealand, by
Hepaticae, Classification of N.Z., by E. Amy Hodgson. 3. (1): 20-32.
Hepaticae, Classification of N.Z., Corrections to, by E. Amy Hodgson. 3. (2): 86.
Hydroids, A Guide to the N.Z. Athecate, by
Isopods, N.Z. Terrestrial by
Holothurians, A Guide to the N.Z., by
Insects, Expeditions, and Dr. Johnson, by
Insects, Collection and Preservation of, by
Kapiti Island, by Heather
Keys:
How to Use Keys for Identifying Organisms. 1. (2): 6.
Botanical:
Lichens, Note on with a Key to the Commoner N.Z. Genera, by H.
Lichens, Crustaceous, A Note on the N.Z., by H.
Lichens, Stictaceae of N.Z., by H.
Mosses, Some Notes on, with Key to Commoner N.Z. Genera, by
Mosses, Correction to Key, by
Hepaticae, Classification of N.Z., Corrections to, by E. Amy Hodgson. 3. (2): 86.
Orchids, N.Z., Checklist of, by
Lichens, N.Z. (Umbilicariaceae), by H.
Lycopods, A Key to New Zealand, by
Zoological:
Asteroids of N.Z., Littoral, by H. B. Fell. 1. (1): 20-33.
Spiders, Common, of the Wellington District, by
Sea Urchins of N.Z., by H. B. Fell. 1. (3): 6-13.
Crabs, Oxyrhyncha, Oxystoma and Lesser, by
Crabs, Brachyura, Corrections and Additions for the Guides to, by
Ophiuroids, N.Z. Littoral, by H. B. Fell. 2. (3): 121-129.
Shags, Identification of N.Z. Mainland, by
Sea Urchins of N.Z., Key to, Additional Species, by H. B. Fell. 3. (1): 42.
Holothurians of N.Z., A Guide to, by
Crinoids, N.Z., by H. B. Fell. 3. (2): 78-85.
Isopods, New Zealand Terrestrial, by
Chitons, N.Z., A Key to the Common, by
Polychaetes, N.Z., A Guide to the Families and Genera of, by
Cephalopoda, The N.Z., by
Cartilaginous Fishes, Lesser Chordates, A Guide to, by
Athecate Hydroids and Medusae of N.Z., A Guide to the, by
Kirk, Harry Borrer, In Memoriam, by F. A. de la Mare. 1. (3): 1-4.
Lancelet, N.Z., The Generic Status of, by
Lichens:
A Note on with a Key to the Commoner N.Z. Genera, by H.
N.Z. Crustaceous, A Note on, by H.
Key to the Stictaceae of N.Z., by H.
Key to Umbilicariaceae, by H.
Liverworts:
Classifications of N.Z. Hepaticae, by E. Amy Hodgson. 3. (1): 20-32. Classification of N.Z. Hepaticae, Corrections to, by E. Amy Hodgson. 3. (2): 86.
Lycopods, A Key to New Zealand, by
Marchant ridge, Tararuas, by
Medusae of N.Z., A Guide to, by
Mollusca — See various groups.
Mosquito Borne Disease and the War in the Pacific, by
Mosses:
Note on Some N.Z., with a Key to the Commoner N.Z. Genera, by
Corrections to Key, by
Museums, N.Z., Biology in, by
Nematodes, The Frog as a Source of Animal Parasites, by a V.U.C. Zoology Class. 5. (1): 12-21.
Nomenclature, Zoological, Type Terminology, by
Nothofagus, Requests for Observation on Flowering of, by
Notidanoidea, Key to Species — see Selachii.
Notornis, Correction, by J. H. S. 2. (3): 115.
Obituary — In Memoriam —
Onychophora — See Peripatus.
Ophiuroids, N.Z. Littoral, by H. B. Fell. 2. (3): 121-129.
Orchids, Checklist of New Zealand, by
Oxyrhyncha, Oxystoma and Lesser Crabs, by
Palaeontology:
Geological History of the N.Z., with Reference to the Origin and History of the Flora and Fauna, by
Vertebrate, in N.Z., by
Flora of N.Z., Fossil, by
Flora, Climate Relations of Fossil and Recent, by W. F. Harris 3. (2): 53-66.
Paper Chromatography, by E. W. Harvey. 5. (3): 100-110.
Parasites — Vegetable Caterpillars, by
Animal, Collection of, by
The Frog, Hyla aurea, as a Source of Animal, by a V.U.C. Zoology Class 5. (1): 12-21.
Gyrocotyle, A Peculiar Parasite of the Elephant Fish in New Zealand by
Peripatus ‘Living Fossil’ and ‘Missing Link’, by Rupert L. Wenzel 3. (3): 98-99.
Photography, Aerial, Use in forest surveys — see National Forest Survey by Ashby Cunningham. 5. (2): 39-48.
Phytoplankton — Seawater Discolouration, by
Phytoplankton, by D. Alleyne Crawford. 1. (1): 15-20.
Plant Quarantines, Changing Conditions and, by
Plant Research Institutions, N.Z., Scope of the Biologist in, by
Platyhelminthes — See various groups.
Plymouth Marine Station Life at, by
Polychaetes, N.Z., A Guide to the Families and Genera of, by
Preservation:
Botanical Specimens, A Guide to the Collections of, by
Zoological Specimens:
Collection and Preservation of (Marine Invertebrates), by
Insects, Collection and Preservation of, by
Protozoa — The Frog as a Source of Animal Parasites, by a V.U.C. Zoology Class. 5. (1): 12-21.
Protozoa — Sea Water Discolouration, by
Protozoa and the Soil, by
Protozoa — See Ciliates.
Psilopsida — Living Fossils of the Plant Kingdom, by
Psilotales — Living Fossils of the Plant Kingdom (Psilotum and Tmesipteris), by
Psilotum — Living Fossils of the Plant Kingdom, by
Rain Forest to Grassland in N.Z., The Conversion of, by E. Bruce Levy. 2. (1): 37-51.
Rata the Killer, by
Rays — See Batoidei.
Research Institutions:
Plant Research, Scope of the Biologist in N.Z. Institutions, by
Plymouth Marine Station, Life at, by
Museums, N.Z., Biology in, by
Cawthron Institute, by Sir
Reviews:
Biology for Australian Students (W. M. Curtis), by P.M.R. 2. (1): 52.
Salamanders at Metamorphosis, Loss of Memory in, by B.M.B. 2. (1): 28.
Samoa, Western, Shark Fishing in, by Siaosi E. Tuioti. 5. (3): 82-85.
Sea Cucumbers — A Guide to the Holothurians of N.Z., by
Selachii, Key to Main Groups, in A Guide to the Lesser Chordates and Cartilaginous Fishes, by
Sea Lilies — N.Z. Crinoids, by H. B. Fell. 3. (2): 78-85.
Sea Urchins, Key to the N.Z., by H. B. Fell. 1. (3): 6-13.
Sea Urchins, Key to the N.Z., Additional Species, by H. B. Fell. 3. (1): 42.
Shags, Identification of N.Z. Mainland, by
Shark Fishing in Western Samoa, by Siaosi E. Tuioti. 5. (3): 82-85.
Sharks, Key to Main Groups — see Selachii.
Shakespearean Entomology, by
Skates — see Batoidei.
Soil:
Conservation, The Place of the Botanist in, by A.
N.Z., Role of Earthworms in, by
Protozoa and, by
Sphenodon punctatus — The Tuatara, by
Spiders of the Wellington District, A Key to the Common, by
Starfish — Key to the Littoral Asteroids of N.Z., by H. B. Fell. 1. (1): 20-23.
Statistical Method, Some Basic Ideas in, by
Stick Insects, by
Swell Sharks — Inflation of the Abdomen in Cephaloscyllium, by
Taxonomy, Zoological — see Nomenclature.
Timber, Seasoned, Biology and Control of Beetles attacking, by
Tmesipteris — Living Fossils of the Plant Kingdom, by
Trematodes — The Frog as a Source of Animal Parasites, by a V.U.C. Zoology Class. 5. (1): 12-21.
Tuatara, The, by
Tauatara — ‘To a Tuatara Alive in My Hand’, by
Type Terminology, by L. T. Rollo. 4. (2): 54-55.
Use of Barrier Cream in the Dissection Laboratory, by
Vegetable Caterpillars, by
Virus, Research Plant, by Cynthia
Whaling Station, Biological Interests at a, by
Woodlice — N.Z. Terrestrial Isopods, by
Vertebrate Palaeontology in N.Z., by
Zoogeography — Role of Earthworms in N.Z. Soil, by
Zoological Specimens (Marine Invertebrates), Collection and Preservation of, by