Some Observations on the Behaviour of Amoeba Proteus

Some Observations on the Behaviour of Amoeba proteus.

By C. W. Parsons, B.A., Department of Zoology, University of Glasgow.

With 9 Text-figures.

ALTHOUGH the body-form of Amoeba proteus shows so much diversity in any healthy culture, the differences that may be observed conform to certain types. If these are assembled, they fall into groups which may be supposed to indicate physiological variation in the organisms ; for they are then reacting in different ways to the stimuli provided by the same external medium, and such behaviour can best be explained on physiological bases. The investigation of the groups is approached in the following pages by two different routes. The first is experimental and consists in a re-examination of the normal behaviour of adult Amoebae in the aquarium medium in which they are best cultured (see Monica Taylor, 11), followed by an analysis of their reactions to simple changes in their environment. The second is deductive. It rests upon a hypothesis relating the degree of activity of these organisms, as measured by the rapidity and ease with which they extend their pseudopodia, with their health. It recognizes the presence of healthy forms, in unfavourable as well as in favourable environments, and puts forward the view that some Amoebae are more adaptable to change of medium than are others. A link is here established with the well-known fact that while the Amoebae in a culture cease to reproduce by fission after a time, many of them do not enter on the cycle of encystment. They die out for no obvious 630 C. W. PARSONS reason. Food remains abundant, overcrowding is avoided, and the pH throughout the culture may be adjusted to give condi- tions normally ideal for multiplication. The attempt is here made to correlate this peculiarity in life history with a con- spicuous variation in body-form, and the types exhibiting this variation are defined. Material.—Adult Amoebae having chromosome blocks in the periphery of their nuclei, from a nourishing sub-culture of Dr. Monica Taylor's culture ' 19 ', type ' B ' (12, pp. 187 and 120). Apparatus included — plunger pipettes, i.e. capillary pipettes fitted internally with drawn-glass rods and sealed with rubber tubing. (See Brooker Klugh, 5.) Solid watch-glasses, 1| in. square; J in. deep for supporting cover-slips in high-power work ; ^ in. deep for general purposes. Sarensen phosphate buffer solutions with indicators, made up to 10 e.c. and 2 c.c. A. Behaviour of Adult Amoebae in Culture Media free from Debris. pH 6-8. The Amoebae are readily seen as white specks if debris con- taining them is taken from the parent culture and placed in a shallow dish on a black background. They may be picked up singly with the plunger pipette and transferred for observa- tion to a solid watch-glass containing clean culture medium. If there is rather less than 2 c.c. of fluid they may be focussed with a Zeiss ' A ' objective (f in.), and their behaviour recorded by notes and drawings taken at suitable intervals. High-power observation of single living Amoebae in a drop of fluid was carried out on a clean glass cover-slip supported in the well of one of the very shallow watch-glasses (£ in. deep). If the drop of fluid is small, the Amoebae may be focussed with a Zeiss ' D ' objective (£ in.), and the drop can be renewed as required from a fine-drawn pipette. When set aside, rapid evaporation may be prevented by placing another solid watch- glass on top of the one already holding the cover-slip, and the Amoebae remain alive in the drop. BEHAVIOUR OF AMOEBA 631 When they are picked up with the plunger pipette the Amoebae withdraw their pseudopodia, and it thus comes about that they are delivered to the solid watch-glasses as rounded masses of protoplasm. If they are kept floating—by gentle agitation of the fluid—they extend numerous blunt pseudo- podia (Text-fig. 1). When allowed to settle, however, they adhere to the glass surface in an orderly fashion. First, one of the pseudopodia reaches the substratum and adheres to it by

TEXT-FIGS. 1, 2.

Tig. 1.—Typical body-form of a floating Amoeba proteusinits aquarium culture medium. The figure shows a radial disposition of pseudopodia of roughly equal size. Fig. 2.—Contact with the substratum is first established by adhesion to it of one of the radial pseudopodia, which is distinguishable now as the major pseudopodium. the tip. Then it receives the remaining protoplasm (Text- figs. 2, 3, and 4) and, leading the organism down to the sub- stratum, it becomes the directive pseudopodium in the new line of advance. The Amoebae adhere closely to the glass surface as a rule, and move rapidly over it in a digitate form. A broader extremity crowned with active pseudopodia lies in the main axis of advance, and a slight constriction commonly marks the ' tail' (Text-fig. 4, t). The ' tail ' is a conspicuous feature of most Amoebae even in experimental media. It is seldom the seat of pseudopodia 632 C. W. PARSONS formation, a point which is emphasized by observing the- effect of mechanical stimulation upon one of these adhering- forms in culture medium. Schaeffer has shown how they respond when a glass needle is agitated near to them by initiating the ' feeding mechanism' (10, p. 229), but if the medium is- disturbed in this way immediately in front of the advancing pseudopodia, an avoiding reaction is induced. The flow of

TEXT-FIGS. 3, 4.

Fig. 3.—Further stage in the transition from a floating condition to one of adherence to the substratum. Fig. 4.—A completely adherent active Amoeba of the type shown floating in Text-fig. 1, with a body-form frequently observed, comprising three main pseudopodia, an elongate body, and a slightly constricted ' tail' (t). Same scale as fig. 1. granules is at first arrested and then reversed. After receding' a little, the Amoeba moves away afresh at an angle less than a right angle from its previous direction of flow. If now left undisturbed the pseudopodia gradually deflect into the original alinement and draw the animal into a path parallel to but slightly removed from its former course. On the other hand, this movement may be checked soon after its commencement by disturbing the medium again in front of the pseudopodia when they begin to diverge. The original course is then BEHAVIOUR OF AMOEBA 638 reversed : not, however, by the formation of pseudopodia at the hinder end, but by movement of lateral pseudopodia into the new position which draw the ' tail' round after them. This behaviour is typical of the most active adult Amoebae in culture media. Many adult Amoebae prove to be relatively inactive when taken from the parent culture. They are frequently more consolidated than the more active forms and may be very much shrunken. There seems, in fact, to be a gradation between the most active and least active Amoebae, marked by a change in their appearance under transmitted light from grey to black. It is notable also that the darker forms tend to lose their adhesion to the sides of the vessel containing them with greater ease than do the active grey ones. This suggests that granu- larity and adhesiveness on the one hand, and readiness of movement on the other, may be complementary features. B. Behaviour of Amoeba proteus in Pure Media. The water used in the following observations was distilled once from Glasgow tap-water in a glass condensing apparatus. A little potassium permanganate and a trace of mineral acid was added before distillation to oxidize impurities. The distillate, called hereunder glass distilled Avater, had a steady pH of 5-8 before prolonged exposure to the atmosphere. The Amoebae may reasonably be separated into three types on the basis of their reactions to this medium. 1. The majority quickly extend numerous pseudopodia to relatively great distances. (Text-fig. 5.) The reaction is the same in well-aerated distilled water, and has no relation therefore to the recognized paucity of oxygen in untreated glass distilled water. On theoretical grounds Mr. C. F. A. Pantin informs me that the ratio of surface to volume in Amoeba is so great, that equilibration with oxygen in the external medium is effected in an extremely short space of time. The small increase of surface produced therefore by great extension of pseudopodia, will not avail to compensate for a law oxygen content in the medium; although of course the great variation in ' per- 634 C. W. PARSONS nieability ' of living protoplasm to oxygen is not disputed (cf. ' Air-bladder of Fishes ').

TEXT-FIG. 5.

The exaggerated floating Jorm ot an active Amoeba suspended in distilled water. The attenuated pseudopodia suggested the dis- tinction of Amoebae that react in this way from others which are less active, and it was convenient to describe them as type ' A ' Amoebae.

Amoebae in glass distilled water do not, as a rule, fix them- selves to the substratum. They float with extended pseudo- podia in a most grotesque manner, and are extremely active BEHAVIOUR OF AMOEBA 685 at first. Their energy is slowly dissipated, however, and in 8-10 hours they become sluggish. As time elapses they shrink and grow darker in appearance, but they may remain alive 5-7 days before cytolysis. The death of the organism is fre- quently preceded by its assumption of a more or less spherical form in accordance with its subjection to the ordinary forces of surface tension. Distension of its outer layers—owing to the decrease in efficiency of the contractile vacuole which normally

Illustrates a body-form not infrequently observed amongst active Amoebae that are floating in pure media. Movement is steadily maintained in the outer layers, which may therefore differentiate sharply as at a, from the endoplasm. Movement in the latter is also vigorous, but it is checked periodically and results in excep- tionally blunt pseudopodia, b. Same scale as fig. 1. counteracts the diffusion of water into the Amoeba may be observed, and the appearance then presented is that of a very large vacuole with a small quantity of granular cytoplasm about it. After this has been realized, the actual cytolysis may be delayed for a period varying in individual cases between a clay and only a few hours. If returned to culture medium within the first 8 hours, or during their active phase, the Amoebae recover rapidly and completely : but if this is delayed until they have become sluggish, recovery is partial and gives rise to dark, slow-moving forms in the majority of cases. A noticeable variation in the ease with which the elongated pseudopodia are extended, is to be seen in these experiments.. 686 C. W. PARSONS Movement in some Amoebae proceeds jerkily. At a, Text- fig. 6, a narrow streak of the clear outer layer is put forward, while the flow of granules goes on in the cytoplasm behind it. The granules accumulate in great numbers as a result and swell out the ectoplasm about them into ' beads '. These disperse suddenly when the pressure releases, to form excep- tionally blunt pseudopodia (b, Text-fig. 6). This type of body- form is described by Gruber (3, p. 256), with the suggestion that it indicates abnormal consolidation of the outer surface layers.

TEXT-FIG. 7.

The poor response to cuange in environment or one of the darker inactive Amoebae that occur in culture media, illustrated by the ragged appearance of 6ne of them in distilled water. It is a typical type ' R ' Amoeba. Same scale as fig. 1.

2. The darker inactive Amoebae, which have already been mentioned because they occur in culture media, do not respond so well to the stimulus of glass distilled water. Only short pseudopodia are extended when they are transferred to this fluid. The outer layers are abnormally distinct in them, and they float with very ragged outlines and limited powers of movement. (Text-fig. 7.) 3. Exceptional Amoebae remain. These rare forms make no response at all to the alteration in environment. They are typically clavate, and adhere lightly to the substratum in culture medium. In distilled water they retain this capacity to some extent. (Text-fig. 8.) BEHAVIOUR OF AMOEBA 687 When these forms are contrasted, the desirability of splitting the active and inactive groups into types is apparent. Active attenuating forms . . type ' A ' (Text-fig. 5). Inactive ragged forms . . type ' E ' (Text-fig. 7). Inactive clavate forms . . type ' C ' (Text-fig. 8). The types ' E ' and ' 0 ' are more abundant in cultures wherein multiplication of Amoebae by fission has been in full progress

TEXT-FIG. 8.

A clavate irresponsive form of Amoeba, which is rare in culture media, figured from distilled water; the type ' C ' Amoeba. for many months. This, correlated with the fact that type ' A ' Amoebae from these cultures are less responsive to the imme- diate effects of distilled water, suggests that somewhat unfavour- able conditions hasten the change from type ' A ' to type ' E '; which under normal vigorous circumstances will proceed very slowly. This does not mean that the types ' E ' and ' C ' are absent from the most flourishing cultures. They may be found in them, but are rare. 688 C. W. PARSONS In glass distilled water containing measurable traces of acid or base, Amoebae react in a manner emphasizing the above point with regard to unfavourable conditions. Their behaviour in these solutions is influenced by four factors : 1. The type of Amoeba, ' A ', ' E ', or ' C '. 2. The duration of experiment. 3. The acid or base employed. 4. The change in pH involved in transferring the Amoebae from the culture medium to the artificial medium. 1, 2, and 3 may be controlled by selection ; 4 requires an adjustment of the pH of the artificial medium, which may be carried out by the following process : 10 c.c. of glass distilled Avater are measured from a burette into each of a dozen clean test-tubes. Ten drops of the indicator proper to the desired range of pH follow in the concentration given in Clark and Lubs's list of indicators (2, p. 80), and drops of a very dilute solution of the pure acid or base selected for experiment. Any one colour given in the same amount of phosphate mixture by the same indicator can thus be accurately matched, and the pH of the Avater in the test-tubes is standardized. If the number of drops of the acid or alkaline solution required to obtain a desired pH exceeds five, a slightly stronger solution should be employed. It was at first desirable to use experimental media free from indicators. The number of drops of acid or base at the giA7en dilution necessary to bring 10 c.c. of glass distilled water to the desired pH Avas calculated by this method, therefore, and then added to 10 c.c. of fresh distilled water. The pH of the resulting solution was checked by removing 2 c.c. of it, adding 2 drops of indicator, and comparing the colour obtained with that of 2 c.c. of phosphate mixture with indicator. It was found, hoAvever, that the presence of so small a quantity of indicator made little difference to the behaviour of the Amoebae. It is not possible to maintain a standardized pH for long observation in the region of the neutral point. In dealing Avith alkali, for example, media in the absence of buffering salts readily absorb CO2 and other acid gases from the atmosphere, BEHAVIOUR OP AMOEBA 639

and tend to become less alkaline with increasing rapidity as the pH decreases. This can only be partially remedied by basing every judgement on a number of observations, by renewing the fluid as often as possible, by recording the pH before and after each experiment, and by making up such solutions in glass distilled water that has been exposed to the atmosphere until the pH has risen to some steady point. In the Glasgow labora- tory it commonly rises from 5-8 to 6-2. For comparison, the records of the behaviour of the Amoebae over a definite range of pH are best obtained simultaneously. A number of solid watch-glasses are placed under microscopes accommodating the solutions of different pH, and to each are then added four or five Amoebae. These are selected from distilled water according to the type required, and transferred to the solid watch-glass after a preliminary wash in the experi- mental solution. If the experiments are started at 20 min. intervals, a general idea of the behaviour of the Amoebae in each watch-glass is gathered by observing them continuously for the first 15 min., and afterwards at intervals determined by their activity. A wide range of pH cannot of course be attempted. Six values are about the maximum that may be covered, and even then it is barely possible to give the necessary attention to the six microscopes when the whole series is under observation. The results of such observation, covering a period of one hour for each watch-glass, are given in Text-fig. 9. The degree of attenuation of the pseudopodia, which attains a maximum in this time as a rule, is expressed by the size of the crosses. The pHs of the experimental solutions are given in the horizontal row of figures, and in this instance the Amoebae selected were of the type ' A ' after a short immersion in distilled water— pH 5-8. It is important to emphasize the fact that they were derived originally from a culture of pH 6-8 ; for Amoebae flourish in cultures considerably more acid and more alkaline than this, and their behaviour in the experimental solutions is determined by the extent of the contrast between the pH of the culture and that of the solution to which they are trans- NO. 280 U U 640 C. W. PARSONS ferred. If returned to the culture medium at the end of an hour, Amoebae that have not been brought to the point of maximum attenuation recover rapidly and completely. Eecovery is also possible beyond this point over the range of pH covered by the diminishing crosses in Text-fig. 9 ; but it then gives rise to forms which approximate to the types ' E' and ' C', and the changes are not therefore completely reversible. If the period of immersion in an experimental solution is pro- longed, recovery becomes progressively less possible. The Amoebae die in the more acid and more alkaline solutions within the first 5 hours. In the less abnormal solutions they become sluggish and consolidated in the same way as they do when floating in distilled water, and will make a partial recovery if returned to culture medium while movement of granules in their endoplasm can still be traced. The time when such move- ment ceases and when the changes become irreversible varies with the acid or base employed, and, to some extent, with the individual resistance of each Amoeba. In acid solutions there is a tendency for this time to be marked by local distensions of the outer layers, which give to an Amoeba a ' beaded ' appearance. In some alkaline solutions, e. g. sodium hydroxide, the cessation of all movement is attendant on the formation of perfectly spherical bodies ; and in both instances the actual rupture of the cell may be delayed two or three days after this has happened. Transference to a new medium in this condition, however, is almost always followed by immediate cytolysis. Ammoniacal solutions, on the other hand, induce cytolysis at similar pHs in 4-6 hours, and in them changes involving sudden cessation of movement may obviate the possibility of adopting a spherical form. The reactions of Amoebae belonging to the types ' E ' and ' C' to these experimental solutions suggest a correlation between their reduced surface areas and their lesser irritability. There is some attenuation of type ' E ' Amoebae in the early stages of immersion, but they round off more readily than do the type ' A ' Amoebae. Type ' C ' Amoebae, on the other hand, BEHAVIOUR OF AMOEBA 641 remain unaffected until they, in common with the types ' E ' and ' A ', round off and cytolize at the higher concentrations of acid or base. It may be concluded that the effect of these solutions is to

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A representation, by the size of crosses, of the degree of attenuation of type ' A ' Amoebae in very dilute pure solutions of various acids and bases.

accentuate the reactions typical of Amoebae to distilled water. They are stimulants ; at fairly well-marked critical concentra- tions (pH 4-6 for acetic acid, 4-8 for hydrochloric acid, &c.) they become injurious to Amoebae derived from a culture of pH 6-8, and their effects are cumulative. Some reagents, e. g. ammonia, bring about early cytolysis, and with others this occurs after a u u 2 642 C. W. PARSONS variable period of delay. It is also noticeable that the cell penetrative fatty acids have a slightly less inhibitory effect upon attenuation in type ' A ' Amoebae; an indication, perhaps, that they involve in them less radical surface changes.

DISCUSSION. 1. Of Adhesion.—It is clear that while the types of Amoebae ' A ', ' E ', and ' C ' differ mainly in respect of mobility, their behaviour in culture media is further compli- cated by the power they possess of adhering to the substratum. When floated in culture media the majority send out spreading pseudopodia all round the central mass of protoplasm as in Text-fig. 1. In pure media they react in essentially the same way (Text-fig. 5), but with excessive attenuation of the pseudo- podia owing to their abnormal environment. The loss of adhesion reveals therefore a characteristic floating form which is accentuated in the grotesque appearances presented by the floating type ' A ' Amoebae ; and it follows that adhesive capacity is an important property of the surface in normal healthy Amoebae. The nature of this surface is a matter of controversy. It is generally recognized that it cannot be the single uniform structure that the term ' ectoplasm ' may be held to imply, but there is no general agreement as to the nomenclature or structure of the layers that are supposed to comprise it. The problem has received the attention of several workers who in recent years have studied amoeboid movement, either with the view of supplementing the surface tension hypothesis, e.g. Schaeffer (9, p. 89), or of replacing it with new theories, e.g. Jennings (4), Mast (6), and Pantin (7). Schaeffer recognizes a fluid surface tension layer capable of work (9, p. 74) over parts of an Amoeba that are not in contact Avith the substratum. He explains thus the movement of grains of soot, carmine, &c, over the surface of an Amoeba in the direction of flow. His hypothesis is supported by experiments with type ' A ' Amoebae in water containing particles of washed carmine. The grains of carmine adhere to and move over the surfaces of Amoebae that are floating in distilled water although BEHAVIOUR OF AMOEBA 643 there is a verj' definite loss in them of the capacity for adhering to a substratum. Some justification may therefore be claimed for the conclusion that the mechanism responsible for fixing an Amoeba to the substratum is distinct from that by which small particles are carried over its surface. The thin tenuous layer called by Chambers a ' pellicle ' (1, p. 279) probably represents the former, while the latter, a thin fluid film, would not be demonstrable separately by microdissection. On this hypothesis, the capacity for adhesion depends upon the condi- tion of a layer beneath the surface tension layer. Circum- stances which may render it unusually fluid will impair this capacity therefore, and it seems reasonable to assign to such a cause the difficulty of inducing adhesion of type ' A ' Amoebae in pure media. In active attenuating forms the so-called ' pellicle ', and any gelating layers associated with it, must be in an abnormally fluid condition ; and once they have lost from this cause the opportunity of adhesion to the substratum, further contact with it is prevented by the buoyancy they acquire from their elongating pseudopodia. Conversely, con- solidation of the outer layers of an Amoeba beyond a certain point will also injure its power of adhesion. The types ' B ' and ' C ' have been described as comparatively immobile forms in culture media, and processes of consolidation will therefore influence their surface layers for a greater period of time than is normally the case. This offers an explanation of a fact of behaviour that has been noticed, namely, that these types readily lose their adhesion in culture media, and seldom adhere to the substratum in pure media although they retain contact with it. '2. Of Attenuated Pseudopodia.—The remarkable activity of type ' A ' Amoebae in pure media directs attention to the elongation of their pseudopodia. Schaeffer calls them ' pseudopodia of position ' to distinguish them from the broad pseudopodia of movement seen in normal adhering Amoebae. The tendency to adopt a ' radiose ' form was figured by Ver- wornfor Amoeba limax (13, p. 185), and obviously presents itself here with the difference that in Amoeba proteus the 644 C. W. PARSONS pseudopodia are always blunt. The reason for this attenuation of pseudopodia lacks explanation however. It is not simply a question of pH, for Amoebae can appear in flourishing cul- tures at pHs as low as 4 (Taylor, 12, p. 139) and at least as high as 7-6. Further, if the pH was ordinarily an important factor in determining the form of these organisms, the type ' A ' Amoebae would only be obtainable from cultures at the most suitable pH, and when the types ' E ' and ' C ' occurred at all in the same cultures, they would be localized in areas which, by reason of the metabolism of other organisms, were beyond the range of favourable pHs. There may actually be a small variation in pH over the bottom of a standing culture, but it is so small as to make no difference whatever to the variety of forms that may be yielded from different areas of it. The colloidal nature of protoplasm is an accepted principle ; and in active attenuating forms the change of phase from the fluid internal sol to the gelated condition of the surface layers must take place very rapidly. Schaeffer points out (9, p. 90) that this is accompanied by dispersion in a high degree of the internal phase of the latter, and depends upon the amount of water in the protoplasm. It seems probable, there- fore, that transference of a type ' A ' Amoeba from culture medium to distilled water is followed by imbibition and diffusion of water through an ever-increasing surface to increase the fluidity of the protoplasm. This, combined with the loss of adhesion and absence of ions essential for normal con- tractility, e. g. calcium ions (Pantin, 8) expresses itself in great attenuation. 3. Of the Eelationship between the types 'A', ' E ', and ' C '.—Their mobility leaves little doubt that the type ' A ' Amoebae are the most healthy forms in culture media. They are more likely to reproduce by fission and to produce encysted young than either of the types ' E ' or ' C ' therefore, and the comparative unhealthiness of the latter may be indicated by the approximation of the type ' A ' Amoebae to their form, after long residence in distilled water. The inadaptability of the types ' E ' and ' C ' to changes in the BEHAVIOUR OF AMOEBA 645 environment, their occurrence in small numbers in flourishing cultures, and their abundance in stale or overcrowded ones, points to the same conclusion. Cultures that are deliberately stocked with these forms fail altogether, and it is for these reasons that it seems probable that the bulk of those Amoebae which fail to enter on the cycle of encystment and which were mentioned at the commencement of this paper, belong to the types ' E ' and ' C '. SUMMARY. 1. The behaviour of the adult Amoeba proteus has been studied in their aquarium culture media, in distilled water, and in dilute pure solutions of various acids and bases of known and graded pHs. 2. Amoebae belonging to three physiological types are described from the cultures. The majority are large, active, digitate forms, which adhere closely to the substratum, and are grey in colour when seen in transmitted light. On transference to pure media they become attenuated, floating forms, to which the name type ' A ' Amoebae has here been applied. The remainder are darker in appearance and less adherent in the culture media. They belong to two types, which from their behaviour in pure media have been separated as type ' R.' and type ' 0 ' Amoebae. 3. The association between the floating form and the capacity for adhering to the substratum is stressed. 4. The problems of adhesion, of attenuation in pseudopodia, and of the relationship between the types of Amoebae are discussed. I wish to express my gratitude to Dr. Monica Taylor, S.N.D., without whose splendid material this investigation could not have been pursued, and my sincere thanks for their kind criticism and advice to Professor J. Graham Kerr, Dr. G. S. Carter, and Mr. C. P. A. Pantin.

March 1926. 646 C. W. PARSONS

BIBLIOGRAPHY. 1. Chambers, Robert.—' General Cytology.' University of Chicago Press, 1924. 2. Clark, W. Mansfield.—' The Determination of Hydrogen Ions.' Wil- liams and Wilkins, Baltimore, 1925. 3. Gruber, Karl.—" Uber eigenartige Korperformen von Amoeba pro- teus ", 'Arch. Protistenkunde', Bd. 23. Jena, 1911. 4. Jennings, H. S.—' Contributions to the study of the behaviour of the lower organisms.' Cam. Inst. Pub. 16. Washington, 1904. 5. Brooker Klugh.—" The Plunger Pipette : A new instrument for isolating minute organisms ", ' Journ. Roy. Mior. Soc.' London, 1922. 6. Mast, S. 0.—" Mechanics of locomotion in Amoeba ", ' Proe. Nat. Aoad. Sci.', vol. 9. Washington, 1923. 7. Pantin, C. P. A.—" On the Physiology of Amoeboid Movement", 'Journ. Mar. Biol. Ass.', vol. 13. Plymouth, 1923. 8. '' On the Physiology of Amoeboid Movement—the effect of Calcium". 'Brit. Journ. Exp. Biol.', vol. 3, 1926. 9. Schaeffer, Asa A.—' Amoeboid Movement.' Princeton University Press, 1920. 10. " Choice of food in Amoeba ", ' Journ. Anim. Behav.' Boston, 1917. 11. Taylor, Monica.—" The Technique of Culturing Amoeba proteus ", ' Journ. Roy. Micr. Soc' London, 1921. 12. " Amoeba proteus : some new observations on its Nucleus, Life- history, and Culture ", ' Quart. Journ. Micr. Sci.', vol. 69. London, 1924. 13. Verworn, Max.—' General Physiology.' Translation by F. S. Lee. Macmillans, London, 1899.