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Some observations of Pelomyxa carolinensis with special reference to mercury orange sulfhydryl staining McQuillan, Loretta M. 1962

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SOME OBSERVATIONS ON Pelomyxa carolinensls with SPECIAL REFERENCE TO MERCURY ORANGE SULFHYDRYL STAINING  by Loretta M. McQuillan, B.A., University of British Columbia 1957  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE FEQUintMENTS FOR THE DEGREE OF Master of Science i n the Department of ZOOLOGY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA 1962  In presenting  t h i s thesis i n p a r t i a l f u l f i l m e n t of  the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference and study.  I further agree that permission  f o r extensive copying of t h i s t h e s i s f o r scholarly purposes may granted by the Head of my Department or by his  be  representatives.  It i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of  o up  Q  y  The University of B r i t i s h Columbia, Vancouver 8, Canada. Date  - iii -  ABSTRACT  Cytochemical studies using l-(U-chloromercuriphenylazo)naphthol-2 (Mercury Orange), a specific reagent for the detection of sulfhydryl groups, were carried out on the giant raulti-nucleated amoeba, Pelomyxa carolinensis. Distribution of -SH groups was compared i n normal well-fed organisms, starved amoebas and i n sodium arsenite-treated individuals. The hypothesis that spindle fibre formation during mitosis involves, i n part, oxidation of -SH groups to -SS- bonds and that peripheral nuclear granules play a role i n spindle fibre formation was investigated. Preliminary investigations on mouse and toad muscle fibres were carried out for the purpose of confirming Bennett's results as well as to test the specificity of Bennett's Mercury Orange technique for -SH. Considerable time was spent i n observing the living amoeba. Included i n these observations was feeding behaviour, reaction to various stimuli and reproduction. Prior to the -SH studies, the general morphology of P. carolinensis was investigated i n considerable detail using various fixation and staining techniques. Likewise, mitosis i n this amoeba was thoroughly studied. Synchronous division of the many nuclei (as observed by Kudo and Schaeffer) was confirmed. Relationship of the amoeba's contour to the stage of nuclear division was studied. This relationship proved helpful i n the study of -SH groups i n the dividing nucleus since i t provided a means of choosing a specifically desired mitotic stage.  ACKNOWLEDGMENT  I wish to extend my gratitude to Dr. Ian McTaggart-Coxran, Head of the Department of Zoology, University of British Columbia, for making available to me the f a c i l i t i e s for carrying out this work. This study was supported Research Fund. I wish to express Dr. Peter Ford for the assistance, has given me throughout the course  by a Grant from the President's my sincere appreciation to direction and encouragement he of this work.  I wish also to thank the members of my Committee: Dr. James R. Adams, Dr. R. Neal Baad, Dr. Cyril V. Finnegan, Dr. Kenneth Graham, and Dr. Sidney H. Zbarsky, who have advised and assisted me i n this endeavour. To Dr. Denys K. Ford, Faculty of Medicine, wao extended to me, for additional study, his laboratory f a c i l i t i e s at the Strong Laboratories, Vancouver General Hospital, I express my thanks. The amoeba and Paramecium cultures were generously donated by Dr. A. A. Schaeffer, Director, Biological Institute, Philadelphia, Pa. and I would like to express my appreciation to him for this assistance. Also, I am grateful to Members of the Faculty, Staff and my colleagues who have assisted me from time to time.  TABLE OF CONTENTS  Page INTRODUCTION Purpose of the Research  1  IZTERA.TURE REVIEW Nomenclature  3  P.carolinensis as an Experimental Animal  h  Arsenic i n Relation to Cell Poisoning v i a -SH....  11  Sulfhydryl Groups  13  Cytochemical Methods for Detection of -SH  16  MATERIALS and METHODS Cultivation of Amoebas and Parameciums  21°  Cytological Studies 1. Routine Histological Staining a) V i t a l Staining b) Permanent Histological Preparations.. 2. Cytochemical Methods for Demonstration of -SH Groups a) Preliminary Experiments with Mercury Orange b) Application of Mercury Orange to P.carolinensis  21; 2$ 29 30 31  Page OBSERVATIONS and RESULTS I.  LIVING MATERIAL General Behaviour Feeding  35  Body Form  37  Correlation of Body Form with Mitotic Events...  39  Conditions which Favour Reproduction and Frequency of Mitosis Duration of Various Stages of Nuclear Division. II.  ill U2  HISTOLOGICAL STUDIES Vital Staining Permanent Histological Preparations Morphology Plasmalemma Cytoplasmic Ground Substance Cytoplasmic Inclusions .... Nuclear Division  Ii3 1*5 U5 U6 U6 h9  III. CYTOCHEMICAL STUDIES Preliminary Experiments with Mercury Orange ...  52  Effects of Sodium Arsenite on Living Amoebas .. Application of the M.O, Method to P.carolinensis Mitotic Nuclei Stained with Mercury Orange  52 55 58  DISCUSSION; I.  LIVING MATERIAL General Behaviour Feeding  60  Body Form  66  Correlation of Body Form with Mitotic Events ...  68  Conditions which Favour Reproduction and Frequency of Mitosis  68  Page II.  HISTOLOGICAL STUDIES Vital Staining  69  Permanent Histological Preparations Morphology Plasmalemma Cytoplasmic Ground Substance.. Cytoplasmic Inclusions........  71  Nuclear Division  71 71 72 77  III. CYTOCHEMICAL STUDIES Preliminary Experiments with Mercury Orange ....  83  Effects of Sodium Arsenite on Living Amoebas....  8U  Application of the M.O. Method to P.carolinensis  8U  Mitotic Nuclei Stained with Mercury Orange  87  SUMMARY BIBLIOGRAPHY  90  LIST OF TABLES  Number  Page  1  Average Duration of Mitotic Stages  U3  2 (a)  Results of Treating 12 Living Amoebas with NaAs03 Followed by Mercury Orange Staining  52  Results of Mercury Orange Staining of P.carolinensis Treated with Sodium Arsenite (NaAs03) Prior to Trichloroacetic Acid Fixation  55  3  Mercury Orange Staining of P.carolinensis.  56  k  Mercury Orange Staining (2li hours) of Dividing Nuclei i n P.carolinensis.....  58  2 (b)  ILLUSTRATIONS  Plate Number  -  Page  Figure 1. Method of Washing Parameciums ... 23 Figure 2. Healthy, normal, living  I  Figure 3. P.carolinensis during process of ingestion. Figure U. Apparent attraction of Parameciums for P.carolinensis.  II  Figure 5. Exploratory behaviour of Parameciums i n vicinity of P.carolinensis. III  Figure 6. Newly formed food vacuole.  IV  Figure 7. Star-shaped, living P.carolinensis. Figure 8. Recently ingested food i n an amoeba.  V  Figure 9. Compact form of P.carolinensis. feeding on small protozoa.  VI  Figure 10 Prophase i n P.carolinensis.  VII  Figure 11 Metaphase i n P.carolinensis.  VIII  Figure 12 Anaphase i n P.carolinensis.  IX  Figure 13 Telophase i n P.carolinensis. Figure lh Interphase i n P.carolinensis.  X  Figure 1$ Frequency of mitosis i n P.carolinensis ....opposite...Ul Figure 16 Schaudinn's Fixation Figure 17 Schaudinn's Fixation  XI  Figure 18 Bouin's Fixation Figure 19 Osmium Tetroxide Fixation  XII  Figure 20 Trichloroacetic Acid Fixation Figure 21 Frozen Preparation  Plate Number XIII  Page  Figure 2 2 . Mitochondria (X850). Figure 2 3 . Mitochondria (X1825). Figure 21. Fat Globules - Sudan Black.  25. Food  Vacuoles - peripheral granules.  XIV  Figure  XV  Figure 2 6 . Crystals i n Living amoeba. Figure 27. Crystals i n Schaudinn-fixed material.  XVI  Figure 2 8 . Nuclear peripheral granules. Figure 29. Nuclear division i n P.carolinensis.  XVII  Figure 3 0 . Nuclei of P. carolinensis. Figure 3 1 . Early Prophase i n P.carolinensis.  XVIII  Figure 3 2 . Late Prophase. Figure 3 3 . Early Metaphase.  XIX  Figure 3U. Ovoid shape - Early Metaphase. Figure 3 5 . Changing shape of mitotic figure. Figure 3 6 . Rectangular shape - Late Metaphase.  XX  Figure 3 7 . Anaphase discs pull apart i n several . directions.  XXI  Figure 3 8 . a) Converging polar fibres, b) Spindle fibres breaking up, i n late anaphase and telophase.  XXII  Figure 3 9 . Sulfhydryl distribution - Normal Figure IiO. Crystals i n food vacuole.  XXIII  Figure i l l . Crystals i n food vacuole. Figure U2. Majority of crystals are rod-shaped. Figure U3. Crystals scattered about i n cytoplasi smaller, square-shaped.  Plate Number XXIV  Figure kk. Sulfhydryl distribution starving.  XXV  Figure hS. M.O. staining of interphase nucleus.  Page  Figure U6. M.O. staining of prophase nucleus. Figure kl. M.O. staining of metaphase nucleus. Figure U8. M.O. staining of anaphase nucleus. Figure U9. Observations and Hypothesis regarding M.O. staining of mitotic nucleus i n Pelomyxa carolinensis.  opposite  IIWRODUGTION  Considerable cytochemical investigations of sulfhydryl groups i n various tissues have been performed within recent years. Except for nitroprusside, however, such studies have not been carried out on protozoa and there are no reports of any for Pelomyxa carolinensis.  It was the object of the present work to detect  and observe the distribution of sulfhydryl groups, by means of a specific cytochemical reagent, i n Pelomyxa carolinensis. Conditions were set up whereby -SH distribution could be studied i n normal, healthy organisms, as well as i n starving and arsenicpoisoned individuals. In order to assess this amoeba as material for cellular research i t was necessary to learn as much as possible about the animal maintained under laboratory conditions, thus considerable time was spent observing living P. carolinensis. The histological studies were performed for the purpose of becoming acquainted with the internal structure of the animal in order to facilitate the interpretation and evaluation of existing literature as well as my own histological and cytochemical observations. A, special interest was taken i n the mitochondria of the animal because of the known correlation between sulfhydryl groups, respiratory enzymes and mitochondria i n multicellular organisms.  -  2  -  The mitotic studies of fixed material were necessary before further investigation regarding the possible role of sulfhydryl groups i n mitosis.  LITERATURE REVIEW Nomenclature Considerable confusion exists regarding the nomenclature of the organism used i n this study. Rosel von Rosenhof discovered an organism i n 1755 which he called "der kleine Proteus".  In 1758  Linnaeus named i t  Volvox chaos, then renamed i t Chaos proteus i n 1767.  The  majority  of zoologists considered Volvox a protozoan, therefore, the binomial name of RSsel's "kleine Proteus" i s Chaos chaos L. Dr. A. A. Schaeffer (1937, 1937a), whose cultures I obtained, discovered Pelomyxa carolinensis Wilson i n New Jersey.  He believed  that P. carolinensis, described and named by Wilson (1900) was Chaos chaos and supplied i t to Turtox under this name. now  Turtox  sells this giant amoeba as Chaos chaos. Therefore, the name  Chaos chaos has been generally accepted by many despite the objections of certain workers who have studied RSsel's observations. Johnson (l93lj and Mast, 1938)  Mast and  are two such investigators tho concluded  that Chaos chaos i s not identical with Wilson's P. carolinensis but that i t i s a myxomycete.  Kudo (19U6, 1952, 1959)  who also examined  Rosel's paper i n conjunction with his own laboratory investigations on P. carolinensis believes that the facts do not support assumption.  Schaeffer s f  He states that, "P. carolinensis i s the valid name of  the organism described and named by Wilson (1900) and that Chaos chaos  -lire-mains an unidentifiable organism of historical interest." In the present study, this giant amoeba i s referred to as Pelomyxa carolinensis Wilson.  P. carolinensis As An Experimental Animal The nutrient requirements of P. carolinensis are as yet undetermined, therefore i t has not been possible to develop axenic cultures of this organism. Kopac (1959) and Prescott (1959) are attempting to solve this problem. Until recent electron microscopic studies fewer references pertaining to experimental studies on P. carolinensis appear i n the literature as compared with some other protozoans, for example Paramecium and Amoeba proteus. could be found.  No reference to histoehemical studies  Most of the experimental work has been i n connection  with behavior, morphological description and physiological investigation. V i t a l staining with Neutral Red reveals cytoplasmic granules known as "neutral red bodies" (Andresen 19lt2, 19u5> 19U6).  These  studies have led to the "vacuome" theory reviewed by MacLennan ( I 9 l i l ) . It i s also supported by earlier workers (Hall, 1930; Hall and Loefer, 1930; Nigrelli and Hall, 1930). Q-950, 1952)  On the other hand, Goldacre  and Torch (1959) believe these particles are an  artifact of fixation subsequent to staining with neutral red.  -  5  -  V i t a l staining with Janus Green B carried out by Mast & Doyle (1935  a,b) and Kassell and Kopak (1953) revealed small particles Studies by Torch (1955)  covering the surface of contractile vacuoles.  and Pappas (1959) have led these workers to believe that these round or oval-shaped bodies are mitochondria.  They have observed  the detailed structure of individual mitochondria. Classical studies on the cytology of P. carolinensis have been carried out by Andresen (19U2, 1956)j Wilbur (19U2, 19U5) and Torch (1955, 1959).  Kudo (19U6, 195l)j Recent electron  microscopic studies on the internal structure of P. carolinensis have extended the classical studies.  Much of what was previously observed  has been confirmed with the electron microscope.  Some of the  assumptions have now been turned into facts whilst others have been proven false. Electron microscopic studies (Borysko & Roslansky, 1959) show that the plasmalemma of P. carolinensis i s a single membrane. Pappas (1959) observed the plasmalemma to be approximately 200 i? thick. o Fine fibrous extensions extend approximately 1700 A from the surface. o o The diameter of these filaments i s i;0 A to 60 A. (Lehmann et a l . , ( l 9 5 6 a,b);  Earlier workers  Manni (1956) and Brandt (1958) ) had  described the plasmalemma as a double-layered membrane but i t i s now believed that the outer layer represents the minute filaments revealed i n Pappas  1  (1959) studies and that these fine filaments may serve as  - 6 -  sites of adsorption of substances from the surrounding environment. The mechanism for such membrane formation proposed by Andresen ( 1 9 5 7 ) i s currently being investigated by electron microscopic studies (Landau, 1 9 5 9 ) . Cytoplasmic ground substance appears homogeneous under the light microscope. However, studies with electron microscope, centrifugation studies of homogenates, chromatographic separation and surface chemical properties of proteins, indicate that submicroscopic particulate structures are contained i n this ground substance ranging i n size from 0.06  to 0.2 microns (Kopac, 1950;  Kassel, 1959). Studies by Kopac (1951) suggest that these submicroscopic components, because of their capacity to undergo f i b r i l l i z a t i o n , may be basically responsible for sol-gel changes involved i n amoeboid movement and cytokinesis. The cytoplasmic inclusions found within P. carolinensis and A,, proteus appear to be similar i n a l l respects, with the exception of the nuclei, P. carolinensis, being multinucleate (Torch, 1959).  Kudo (I9k9,  19U7,  195U)  has described the nuclei  and nuclear division in Pelomyxa carolinensis. Hinchy (1937)  f  Schaeffer ( 1 9 3 8 , 1 9 U 6 )  and Short ( 1 9 U 5 , 1 9 U 6 )  have also studied mitosis  in this organism. Electron microscopic studies show a definite double-membrane  - 7 -  structure of the nuclear envelope. o Pores having a diameter of approximately 600 A were observed by Pappas (1959) within the nuclear envelope and are believed to be formed when the inner and outer membranes are joined with one another.  Since these nuclear pores do not appear to be patent,  certain workers feel that they might more appropriately be termed "annuli" (Freider et a l , 1956; Rebhun, 1956; Gall, 1956 and Swift,  1956). In the three species of amoebae studied (i.e. A. proteus, H. rysodes and P. carolinensis) the pores occupy 15 to 20 percent of the nuclear surface which i s approximately twice that estimated for mammalian cells (Watson, 1955). Immediately within the nuclear envelope of P. carolinensis " o i s found a loose network of f i b r i l s which are approximately 60 A to o 70 A thick but the depth of this intraperinuclear f i b r i l l a r network varies a great deal.  Nucleoli are found just within the nuclear  envelope whilst within the more central areas of the nucleoplasm are found clusters of helices.  At present, studies are i n progress  concerning the appearance and distribution of these helices during the stages of nuclear division (Pappas, 1959). Electron microscope studies have shown that the peripheral granules (nucleoli) contain small clusters of dense granules (Borysko and Roslansky, 1959). Torch (1955) carried out a careful study of the mitochondria of P. carolinensis for the purpose of identifying and describing them  - 8 -  as a step toward determining their possible significance. Borysko & Roslansky (1959) and Pappas (1959) during electron microscopic studies noted a complex internal structure of mitochondria which consists of a system of pleated membranes, numerous small granules and amorphous material.  Serial sections indicate that  many of the mitochondria are serpentine filaments rather than spheres or rods. The studies of Torch, as well as that of other workers (Joyet-Lavergne, 1926,  1928,  1929, 193U,  1935;  Cowdry & Scott,1928;  and Weiss, 1950), indicate that the mitochondria i n protozoa  may  function i n cellular respiration. Torch (1959) described "crystal vacuoles" around food vacuoles which are referred to as "vacuole refractive bodies" and appear as particles possessing darkened rims surrounding a colourless interior.  Brown (1930) and Mast and Doyle (1935) believed  them to be Golgi material because they give positive results with osmium techniques.  But recent studies on centrifuged material and  electron microscopic identification of structures other than these bodies, similar to vertebrate Golgi have led to a new interpretation (Cohen, 1957  and Pappas, 1959).  Further, MacLennan (19U0) has shown  that the Golgi element cannot be identified on the basis of osmium reduction alone.  Pappas (1959) noted a thick layer of granular  material i n close association with the limiting membrane of newly formed  - 9 -  food vacuoles.  Older food vacuoles do not possess this granular  ring of material around them.  Because of this close association  with newly formed food vacuoles they have been assigned a digestive role. Mast and Doyle (1935 a, b) are of the opinion that these homogeneous spherical bodies contain fatty acids and other l i p i d s . Refractive bodies dispersed throughout the cytoplasm are described by Mast and Doyle (1935  a).  Each i s composed of three  morphological components: a relatively fluid cortical layer i n which l i p i d s and proteins have been demonstrated, an inner brittle shell and an innermost apparently f l u i d core. were confirmed by Pappas (195U).  These observations  Heller and Kopac (1955  a, bj  1956)  and Pappas (l95h) have been able to demonstrate organic phosphate, calcium and magnesium i n relatively high concentration, as well as other minerals i n lower concentration, i n the fluid portion of these particles. Fat globules present i n the cytoplasm were shown by Mast and Doyle (1935 a) to be composed largely of neutral fat. confirmed these findings.  Pappas (195U)  Pappas also observed i n A. proteus  that although the glycogen disappeared after 3 days of starvation, as much as one third of the normal content of l i p i d droplets s t i l l remained i n the cytoplasm of most of the organisms at the time of their death due to starvation.  In centrifuged amoebas (Torch, 1959)  the fat  droplets locate themselves at the extreme end of the centripetal pole.  - 10 -  During the i n i t i a l redistribution of the cytoplasmic components the other inclusions (i.e. crystals, food vacuoles, nuclei and mitochondria) begin streaming towards the centripetal pole but the fat droplets remain relatively stationary and clumped.  When redistribution  of the cytoplasmic components i s completed (approximately one hour) the fat globules can be seen circulating en masse within certain individuals. Crystals were not seen directly with the electron microscope but Pappas (1959) states that vacuoles that had contained crystals could be readily identified by the absence of embedding material i n the space that formerly contained the crystals.  These negative  images corresponded to the two types of crystals present i n the l i v i n g amoebas, namely, platelike and truncated bipyramidal. Mast and Doyle (1935 b) and Wilbur (19^5) believed that these crystals originated i n food vacuoles and thus represented a food reserve. However, Andresen and Holter (19U5) have shown that i n P. carolinensis these bodies do not decrease i n numbers during starvation. These workers further claim that the crystals arise i n the cytoplasm and are found only incidentally i n the food vacuoles as a result of coalescence. The contractile vacuoles as seen by the electron microscope (Pappas 1959)  show a vacuolar manbrane surrounded by a densely packed  layer of small, round vesicles believed to be secretory i n function.  - 11  -  These vesicles i n turn are surrounded by mitochondria. (19U6),  Lehmann et a l  Bairati and Lehmann (1956) and Pappas and Brandt (1958)  have also carried out studies on these vesicles i n an attempt to determine their function and relationship to the contractile vacuole. Gatenby et a l (1955), Dalton and Felix (1956) and Pollister and Pollister (1957) discuss the contractile vacuoles with respect to the possibility of their representing Golgi apparatus. Studies of the food vacuoles of P. carolinensis carried out by Borysko & Roslansky (1959) revealed that the lumen of the vacuole i s continuous with several long channels that extend radially away from the vacuole for long distances.  They postulate  that these channels extend to the exterior of the amoeba and thus provide an escape route for water ingested with food organisms.  Arsenic i n Relation to Cell Poisoning via -SH Ehrlich, as early as 1909 thought the toxicity of arsenic was due to a reaction with receptor groups present i n the protoplasm.  He suggested that the groups involved may be hydroxyl  or sulfhydryl. Voegtlin (1925) and co-workers (1923, 1925) while studying the trypaniacidal action of arsenicals i n rats, found that arsenicals must be i n the tri-valent form (as i n arsenoxide R-As=0) i n order to exert a toxic effect on the c e l l .  When rats were injected with  arsenoxide the number of parasites f e l l .  However, i f glutathione,  - 12 -  or some other similar -SH compound, was injected together with the arsenoxide the toxic effect could be inhibited.  Thus,  Voegtlin postulated that the toxicity of arsenoxide on trypanosomes was due to the blocking of -SH groups i n the c e l l .  Since then  more evidence has accumulated along these lines (Szent-Gyorgyi, 1930; Labes, 1929; Cohen et a l , 1931; Rosenthal, 1932;  Glahn et a l , 1938;  Hitchcock, 19U6) and i n vitro studies show that the toxic effects of arsenic are brought about by inhibition of metabolic enzymes the pyruvate oxidase system i n particular - through the blocking of -SH groups (Pillae, 1938).  Peters (19U0, 19U9) demonstrated the  inhibition of the pyruvate oxidase system i n vivo.  He found an  increase i n blood pyruvate level at an early stage of arsenic poisoning (lewisite) which he attributed to the effect of arsenic on -SH groups.  However, glutathione and similar monothiols  could not reverse the effect of lewisite on the pyruvate oxidase system. Baron (19U7 a, b) found that lewisite had the greatest affinity (amongst the trivalent compounds) for -SH groups and inhibited a l l sulfhydryl enzymes except d-amino acid oxidase, yeast carboxylase and transaminase.  Inhibition was reversed by BAi  (British anti-lewisite) i n a l l cases. Stocken and Thompson (19U6, 19U8) found that arsenic combined with the thiol groups of protein i n the ratio:  1 arsenic  to 2 -SH groups and hence postulated the "Dithiol Theory" which may  - 13 -  be illustrated as follows: S-R•»  R-As  Whittaker (19U7) supports this theory and Danielli (19U7) states that BAX reverses the above reaction. Sulfhydryl Groups Soluble t h i o l groups, distributed throughout the c e l l , and fixed sulfhydryl groups attached to side chains of proteins, serve important functions i n the regulatory mechanisms of cellular respiration as well as i n the performance of the processes leading to c e l l division and growth. Glutathione, a tripeptide - (2T-glutamylcysteinylglycine) i s the most representative example of such soluble thiol groups. The biological functions of glutathione have not yet been definitely established.  Functions attributed to this substance date back  to 1888 when i t was discovered by deRey Pailhade. and rediscovered i n 1921 by Hopkins. to be a catalyst for cell respiration.  It was forgotten  It was f i r s t postulated The role of glutathione  in regulation of respiration i s the result of i t s low oxidationreduction potential.  Glutathione also exerts a protective or  reactivation role towards metabolic enzymes which contain -SH groups Whenever these -SH groups are attacked by  -  lU -  destructive agents, glutathione restores them by either withdrawing heavy metal or by reducing the oxidized sulfhydryl group (Barron, 1951). Some workers i n the f i e l d of c e l l division believed that the -SH groups involved belonged to glutathione (Shearer, 1922; HammetJ^ 1929; Dulzetto, 1931; Rapkine, 1931; Rapkine et a l , 1931; Binet and Weller, 1936;  Barron et a l , 19U8;  Bolognari, 1952; Mazia, 1952,  Infantellina and LaGrutta,  19$k a, b; 1955;  19U8;  Backstrom, 1956;  Stern, 1956). Others f e l t that both fixed and soluble -SH groups play a part i n c e l l division - probably i n spindle formation as well as i n plasmotomy (Binet et a l , 1937; Ghosh, 1937; Vincent, Colien, 1938;  1937;  Williams, 191*7; Ecker and Pillemer, 1938; Watkins and  Wormall, 1938). S t i l l others felt that glutathione does not play the leading role but a rather more passive one .serving as a respiratory carrier (Mapson and Moustafa, 1956)  or as a regulator of the  oxidation-reduction level i n the c e l l (Barron et a l , 19U8)  and i t was  therefore f e l t that the key to the solution of division activity should be sought for in the sulfhydryl (r.SH) disulfide (-SS-) transformation occurring i n the complicated protein systems which are required for the formation of the spindle apparatus. The manner i n which -SH groups take part i n c e l l division i s poorly understood and the elucidation of this problem i s one of  - 15  -  the major tasks of biochemical cytology (Brachet, 19h0; 19hl; Mazia and Zimmerman, 1958;  Mazia, 1958;  1959;  1957;  I960; Zimmerman, I960),  as well as others whose work i s referred to below. Until the work of Saiki (1959) l i t t l e progress was made i n this regard since Rapkine f i r s t suggested this function of -SH groups. Saiki employed several methods for quantitative determination of glutathione - among which was the titration method improved by Kuroiwa (1953) as well as the nitroprusside method proposed by Grunert and Phillips (1951).  His results showed that glutathione  leaves sea urchin eggs within 2 minutes after addition of trichloroacetic acid, indicating that the positive sulfhydryl tests obtained by the many experiments must be due to protein-bound -SH groups of the egg protoplasm.  The work of Neufeld and Mazia (1957) and Mazia  (1957) f a l l s i n line with this concept, for they also found that neither glutathione nor ascorbic acid fluctuate i n dividing sea urchin eggs. Recent cytochemical investigation of the distribution of free sulfhydryl groups i n Clypeaster japonicus by Kawamura and  Dan,  (1958) and Kawamura (i960) also indicate that theories of c e l l division i n which glutathione i s instrumental may not be valid. These workers, as well as Mazia (1961 a, b), now feel that the sulfur-containing groups need not necessarily be fully oxidized -SS- bonds similar to those found i n such stable structures  - 16 -  as hair and vulcanized rubber. Cytochemical Methods for the Detection of -SH Groups Cytochemical methods for demonstration of -SH groups rely on the fact that sulfhydryls react with almost a l l protein reagents (Olcott and Fraenket-Conrat, 19U7).  That the reaction has occurred  i s evident upon inspection because of the chromogenic moiety i n the reagent.  The latter may be an oxidizing agent bearing a  reactive halogen group or a reactive heavy metal on i t s molecule. Such reagents have been sought since the beginning of the century (Glick, 19U9; 1953 a, b;  Pearse, 1953, 1960j  195U a, b;  Casselman, 1959;  Barrnett and Seligman, 1952,  Brachet, 1957;  Mellors, 1959;  Baker, 1958j  Davenport, I960).  MSrner, i n 1902, f i r s t used the Nitroprusside reaction for identifying cysteine (Morner, 1921).  Others later employed i t  to demonstrate sulfhydryl groups i n plant and animal material (Gola, 1902;  Buffe, 190U;  Heffter, 1908).  been used by many workers (Kaye, 1921*j Joyet-Lavergne, 1928j  Since then i t has  Walker, 1925J  Percival and Stewart, 1930;  Giroud, 1931;  Giroud and Bulliard, 1933;  Rapkine, 193U;  Bourne, 1935;  Hammett and Chapman, 1938;  Serra, 19U6).  Some of the aforementioned  have modified the original technique i n various ways.  Lison, 1936;  X-ftien thiols  react with Nitroprusside i n the presence of ammonium hydroxide and ammonium sulfate, a purple colour i s produced.  Nothing i s known of  - 17 -  the chemistry of the reaction, however, this technique has been extensively treated i n the literature (Kay, 192hj Giroud and Bulliard, 1933] Lopes, 19U5j  Walker,  Fugita and Wumata, 1939;  Mescon and Flesch, 1952;  1925;  Serra and  Rudall, 1952;  Adams, 1956;  Pearse, 1961). Golodetz and Unna (1909) used the Ferricyanide method to demonstrate "reduction sites".  Chevremont and Frederic (19U3)  later applied the i n vitro ferric ferricyanide reduction test described by Mason (1930) to the demonstration of thiols i n tissue. The method has since been used successfully by Frederic  (l9h9),  Yao (19U9), Hardy (1952) and Rudall (1952), whilst some workers feel that the need for caution and many controls before safe conclusion can be reached i s a considerable disadvantage ( L i l l i e and Burtnsr, Findlay, 1955).  1953;  Barron (1951) points out that the inability of  ferricyanide to oxidize a l l the -SH groups of denatured protein results i n an ill-defined end-point i n the colour reaction. Adams (1956) has carefully considered the histoehemical interpretation of this method. Bennett (l95l) demonstrated thiol groups i n certain tissues by means of a red mercaptide-forming sulfhydryl reagent ( l - [^-chloromercuriphenylazq] -2-naphthol). referred to as Mercury Orange.  This reagent i s commonly  Bennett and Yphantis (19U8)  synthesized Mercury Orange while attempting to develop a quantitative  - 18 -  analytical cytochemical method for measuring sulfhydryl content of tissue components too small for conventional microchemical procedures. The technique i s a modification of the i n vitro phenyl mercuric chloride (p-chloro-mercuri-benzoic acid) sulfhydryl reaction of Hellerman, Perkins and Clark (1933).  The red-coloured azo  derivative of Hellerman s reagent retains the specificity for 1  mercaptans displayed by phenyl mercuric chloride and also, because of i t s chromogenicity, acts as an optical tag which dilineates the sites where i t i s bound to tissue components by mercaptide linkage. The basic reaction iss  R-SH + Cl-Hg-<^-N  6H  t  R-S-Hg-<^-N  + HCl HO  Protein containing Groups  Mercury Orange  Compound responsible for Characteristic Colour = Chromogen  Since Bennett (1951) f i r s t used Mercury Orange for qualitative work certain conditions have come to be appreciated not previously considered which must be met i f a l l , or most, of the -SH groups of a tissue are to be demonstrated cytochemically. Hence, Mercury Orange has not enjoyed the popularity of the BarrnettSeligman Method (Barrnett et a l , 1955J Seligman, 19k9',  Ravin et a l , 1952 j Ashbel and  Seligman et a l , 19U9)..  But, when these conditions  are satisfied, Mercury Orange possesses the characteristics of an ideal  - 19 -  cytochemical reagent (Bennett and Watts, 1958).  Pearse (1961)  regards both methods as possessing equal usefulness for cytochemical work. Cafruny et a l (1955) observed, from sections which stained sufficiently to allow photometric determination, that amounts of t h i o l in various c e l l types were comparable by the two methods, and feels therefore, that the two methods are comparable. The success of the Barrnett and Seligman Method (1952, 1953 a, b; 195U a, b) i s attributed to the fact that the reagent (2,2 -dihydroxy-6,6'-dinaphthyl disulfide or DDD) contains a ,  disulfide group which reacts with sulfhydryl groups and no other groups at alkaline pH.  DDD has been used to demonstrate histo-  cheraically both sulfhydryls and disulfides or disulfides alone (Cafruny et a l , 195U, 1955).  1 ^ ) Q - S - S ^ 0  +  Protein-SH  The reaction i s as follows:  OH ?  P r - S - S ^ O O ^  (I)  (II)  "Reagent"(2,2'-dihydroxy6,6' dinaphthyl disulfide) OH H3CO  OCfo  OH  J  {  )  Pr (IV) Coloured Product  +  HS(III) Tetrazotized diortho-anisidine  - 20  (I) Reagent (2,2'-dihydroxy-6,6' dinaphthyl disulfide) when used i n excess at pH 8.5 reacts with active -SH groups of fixed tissue proteins to form a colourless substance (II) plus by-product (III). The colourless oxidation product (II) i s insoluble i n both water and ether-alcohol therefore excess reagent (I) as well as by-product (III) can be washed out of the tissue with organic solvents.  Subsequent  treatment of the tissue with tetrazotized di-orthoanisidine results in the rapid development of a red colour (Monocoupling) or a blue colour (di-coupling) at the site of protein-SH groups (IV). Monocoupling (red or pink) indicates sparse, widely separated -SH groups whereas di-coupling (blue) indicates a greater concentration of -SH groups.  - 21 -  MATERIALS AND METHODS  Cultivations of Amoebas and Parameciums Cultures of P. carolinensis and P. multimicronucleatum were set up i n November, 1958.  The amoebas were maintained by  subculturing i n Syracuse watch glasses twice weekly.  Approximately  12 amoebas were placed i n thoroughly cleaned watch glasses containing 1  fresh stream water and a l"-long piece of timothy hay.  The timothy  hay for this purpose was allowed to mature before picking and then dried.  Before use, i t was boiled i n stream water for 10 minutes  to k i l l any undesirable organisms which might be attached thereon. Before placing the small stick of hay into the watch glass, a l l of the juice was poured off. temperature i n diffuse light.  The cultures were kept at room The watch glasses were stacked one  on top of the other and the top one covered by a clean inverted watch glass.  The amount of water i n the glass was not sufficient to  wet the bottom of the glass placed above i t . An adequate supply of Parameciums was kept on hand as a constant source of food for the amoebas by subculturing periodically in a timothy hay infusion.  The infusion was made up i n wide-  mouthed 250-cc Erlenmeyer flasks.  The flasks were f i l l e d to within  5 - 6 cm of the mouth with fresh stream water into which was placed approximately ten 1-inch sticks of timothy hay. 1  The flask was  Distilled water and n i t r i c acid (cone.) 1:1, rinsed several times and dried thoroughly.  - 22 -  covered with a clean inverted 100-cc beaker and the mixture allowed to boil for IS minutes.  After cooling, i t was  with approximately 10 cc of a heavy Paramecium culture.  innoculated Within one  week to ten days, this culture was ready to use for feeding the amoebas. Considerable time was necessary before growth i n these cultures became luxuriant and i t seemed to me that by increasing the amount of timothy hay, i t should be possible to develop thicker cultures.  But, this did not prove to be the case.  An excessive  amount of hay had a deliterious effect upon the organisms.  Rather  than a heavy culture developing, the Parameciums failed to multiply appreciably and eventually the infusion became cloudy and contained many dead organisms.  The problem, therefore, of providing sufficient  food for the amoebas was handled by maintaining a sufficient number of individual flasks of cultures.  More hay and water could be  added from time to time and, i n this manner, cultures were kept i n good condition for months. The Parameciums were washed before adding them to the amoeba cultures.  A small amount of cotton was placed within a  15>-cc conical centrifuge tube and pushed down just beyond the bend; stream water was poured i n t i l l i t reached the top of the cotton and the remainder of the tube was f i l l e d with Paramecium culture (Figure l ) .  Paramecium culture  Figure 1 - Method of washing Parameciums before feeding to P. carolinensis. A small amount of cotton was placed just beyond the bend of a 15-ec centrifuge tube. Stream water was poured i n to top of the cotton and remainder of tube was f i l l e d with Paramecium culture. Tubes were spun i n a hand centrifuge.  Cotton wool Fresh stream water  The tubes were spun i n a hand centrifuge (50 turns of the handle during 2 minutes).  If spun too rapidly, the Parameciums sent out  many trichocysts and died.  I f spun too slowly, they did not  pass through the cotton and therefore failed to become concentrated i n the fresh water beneath.  When the organisms had been brought  down, i t was possible to invert the tube without the cotton plug becoming displaced and the old culture fluid,containing only dehris, could be poured off.  When the cotton was carefully removed, by means  of a flamed glass rod, the Parameciums were sufficiently clean to be fed to the amoebas. During routine cultivation of P. carolinensis, 500 to 800 Parameciums were added to each watch glass.  Five or six watch  glasses of amoebas were sufficient to keep on hand for carrying out various studies.  During studies on mitosis the feeding procedure  - 2k -  described above, but without removing amoebas from watch glasses, was repeated approximately I4. to 6 hours before the organisms were required.  Some water was removed and fresh stream water added.  This ensured richly dividing material.  To obtain desired stages  for experimental purposes the cultures were scanned at intervals during the day and individuals selected according to body form.  Cytological Studies 1.  Routine Histological Staining a)  V i t a l Staining At the commencement of t his study, v i t a l staining was employed i n an attempt to observe the l i v i n g amoebas i n more detail.  For example, the nuclei i n the living  organisms were not visible under the stereoscopic binocular microscope nor under low power of the light microscope. High power could not be used without disturbing the animals (i.e. the watch glass could not be placed under the high power objective and i t was necessary to transfer the amoebas to a depressed microscope slide). The f i r s t concentration of Neutral Red used was 1:1,000. It proved too strong but the results were interesting. A 1:100,000 dilution of Neutral Red and a 1:200,000 of Janus Green B (in culture media) was next tried.  It i s  - 25 generally stated that concentrations of this order are non-toxic, however, i t seems reasonable that normal physiology must be disrupted to a certain extent by "vitaT!* stains since cells exposed to such staining are eventually k i l l e d . Thus, after a few t r i a l s , v i t a l staining was abandoned for methods such as phase contrast and water mounts.  Permanent Histological Preparations Routine histological techniques, known to demonstrate particular components of the animal c e l l were modified i n order to render them suitable for the morphological investigation and mitotic studies performed on P. carolinensis (McClung, 1950 j Gatenby & Beams, 19515 Ford, 1958j Baker, 1958j Davenport, 1960j Armed Forces Inst. Tech., I960; Pearse, 1953 and 196l). The various fixatives used were Bouin's, Schaudinn's, Osmium tetroxide, Formol Saline, Trichloroacetic Acid, Zeigwalner's and Carnoy.  Attempts at freezing the  amoebas before staining were made using dry ice and liquid air. Ehrlich s and Heidenhain' s iron haematoxalin and 1  Grenacher's Borax carmine were employed for nuclear studies. Formalin calcium fixation with Sudan Black stain was  -26used to demonstrate l i p i d s . For mitochondria, Regaud s Method with Regaud as 1  well as neutral formalin fixation was used.  Staining was  carried out using Janus Green B, Regaud*s Method for mitochondria, Heidenhain's Iron Haematoxylin (Champy Fixative) and Altman's Acid Fuchsin. In making permanent histological preparations several methods of handling the amoebas were employed. A modification of Kirby's (l9f?0) Method was used for the f i r s t whole mounts that were prepared.  The amoebas were  allowed to become attached to the bottom of a Syracuse watch glass, then most of the water was pipetted off before pouring hot (65°C) Bouin's Fluid onto the amoebas.  The  f i r s t alcohol i n the hydration series was poured i n with the Bouin's.  The animals were then taken through a series  of alcohols (85$ - $0%).  During this procedure the  amoebas were pipetted individually and the alcohol was contained i n black glass stenders which showed up the white amoebas well.  The amoebas i n the staining solution could  not be seen with the naked eye but when placed on the stage of a dissecting microscope the light passing through the glass from the bottom allowed them to be observed sufficiently well to be handled.  After differentiating, the animals were  - 27  -  cleared i n clove o i l , terpineol, o i l of wintergreen or l i l a c o i l and mounted i n Permount. The above manner of handling the material "was adequate for observing characteristic "body" shapes, pseudopods and some cytoplasmic inclusions (i.e. droplets, crystals, vacuoles).  However, because of the thickness of the  preparations, i t was not satisfactory for demonstrating nuclear structure nor for observing mitotic nuclei.  Thus, for  visualization of the nucleus, both during interphase as well as during the various stages of mitosis, the amoebas were fixed in the shape i n which they happened to be at the time.  Certain fixatives (formol saline, Schaudinn s, osmium 1  tetroxide and trichloroacetic acid) had an instantaneous effect without being heated.  Following fixation, the amoebas  were handled by means of pipettes, individually, as described i n the previous method.  Subsequent to staining they  were embedded i n small wax blocks, sectioned at It u, 5 u and 10 u, mounted on slides which were very clean and therefore required no egg albumin nor PSA\.  They were  then cleared i n xylene and mounted i n Permount.  Ball-  point pen lines were drawi beneath the very tiny sections i n order to facilitate finding them under the microscope. Since sectioned material provides preparations of uniform  - 28 -  thickness i t seemed necessary to employ i t for purposes of comparing intensity of Mercury Orange staining i n various stages of mitosis.  However, I discovered that the  Mercury Orange staining of the cytoplasm was poor.  (This i s  i n part due to the fact that Mercury Orange does not produce an intense colour i n thin preparations.  Although, i n the  past this was considered to be a disadvantage, i t i s now believed that this property, rather, indicates the specificity of the reagent (Pearse, 1961).  Therefore,  i n addition to serial sections I also prepared a large number of squashes for mitotic as well as cytochemical study. Squashes were prepared as follows.  One amoeba was  placed on a slide i n a small drop of culture f l u i d .  Strips  of f i l t e r paper were used to absorb most of this water. Dropping fixative directly onto the amoeba and then lowering the coverglass damaged the preparation considerably since this resulted i n fixation prior to flattening of the animal. Therefore, a method adopted from bacteriological study techniques was employed.  A drop of fixative was placed on  the coverglass •snich was inverted and lowered onto the amoeba. Such simultaneous fixation and flattening caused no apparent damage to the internal structures.  In many  instances the outer limiting membrane of the animal remained intact.  For nuclear observations there was no particular  - 29 -  advantage i n maintaining an intact membrane, therefore a slight amount of pressure was applied to the coverglass to further flatten the amoeba, vhile s t i l l maintaining intact nuclei. Once the correct degree of flattening was obtained, the coverglass was floated off with whichever f l u i d the particular method required.  With the coverglass floated  off, the amoeba remained attached to the slide (or i n manyinstances to the coverglass) and the latter could then be handled i n the usual.manner during dehydration, staining, clearing and mounting procedures.  2.  Cytochemical Methods for Demonstration of -SH Groups In selecting a suitable cytochemical reagent, certain criteria had to be satisfied by the chromogenic agent: (1)  The reagent should be specific for t h i o l groups.  It  must combine with thiols so as to demonstrate their site but should not react with any other groups i n the c e l l or tissue, or i f capable of such binding, convenient methods should be available for detaching the molecules bound nonspecifically without removing any from the t h i o l groups. (2)  The reagent should be monofunctional.  Each molecule  should possess only one group through irfhich the molecule might be bound to a tissue component.  - 30  (3)  The equilibrium constants of the binding reaction should favour strongly the bound state.  (U)  It should have a relatively small molecular size, so as to minimize steric hindrace to binding.  (5)  The reaction should not be affected by any of the histological procedures necessary for the preparation of permanent microscopic slides.  (6)  The colour should be sufficiently intense to be seen microscopically.  (7)  The colour should remain for a considerable time i n order to obtain a stable preparation which can be used as a permanent record of -SH content of the c e l l .  (8)  The reagent should not destroy the cytological quality of the preparation.  (9)  It should be available i n very pure form.  a)  Preliminary Exp eriments with Mercury Orange ' Bennett's Reagent, Mercury Orange, (l-(u-ehloromercuri-phenylazo)-2-Naphthol) was believed to satisfy a l l of the above c r i t e r i a .  Nos. (2) to (9) were apparent from the  literature and the chemical structure.  However, I felt  - 31 -  i t desirable to prove No. (l) i n our laboratory prior to using i t i n this study.  Preliminary experiments were  performed, therefore, on muscle tissue i n order to test the specificity of the reagent and to confirm the results of Bennett (19S>1).  Toad and mouse muscle fibres fixed  2k hours i n $% Trichloroacetic Acid, washed and dehydrated, were teased and immersed i n the Mercury Orange reagent (a saturated solution i n butanol containing 6 mgs/litre). Mouse muscle fibres were l e f t i n the solution for 7 hours and toad fibres for 2k hours.  Then they were washed  in butanol 2k hours, cleared i n xylene and mounted i n Permount.  These served as the controls.  The experimental  group were fixed similarly but prior to immersion i n Mercury Orage they were l e f t i n 1%, 5% and 10$ sodium arsenite (NaitsO^) for 2k hours. Application of Mercury Orange to P. carolinensis After confirming Bennett's experiments as above, techniques were developed for adapting this method to P. carolinensis.  Mercury Orange was used i n the same  concentrations and solvent as for preliminary muscle experiments (i.e.  6 mgm/litre i n butanol).  Amoebas  taken directly from healthy cultures were fixed with 5% TCA, washed i n d i s t i l l e d water,- dehydrated and rinsed i n butanol, then allowed to remain i n Mercury Orange reagent for  - 32 -  varying periods of time ( i . e . 3 , U, 8 , 2k and U8 hours). They were washed i n pure solvent 2U hours, cleared i n clove o i l , o i l of wintergreen or tnrpineol and mounted i n Permount on clean slides with no mounting media.  Some  of the amoebas were subjected to sodium arsenite both i n the l i v i n g state and subsequent to TCA fixation, but prior to Mercury Orange staining.  Mercury Orange was also  applied to "starving" individuals. The method chosen for the application of arsenic was the direct immersion of the specimen into the test solution. This i s a common procedure employed for application of a chemical agent to the l i v i n g c e l l for studying i t s effect but some of the problems involved should be borne i n mind. They ares  (l) modification of culture media;  (2) diffusion of substance into the organism, and (3) chemical solubility of the test substance.  The chemical  solubility of arsenic was a problem at the outset because I chose AS2O3 (Arsenic Tri-Oxide).  To overcome this, 1 gram  of AS2O3 was added to 10 ml of glycerine.  It was hoped  that glycerine would be sufficientty inert to have a minimiim deleterious effect upon the physiology of the amoeba.  The mixture was heated until clear.  After cooling,  90 ml of stream water were added to i t giving a 1% stock solution.  This method was not satisfactory because the  - 33 -  maximum concentration of arsenic which could be used was 1% and i t was desired to try concentrations of 1% and greater since concentrations below 1% did not affect the intensity of the -SH stain.  Increasing the proportion  of glycerine was not desirable since the results would probably be less significant i f a great quantify of extraneous substance was introduced along with the arsenic.  The  soluble sodium salt, sodium arsenite (NaAsO^) was therefore tried on l i v i n g material i n concentrations of 0.01 gm %, 0.5 gm % and 1.0 gm %.  Although some changes were  observed at concentrations of less than 1.0 gm %, death of the organisms did not occur - nor was there any difference between these amoebas and untreated ones with respect to sulfhydryl staining.  On the other hand, concentrations of  1.0 gm % and above caused disintegration of the membrane and the animals could not be used for cytochemical studies.  Thus,  i t was decided to use 1.0 gm %, 5.0 gm % and 10.0 gm % solutions of sodium arsenite on fixed material, as a means of verifying the specificity of Mercury Orange and to determine whether arsenic has an affinity for -SH i n protozoa as has been found i n i n vitro studies. In applying Bennett's Mercury Orange method, some sections were prepared as well as many squashes. For the study of changes i n -SH content of P. carolinensis  - 31* -  during nuclear division, the same procedures of feeding and selection of individual amoebas as described for making permanent histological preparations of mitotic stages were used but the cytochemical reagent was used (as described above} i n place of routine stains.  PLATE I  Figure 2 - Photomicrograph of healthy, normal living F.carolinensis placed i n clean watch glass containing fresh stream water immediately begins to send out pseudopodia and attaches i t s e l f to the substratum. X 95  Figure 3 - QDC« attached to the substratum P. carolinensis begins ingesting food organisms. Recently ingested Paramecium i n food vacuole i s seen here. Note the thin, delicate-appearing membrane of the amoeba compared with the dense, thick, ciliated pellicle of the entrapped Paramecium. X 95  - 35 -  OBSERVATIONS & RESULTS  I.  LIVING MATERIAL General Behaviour Feeding Unless undergoing mitosis, healthy, normal, living P. carolinensis became attached to the bottom of the glass containing fresh stream water (Figure 2).  The amoebas  attached themselves whether or not Parameciums were added, provided the glassware was meticulously cleaned and dried prior to adding fresh water.  If food was withheld  indefinitely, however, they became detached once more. Likewise, when the food organisms were depleted or when environmental conditions were not optimum the amoebas became detached once more. P. carolinensis i s a voracious feeder.  Almost immediately  following attachment to the substratum i t began ingesting food organisms. Frequently an individual amoeba was seen to contain several Parameciums at one time.  Each Paramecium  was contained i n a separate food vacuole (Figure 3) usually, but two Parameciums i n one food vacuole have been observed. When feeding on smaller protozoa (Chilomonas, Menoidium, Chilodon and Colpitium) one food vacuole contained many  PLATE II  Figure k - Photomicrograph illustrates the apparent attraction of Paramecium to the amoeba. This phenomenon seems to be a significant factor in feeding of P, carolinensis. X 95  Figure 5 - Parameciums have just been added to the watch glasses containing fresh stream water, a stick of boiled timothy hay and P. carolinensis. The characteristic exploratory behaviour of the Parameciums i s evident. X kO  - 36 -  organisms but the vacuole remained approximately the same size as those which were formed about one Paramecium. Thus, there appears to be an average size that a food vacuole attains. Although the amoebas move by means of pseudopodia, this movement i s too slow to be a significant factor i n the pursuit of food. Rather, i t was observed that the Parameciums are attracted to the amoebas (Figure U). Parameciums, when f i r s t put into a watch glass containing P. carolinensis, swam around exploring various bits of organic material i n the culture.  In this respect their  behavior was similar whether they encountered bits of timothy hay or an amoeba (Figure 5). A l l Parameciums coming i n contact with a groping pseudopod were not ingested. Whether or not ingestion took place depended on a co-ordination of events.  The Paramecium  usually crawled under a part of the amoeba and remained sufficiently long for the amoeba to move i t s pseudopodia about in a position such that cytoplasm flowed over top of the Paramecium completing the vacuole (Figure 6). The Paramecium usually did not move away u n t i l the bottom of the vacuole began f i l l i n g i n . Once the vacuole was formed the previously almost stationary Paramecium began frantic movements  Figure 6 - Newly formed food vacuole. Photomicrograph of living material was taken soon after pseudopodia had surrounded the Paramecium, the cytoplasm had flowed over the top, and the "floor * of the food vacuole was forming. The Paramecium was stationary at this time as indicated by the clarity of c i l i a and the fact that i t was not yet swimming i n a curled position as seen i n Figure 3 (i.e. a later stage of food vacuole). X 200 1  - 37 -  which continued u n t i l i t was swimming continuously round and round i n the vacuole i n a curled position corresponding to the round shape of the food vacuole. An ingested Paramecium circled continuously within the new food vacuole for approximately 1$ minutes before slowing down.  Eventually i t became less motile and  by 2 to 6 hours was usually i n pieces.  Upon ingestion of  a Paramecium, small dark granules i n the cytoplasm were seen streaming towards the point of ingestion.  On formation  of the new food vacuole these granules "lined" i t s periphery. As digestion of the Paramecium progressed the granules diminished i n size and by approximately 3 hours could not be detected.  Body Form Relation to Physiological State Degenerating amoebas undergo alterations of body form from f l a t , attached, with surface wrinkles, indicating health and normal function (Figure 2), to a perfect sphere which precedes "death". Of a less permanent nature i s the change i n form when i t i s irritated.  When disturbed i t tends to  round up. Club-shaped organisms were often seen i n older  Figure 7 - Photomicrograph of "star-shaped" l i v i n g P". c a r o l i n e n s i s . Individuals of t h i s shape were numerous when food was scarce or absent. X u5  Figure 8 - Living P. carolinensis which has ingested food and i s beginning the process of digestion assumes a more compact form. X 90  - 38 -  cultures where food was becoming scarce. Star-shaped amoebas (Figure 7) were numerous when l i t t l e food remained (i.e. 10 to 20 hours after addition of Parameciums) as well as immediately upon transferring organisms from old, barren cultures into fresh cool stream water before any food was given (and before attachment to the substratum). Amoebas which had ingested food and were beginning the process of digestion assumed a compact form, more or less irregular i n outline but tending towards a spherical shape with many short pseudopodia firmly attached to the substratum (Figure 3 and Figure 8). The type of food organisms influenced the shape of P. carolinensis.  When feeding on very small protozoa  i t was more regular i n shape with more rounded contours than when feeding on larger organisms such as Parameciums. (Figure 9 ) . The accumulation of excess waste products seemed to cause death to the amoebas more rapidly than did lack of food.  I f food was added periodically without changing  or adding fresh water, the amoebas soon died i n spite of the presence of adequate food.  I f unhealthy,  detached amoebas, tending towards the smooth spherical shape (which signifies degeneration) were removed from  PLATE V  Figure 9 - (a) and (b) are photomicrographs of living amoebas to illustrate the compact regular form assumed when feeding on small protozoa. X 110  - 39 -  their environment and placed into fresh water they immediately spread out and assumed a healthy appearance. The pH of the media i n which degenerating individuals were numerous was nearer to alkaline (6.5) than was the pH (5.2) of culture media containing healthy organisms undergoing growth and reproduction. Amoebas put into fresh water without added food organisms gradually decreased i n size and became a great deal more transparent.  Within 8 to 9 weeks the  membrane disintegrated and the small amount of cytoplasm remaining was released.  In contrast to this, when an  amoeba i s exposed to injurious chemicals or other toxic conditions, the membrane breaks down rapidly and the contents are released explosively. Correlation of Body Form with Mitotic Events There was another unattached, roughly spherical form, with numerous wart-like pseudopodia which was not due to unfavourable environment.  This morphological change  occurred, as a rule, i n amoebas which had been feeding for some time under healthy environmental conditions.  Visible  cytoplasmic streaming stopped, or decreased, as did ingestion'. But, often one s t i l l observed Parameciums within food vacuoles of these amoebas. Amoebas possessing the shape shown i n Figure 10 contained early prophase nuclei.  This stage was d i f f i c u l t to detect  Nuclear Stage  X 1250 (a) E a r l y Prophase  (b) Late Prophase  X 500  Figure 10 - Amoebas having the shapes shown diagraraatically on the l e f t contained prophase n u c l e i . Nuclear stages are shown i n photomicrographs of h i s t o l o g i c a l preparations on the r i g h t .  PLATE VII Body Form  Nuclear Stage  (b) E a r l y metaphase. X 800  (c) E a r l y metaphase, Later than (b).  X  950  (d) More advanced metaphase. X95>0  Figure 11 - Amoebas having the shapes shown diagramatically on the l e f t contained metaphase n u c l e i . Nuclear stages are shown i n photomicrographs of h i s t o l o g i c a l preparations on the r i g h t .  Nuclear Stage  Body Form  X 1200 (c) Late Anaphase Figure 12 - (a) The most symmetrically spherical and compact form ("berry form") contains early to mid-anaphase nuclei. Very l i t t l e activity was observed i n the pseudopodia during this stage, (b) and (c) Pseudopodia began to change shape and enlarge during late anaphase.  PLATE IX  Body Form  Nuclear Stage  Figure 13 - Telophase. The plates are progressing towards interphase n u c l e i . Body shape resembles l a t e anaphase. X 1200  Figure l U - Interphase. Constriction of the body with cytoplasmic bridges indicates interphase nuclear stage has been reached. Plasmotomy usually follows the above sequence of events but occasionally the constrictions disappear and the amoeba s e t t l e s down to feed once more. X 800  - Uo -  with certainty since the shape differed l i t t l e from elongated forms i n which interphase nuclei occur.  The  body was irregular i n outline and the amoeba was f a i r l y spread out. Pseudopodia were rounded and rather shortj their size and shape changed slowly.  Cytoplasmic stream-  ing was slow and directed towards the centre of the body. Body form of an amoeba containing late prophase nuclei i s shown i n Figure 10b.  The organism continued to  contract and exhibited many short rounded pseudopodia until i t became compactly rounded with short, round pseudopodia extending i n a l l directions. Figure 11 illustrates the shape assumed when the nucleus was undergoing metaphase. The body was contracted s t i l l further and very l i t t l e change could be detected i n the numerous, short, round pseudopodia. The most symmetrically spherical and compact form was exhibited during early anaphase as seen i n Figure 12a. It had the appearance of a blackberry. During late anaphase (Figure 12b and c) pseudopodia began to change shape and enlarge.  Increased cytoplasmic  activity was noted. Telophase resembled late anaphase (Figure 13).  The body  form i n which young interphase nuclei occurred was similar to  2  3  U  5  6  Days  Figure 15 - Within 2)i hours, approximately one-quarter of the amoebas underwent plasmotomy into two or three individuals. By the second day, another one-quarter divided, followed by another one-fifth or one-sixth by the third day. After the third day, unless fresh food was added, very few amoebas divided and the remaining individuals assumed an unhealthy appearance.  - la -  late anaphase and telophase (Figure ll;).  In addition,  constriction of the body became evident and cytoplasm could be seen streaming back and forth through one or more bridges. Plasmotomy into 2 to 6 daughters usuallyfollowed the above observed sequence of events. However, this was not always the case. At times such an individual would settle down and begin feeding once more, then some time later the above events would be repeated before plasmotomy finally occurred.  Conditions which favour Reproduction and Frequency of Mitosis The nuclear details of dividing amoebas were best observed and recorded by making permanent histological preparations.  However, studies of living organisms lead  to observations relating to frequency of nuclear division and conditions favouring mitosis and plasmotomy. No periodicity i n division was found.  Regardless of  the time of day, i f £0 to 100 Parameciums per amoeba (and fresh water) were added to the culture, the animals went about feeding for approximately h to 6 hours, after which time pre-plasmotic individuals (undergoing nuclear division) appeared i n abundance. Figure 15 represents the general trend i n reproduction of P. carolinensis and i s the result of 33 months of observation. Within 2li hours, approximately one-quarter of the amoebas underwent plasmotomy into 2 or 3  - 1*2 -  individuals.  Plasmotomy into k to 6 daughters was  on rare occasions.  observed  By the second day another quarter  divided, followed by a further one-fifth or one-sixth by the third day.  Within three days, therefore, there was  generally a two to three-fold increase i n organisms and often the yield was greater.  After the third day, unless  fresh food was added, very few amoebas divided and the remaining individuals assumed an unhealthy appearance. Division appeared to be related to size of the amoeba in that the interphase period was longer i n smaller animals. Body size of parent was not an apparent factor i n determining the number of daughters into which i t would divide.  Duration of Various Stages of Nuclear Division These times could not be determined for undisturbed organisms since i t was impossible to place the culture dishes under sufficiently high power to observe the nuclei. Therefore, the data appearing i n Table 1 applies to organisms which, because of their characteristic shape were believed to be undergoing mitosis.  They were removed from  the culture glass to microscopic slides for observation as described i n Materials and Methods (Page 21)  - U3 -  Table No. 1  AVERAGE DURATION OF MITOTIC STAGES  Mitotic Stage of Nucleus Early Prophase  (Figure 10a)  Late prophase to metaphase  (Figure 10b-ll)  Metaphase  (Figure 11)  Anaphase  (Figure 12)  Late anaphase through (Figure 12c, telophase to through 13 interphase to lU)  Average Time Taken k$ minutes 8 minutes A few seconds 5 minutes  30 to U5 min.  Plasmotomy was usually complete within 15 to 25 minutes after the young interphase nuclei appeared. When plasmotomy resulted i n more than two amoebas, the event required slightly longer. The distribution of nuclei amongst the daughters was random, but the variation i n size of newly divided amoebas was not great.  II.  HISTOLOGICAL STUDIES Vital Staining When placed i n a concentration of Neutral Red of 1:1,000 a large vacuole began to form at one end of the amoeba. This  - hh was later pinched off.  The process was repeated several  times during the time the organism remained i n the staining solution.  Eventually the entire amoeba ruptured and  disintegrated.  4s a rule, when animals were transferred  from the stain to fresh water during the vacuole-forming stages existing vacuoles were pinched off and the amoeba appeared normal once more, with respect to form and function. In concentrations of 1:100,000 no immediate toxic effects were observed.  The material within food vacuoles stained  bright red after 1$ to 20 minutes whilst the fluid was coloured pale reddish orange (pH 5 - 6 ) .  There also  appeared, vacuoles of various sizes (2 - 10 u i n diameter) which contained small bright red stained inclusions. vacuolar fluid was not stained.  The  The number of granules  contained within each vacuole varied and d i d not appear to be correlated with the size of the vacuole. showed very definite Brownian movement.  The granules After 3 or k days  i n the staining solution, large vacuoles containing these granules almost completely f i l l e d the amoebas, resulting i n the occlusion of a l l other cytoplasmic inclusions. Finally the amoebas began to degenerate. In material stained with Janus Green B, large spherical structures believed to be contractile vacuoles covered with small, oval-shaped and round particles were observed.  These  Figure 16 - Schaudinn's Fixation. Note the thin, delicate outer limiting membrane of the amoeba compared with the thick, dense, ciliated pellicle of the ingested Paramecium. X UOO  Figure 17 - Schaudinn's Fixation. Appearance of ground substance i s somewhat "stringier" than Osmium Tetroxide-fixed and frozen material (Figures 19 and 21) but resembles the latter two methods of fixation more closely than does Bouin's.  - kS are further discussed and illustrated below. Permanent Histological Preparations Material fixed with Schaudinn's, TCA and Osmium Tetroxide was superior to that fixed with Bouin's. Using frozen material as a criterion, Osmium Tetroxide caused the least post-fixation changes i n the cytoplasm. Heidenhain's Iron Haematoxalin staining gave a somewhat clear picture of nuclear detail than did Grenacher's Borax Carmine, but the latter was satisfactory for making quick preparations for nuclear counts or other studies where detailed observation was not necessary. For such purposes Schaudinn's fixation was used since i t eliminated the extreme caution necessary when working with the highly toxic Osmium Tetroxide. Figures 16 to 38 illustrate some morphological details of P. carolinensis observed i n the histological material. Morphology Plasmalemma Studies with the light microscope revealed l i t t l e regarding the structure of the outer limiting membrane. It i s apparent from Figures 16 and 18b however, that i t i s a relatively thin, simple membrane compared with the thick, dense, double, ciliated pellicle of the entrapped Paramecium.  PLATE XI  (a)  X UOO  (b)  X  1000  Figure 18 - Bouin's F i x a t i o n , (a) Cytoplasm appears more dense and fine-grained than i s the case with other f i x a t i v e s t r i e d . Magnificat i o n i s the same as Figures 16, 17, 19, 20 and 21. (b) Higher magnification. Note the plasmalemma and clear area between the l a t t e r and main body o f the cytoplasm.  Figure 19 - Osmium loose appearance. ground substance. is striking.  tetroxide f i x a t i o n . Ground substance has a Small granules may be seen adhering to the The s i m i l a r i t y t o frozen material (Figure 21) X UOO  Figure 20 - Trichloroacetic acid f i x a t i o n . X liOO  Figure 21 - Frozen preparation c l o s e l y resembles l i v i n g material. X 1;00  - U6 Photomicrographs of l i v i n g material (Figures 3, U, 6, 8) also illustrate this comparison.  The clear area between  plasmalemma and main body of cytoplasm i s illustrated especially i n the advancing end of pseudopodia (Figures 3, U, 6, 7, 8). Cytoplasmic Ground Substance There were variations i n the appearance of the ground substance depending upon the fixative employed.  For example,  Bouin's Fluid (Figure 18a) gave a more dense, fine-grained picture than Schaudinn's (Figures 16 and 17), TCA (Figure 20) or Osmium tetroxide (Figure 19).  The latter three resulted  i n a looser appearance with small granules adhering to the ground substance similar to frozen material (Figure 2L). It i s interesting to compare the fixed material described above with the living cytoplasm (Figure 6). Cytoplasmic Inclusions Mitochondria, vacuole refractive bodies, fat droplets, glycogen (carbohydrate), crystals, contractile vacuoles, food vacuoles and nuclei were observed. Mitochondria - Rod and oval-shaped dark particles observed i n living animals under high dry (Xlr5C-) and o i l immersion (X9l*0) were thought to be mitochondria.  Other cytoplasmic  inclusions which were approximately the same size, resembled these particles.  On more careful observation, however, a  PLATE XIII  F i g u r e 22 - C h a r a c t e r i s t i c accumulat i o n o f m i t o c h o n d r i a around c o n t r a c t i l e vacuoles. Regaud's Method f o r mitochondria. X 850  F i g u r e 23 - H i g h e r m a g n i f i c a t i o n o f f i g u r e 22. X 1825  4  F i g u r e 2U - F a t d r o p l e t s i n P. c a r o l i n e n s i s a r e v e r y numerous as seen i n t h i s Sudan B l a c k F o r m o l Calcium preparation. X 1|00  - I n -  difference i n the refractability between the two types was detected.  The latter were highly refractile and appeared  bluish-green by transmitted l i g h t , whereas those believed to be mitochondria had a refractive index similar to that of the cytoplasmic ground substance and appeared d u l l .  Despite  this distinction, i t was deemed necessary to employ histological methods specific for mitochondria and thus confirm that the particles were indeed mitochondria. Furthermore, permanent preparations were desired for record purposes. Although many of the mitochondria were rod-shaped i n living amoebas, they appeared round (approximately 1.5 to 2.5 u i n diameter) i n fixed material. With Regaud's method, the particles appeared dark grey or black within the lighter grey cytoplasm.  Although distributed f a i r l y evenly throughout the  c e l l , their characteristic accumulation i n a single layer around the contractile vacuoles was quite apparent (Figures 22 and 23). A similar picture to that described above resulted with Heidenhain's Iron Haematoxylin when Champy fixation was used. With osmium chromium fixation, however, the mitochondria stained intensely black. Although this blackening was desirable for observing histological detail, i t resulted, i n this case, i n difficulty i n distinguishing the mitochondria from other inclusions of similar size and shape. Fat Droplets - These inclusions were clearly demonstrated  PLATE XI?  (b) X1200  (c) X1200  Figure 2$ - Small, dark granules similar to those described i n living PT carolinensis are seen forming a narrow rim around the periphery of food vacuoles, (a) and (b) were fixed i n Schaudinn's (c) was fixed in Bouin's. Staining i n a l l three figures was with Grenacher's Borax Carmine.  - U8 -  by the Sudan Black formol-calcium technique (Figure 2k), Glycogen (carbohydrates) - Since existing histological procedures for detection of "glycogen" are not specific, any carbohydrate material i n the food vacuoles as well as i n the cytoplasm took the stain when Best's Carmine and Periodic-acid Schiff was used.  As might be suspected, less carbohydrate was  present i n starving individuals. Contractile Vacuoles - These could be readily identified i n the histological preparations because of the surrounding layer of mitochondria, but l i t t l e else regarding structure or function could be determined. Food Vacuoles - Small, dark granules, similar to those described i n living amoebas, were also seen forming a narrow rim around the periphery of young food vacuoles i n fixed material (Figure 2 5 ) . Around the older food vacuoles, small, empty vesicles were seen.  In starved individuals there remained only a  few older vacuoles f i l l e d with solid-appearing particles (Figure 1+U).  The appearance of food vacuoles i n both normal  and starved amoebas i s discussed later under Mercury Orange studies (page 5 6 ) .  The membrane of the food vacuoles appeared  identical to those surrounding the amoeba i t s e l f as well as those surrounding small or large cytoplasmic vesicles (Figures 16a and Crystals -  18b). These particles, seen both i n l i v i n g (Figure 26)  PLATE XV  Figure 26 - Crystals seen i n l i v i n g Pelomyxa carolinensis. X" 200  Figure 27 - Crystals seen i n Schaudinn's-fixed material. Size and shape of these c r y s t a l s i s remarkably uniform. X 1U00  PLATE XVI  Figure 28 - Large peripheral granules are characteristic of the interphase nucleus. A few granules are also seen distributed throughout the nucleoplasm. Granules stained intensely with Heidenhain's Iron Haematoxylin and with Mercury Orange. X 1^00  Figure 29 - Nuclear d i v i s i o n i n P. carolinensis i s m i t o t i c . With few exceptions, n u c l e i undergoing mitosis do so synchronously. This amazing uniformity of timing i s well i l l u s t r a t e d i n anaphase where plates are the same distance apart. X U00  - U9 -  and fixed materials (Figure 27) were better appreciated when observed by phase contrast which showed their crystalline shape, colour and texture very well. Nuclei - The results of nuclear counts made on 5>0 amoebas showed that the nuclei contained i n any one individual may vary between 1$ to lf>0.  The vesicular-shaped nucleus  of P. carolinensis was approximately 2k x 10 p. i n diameter. The interphase nucleus was characterized by large granules within i t closely applied to the nuclear membrane.  In  cross-section, these granules appeared as a ring around the periphery (Figure 28).  A. few of these granules were also  distributed throughout the nucleoplasm.  Reidenhain's  Iron Haematoxalin stained the peripheral granules intensely black whilst the chromatin appeared less intense (Figure 30). The same was true of Mercury Orange Staining.  This i s  discussed later (page $9 ) and illustrated i n Table 3 and Figure 39. Nuclear Division Nuclear division i n P. carolinensis was mitotic. Except for minor variations on rare occasions, a l l of the nuclei underwent the same stage of mitosis synchronously.  This  amazing uniformity of timing i s well illustrated i n anaphase (Figure 29) where the plates are the same distance apart. In figure 30 are seen 3 nuclei which were slightly more advanced  PLATE XVII  Figure 30 - Three n u c l e i i n this photomicrograph are i n l a t e prophase whilst the others are s t i l l i n interphase. This phenomenon was very r a r e l y observed. Note the well-staining peripheral granules i n most n u c l e i . Heidenhain's Iron Haematoxylin s t a i n . X lj.00  Figure 31 - Early prophase. The beginning of prophase i s characterized by migration of the peripheral granules towards the c e n t r a l area. Schaudinn's f i x a t i o n and Grenacher's Borax Carmine staining. X 12^0  f i g u r e 32 - L a t e prophase. Some p e r i p h e r a l g r a n u l e s remain c l o s e t o t h e n u c l e a r membrane. Schaudinn's f i x a t i o n and Grenacher's Borax Carmine s t a i n i n g . X 1500  F i g u r e 33 - E a r l y metaphase. P e r i p h e r a l g r a n u l e s become s m a l l e r w h i l s t s p i n d l e f i b r e s i n c l o s e c o n n e c t i o n w i t h them become more c o n s p i c u o u s . N u c l e a r membrane i s s t i l l p r e s e n t . Schaudinn's F i x a t i o n and Grenacher's Borax Carmine. X 95>0  PLATE XIX  K . 1  1  Figure 3U - Metaphase has progressed further than Figure 33. Peripheral granules and nuclear membrane are not seen. Schaudinn's f i x a t i o n and Grenacher's Borax Carmine Staining.  X 950  Figure 35 - As metaphase progresses and the nuclear membrane disappears the shape of the mitotic figure changes from ovoid towards rectangular. Schaudinn's f i x a t i o n and Grenacher's Borax Carmine s t a i n i n g . X 950  Figure 36 - Late metaphase. Note rectangular shape of mitotic figure, complete lack of peripheral granules and nuclear membrane, Schaudinn's f i x a t i o n and Grenacher's Borax Carmine staining. X 1750  - 5o -  than the rest. They had progressed to prophase (Figure 31) slightly before the others. Enlargement of the nucleus prior to mitosis, as described i n the literature, was not observed. The beginning of prophase was characterized by migration of the large achromatic peripheral granules towards the central area i n which chromatin granules and filaments are distributed (Figure 31).  Some peripheral granules,  however, remained close to the nuclear membrane, until late prophase (Figure 32).  As prophase progressed towards early  metaphase the achromatin granules became smaller and less distinctly outlined whilst spindle fibres i n close connection with them became more and more conspicuous (Figure 33).  The  fibres were mainly oriented at right angles to the discoid mass of chromatin. At this stage the fibres usually did not reach the nuclear membrane but extended as far as the diminishing peripheral granules. The nuclear membrane, although thinner, was visible well on into late prophase and very early metaphase (Figures 32 and 33) but disappeared during metaphase. Coincident with the disappearance of the nuclear membrane, the shape of the metaphase figure changed from ovoid to rectangular.  This transi-  tion i s depicted i n Figures 3k, 35 > and 36. The complete lack of peripheral granules was observed i n the rectangular figure (Figure 36).  PLATE XX  (a)  (b)  X UOO  X 1000  Figure 37 - Anaphase, (a) Anaphase discs pull apart i n many directions at once, (b) Polar fibres end bluntly during early anaphase and chromatin plates are arranged parallel to each other.  PLATE XXI  (a)  X 1200  (b)  X 1200  Figure 38 - Late anaphase to telophase, (a) and (b) As the plates pull further apart they appear arched and the polar fibres converge towards a point. As telophase i s reached the interphase fibres begin breaking up into granules. Newly formed nuclei are triangular-shaped at f i r s t , then oval-shaped, and finally discoid-shaped.  - 51 -  It was interesting to note that the anaphase discs pulled apart i n many directions at once (Figure 37a). Although individual chromosomes were too small to be observed singly, much less detail was seen i n the chromatin plate during anaphase than at any of the previous stages. Compare metaphase (Figures 33 to 36) with anaphase (Figures 12b, 29, 37 and 38). and stains tried.  This was true of a l l fixatives  In the anaphase photo-micrographs  the chromosomes often appear as a fused dense mass.  As  the 2 anaphase plates pulled apart, spindle fibres appeared between them and the previously existing (outer) spindle fibres became the so-called polar fibres.  These polar  fibres ended bluntly during early anaphase (Figures 12a and 37b) but from late anaphase through telophase the fibres converged towards a point (Figures 12b, 13 and  38).  During early anaphase the chromatin plates were arranged parallel to each other (Figures 12a and 37b) but as the plates pulled further apart they became arched (Figures 13 and 38a).  As telophase was reached the inter-plate fibres  began breaking up into granules.  After separating, each  plate became surrounded by a delicate membrane. These newly-formed nuclei were irregular i n outline (Figures 13, and 38b) triangular-shaped at f i r s t , then oval-shaped;  Ik  and  finally they assumed the discoid shape of the interphase nucleus.  - 52 As the young nuclei were growing larger the chromatin granules and filaments became less conspicuous whilst the peripheral achromatic granules grew larger and more numerous until the characteristic appearance of the mature interphase nucleus was reached.  III. CYTOCHEMICAL STUDIES Preliminary Experiments with Mercury Orange on Toad and Mouse Muscle. Untreated muscle tissues stained reddish-orange whilst arsenic-treated fibres remained colourless when subjected to the sulfhydryl reagent. Effects of Sodium Arsenite on Living Amoebas Results of experimental work which was repeated every day for one week appear i n Table 2 (a). Table No. 2 (a) RESULTS OF TREATING 12 LIVING AMOEBAS WITH NaAsOo FOLLOWED BY MERCURY ORANGE STAINING Conc.NaA.sO3 Time (gms$) Exposed 0.01  Motility  12:00 a.m. Decreased at to 3:00pun. end of 3 hrs.  Shape  Results with M.O. staining (2lx hrs.)  Normal  Same as untreated (++++)  -  53 -  Table No. 2 (a) (cont'd) RESULTS OF TREATING 12 LIVING AMOEBAS WITH NaAsO, FOLLOWED BY MERCURY ORANGE STAINING J  Conc.NaAsO^ (gms%) 0.5  Time Exposed  Motility  Shape  Results with M.O. staining (21; hrs.)  12:00 a.m. Immediate increase i n cytoplasmic streaming. Pseudopodia began to elongate and assumed bizzare shape. 12:20 p.m. Animal darker i n centre as cytoplasm streamed i n this direction (similar to mechanical irritation). Tending towards a spherical shape. 1:00 p.m. A l l the amoebas were well attached to substratum.  Normal  Same as untreated (++++)  - 5k -  Table No. 2 (a) (cont'd) RESULTS OF TREATING 12 LIVING AMOEBAS WITH NaAsC^ FOLLOWED BY MERCURY ORANGE STAINING Cone.NaAsO^ (gms$) 1.0  Time Exposed  Motility  Results with M.O. staining (2k hrs.)  Shape  12:00 a.m. Immediately became immobile. 12:10 p.m. Cytoplasm streaming began. The few pseudopodia assumed bizzare shapes. yellowish / vacuole /  12:35 P.m.  wide clear area Yellowish vacuoles formed throughout the cytoplasm. Broad, clear area between cytoplasmic inclusions and plasmalemma.  1:05 p.m.  Disintegration of outer limiting membrane and contents of animal flowed out.  Could not be stained.  PLATE XXII  (a) X 1*00  (b) X 800  Figure 39 - Normal, well-fed P. carolinensis stained with to demonstrate -SH d i s t r i b u t i o n .  M.O.  Figure 1*0 - Small, round food vacuole packed with c r y s t a l s . TCA f i x a t i o n and Mercury Orange Staining. X ll*00  - 55 -  c  Application of the Mercury Orange Method to P. carolinensis Untreated amoebas were coloured orange ( U ) ; those treated +  with 1 gn$ concentrations of arsenic (2U hours) were faintly tinted (+); those immersed i n 5 gm$ solutions (2lx hours) showed only a trace of colour; whilst those subjected to 10 gm$ arsenic (2U hours) remained completely colourless (Table 2 (b) ).  Table Mo. 2 (b) RESULTS .OF MERCURY ORANGE STAINING OF P. carolinensis TREATED WITH SODIUM ARSENITE (NaAsOo) PRIOR TO TRICHLOROACETIC ACID FIXATION Cone, of NaAsO^  Time Exposed  Color Intensity (M.O. Stain)  Non-treated  0 hours  1 gm#  2k hours  .+  $ gn$  2k hours  +  10 grt$  2k hours  0  ++++  Using the intensity of colour produced as a criterion, sulfhydryl groups were found uniformly distributed throughout the entire animal but their concentration was greater i n some areas.  These results (for normal, well-fed amoebas) appear i n  Figure 39 and Table 3.  PLATE XXIII  Figure hi - In this preparation the food vacuoles containing crystals are so close to the surface of the amoeba as to lead one to believe they are being extruded. Trichloroacetic acid fixation and Mercury Orange staining. X lUOO  v  .  -  Figure 1*2 - Crystals seen i n a larger food vacule appear to be limited to the Paramecium whose outline can s t i l l be seen within a food vacuole. Majority of crystals are rod-shaped. T.C.A. fixation and M.O. staining. X luOO  Figure U3 - Crystals scattered about in the cytoplasm are smaller and tend to have a square shape. T.C.A. fixation and M.O. stain. X 1U00  PLATE XXIV  Figure UU - Starved Pelomyxa carolinensis stained for sulfhydryl groups by Bennett's Mercury Orange method. Much s t r u c t u r a l d e t a i l i s seen but ground substance i s more intensely stained compared with normal well-fed individuals i l l u s t r a t e d i n Figure 3 9 . X U00  - 56 -  Table No. 3 MERCURY ORANGE STAINING OF Pelomyxa carolinensis Colour Intensity  Plasmalemma (appears as a very narrow line inside of which i s a clear area, with the cytoplasm innermost.) Ground substance  Normal, Well-fed (Figure 39)  Starving (Figure kh)  +++  ++  ++  ++++  0  Cytoplasmic crystals Interior Rims  ++++  0 ++++  Interphase Nuclei Nuclear membrane Peripheral granules Chromatin Nucleoplasm  +++ ++++ +++ ++  +++ ++++ +++ +++  Contractile vacuoles Interior Rims  + ++++  + ++  Food Vacuoles Food inclusions Crystals* - Interior - Rims - Fluid  ++++ 0 ++++ 0  Only a few small vacuoles f i l l e d with crystals remained.  •fcSome smaller food vacuoles, perfectly round and only slightly larger than the nuclei were l i t e r a l l y "stuffed" with  - 57 -  these rectangular, square, and round-shaped crystals (Figure 1*0). These smaller food vacuoles were similar to the larger ones i n every respect except for the numerous crystals and small, round size of the latter.  Similar crystals appeared within the larger,  variously shaped food vacuoles but i n fewer numbers (Figures 1*1 and 1*2). The crystals described above appeared considerably larger than those scattered about i n the cytoplasm (Figure 1*3). These crystals appeared to be inside the food vacuole and not merely adhering to the outside for they were never seen on the borderline of the vacuole nor were they apparent on nuclei (Figures 39, 1*0 and 1*2). In some preparations these vacuoles stuffed with crystals were so close to the surface of the amoeba as to lead one to believe they were being extruded (Figures 1*0 and 1*1). Although these crystals were distributed as described above i n material stained by other methods, they were never so clearly visible as i n Mercury Orange stained individuals.  (Compare  Figure 39, TCA fixation and Mercury Orange stain with Figure 19, Osmium Tetroxide fixation and Grenadier s Borax Carmine Staining.) 1  Results for starved amoebas appear i n Figure 1*1* and Table 3, on page 56. Much less structural detail was observed but the ground substance was more densely stained and granular i n starved organisms.  Food vacuoles were rarely seen and those present were  exceedingly small. There were fewer contractile vacuoles, their  - 58 -  rims were paler and they contained crystals only.  Nuclei showed  up more distinctly i n the starved preparations, but peripheral granules were considerably smaller. Mitotic Nuclei Stained with Mercury Orange The results of Mercury Orange staining of amoebas undergoing mitosis appear i n Table k below.  Table No. h MERCURY ORANGE STAINING (21; hours) OF DIVIDING NUCLEI i n P. carolinensis  Colour Intensity  Mitotic Stage  Nuclear Peripheral NucleoSpindle plasm Chromatin Fibres Membrane Granules Interphase (Figure h$)  +++  ++++  ++  Prophase (Figure U6)  ++  ++  ++  Early + Late -  Early + 0*  Metaphase (Figure U7) Anaphase (Figure J48)  Early + Late  -  -  0*  +++  0  0**  +++(+)  ++++  ++  Polar: ( ) Interdisc: ++ + + +  * No membrane, therefore, nucleoplasm appears to blend with the rest of the cytoplasm. •SB* No spindles yet formed.  +  Figure k$ - Interphase nucleus stained with Mercury Orange. Intensely s t a i n ed peripheral granules indicates abundant -SH groups i n these structures. X1500  Figure U6 - Prophase nucleus stained with Mercury Orange. Most of the peripheral granules have migrated to the central portion. Prophase n u c l e i were d i f f i c u l t to detect i n these preparations since there was not much contrast between the cytoplasm and the n u c l e i . X225>00  Figure kl - Metaphase n u c l e i stained with Mercury Orange. Were i t not for the chromosomes, these n u c l e i would be d i f f i c u l t to detect. X1000  Figure U8 - Anaphase n u c l e i stained with Mercury Orange. Polar f i b r e s and chromosomes are well seen. Interplate fibres are f a i n t l y stained approximately the same i n t e n s i t y as metaphase spindle f i b r e s . XluOO  - 59 -  Interphase nuclei were easily detectable because they were outlined by intensely stained peripheral granules (Figures 39 and 1*5).  Once the peripheral granules had migrated to the  centre of the nucleus they did not stain as intensely with Mercury Orange. No granules could be detected by late metaphase and anaphase (Figure 1*8).  Staining of the nuclear membrane was  similar to that of the peripheral granules.  (Note that by late  metaphase and anaphase no nuclear membrane i s visible (Figure 1*8)). Mercury Orange staining of the nucleoplasm of interphase and prophase nuclei was of approximately the same intensity (Figures 1*5 and 1*6).  In stages where the nuclear membrane was absent, the  nucleoplasm could not be detected with certainty.  Chromatin  staining was approximately the same i n interphase and prophase nuclei (Figures 1*5 and 1*6).  As chromosomes became visible, stain-  ing was more intense, with the chromosomes appearing darker i n anaphase (Figure 1*8) than i n metaphase (Figure 1*7).  Spindle  fibres were never observed i n .interphase or prophase (Figures 1*5 and 1*6).  Metaphase and interdisc spindle fibres were faintly  stained whilst polar fibres i n anaphase were deeply stained (Figures 1*7 and 1*8).  0  - 60 -  DISCUSSION  I.  LIVING MATERIAL General Behaviour Feeding The explanation of why Parameciums (when added to a watch glass containing fresh culture media and P. carolinensis) swim towards the latter, was not revealed by this study, nor have I found any reference to this phenomenon i n the literature.  Whether i t i s due to a "stickiness", or  to ionic phenomena of the amoeba's outer membrane, or to the fact that Parameciums feed on bacteria i n the vicinity of the amoeba i s not known. Kudo (1959) observed Parameciums feeding on bacteria around amoebas, and Roslansky (1959) obtained electron photomicrographs of ingested bacteria i n Paramecium food vacuoles.  (The rod-shaped particles which I observed  i n food vacuoles could be bacteria since they resemble the latter i n size and shape and show up extremely well with -SH Mercury Orange staining.  However, specific bacterial  staining was not carried out, hence I have no positive evidence to indicate that these are indeed bacteria.)  - 61 -  The explanation that Parameciums are feeding on bacteria does not account for the apparent struggle to extract themselves voluntarily.  However, the apparent struggle may  merely be a sluggishness due to abundant food i n the vicinity, since Parameciums also exhibit a sluggishness around bits of timothy hay. It i s known that many cells have an outer coating of mucoprotein which has an important function i n pinocytosis (Halter, 1959;  Robertson, I960).  Pinocytosis and  ingestion are considered similar phenomena, therefore, the amoeba might well have a "sticky" property which explains why Parameciums require some effort to extract themselves from the v i c i n i t y of the amoeba.  deRobertis et a l (1957)  states that the cytoplasmic matrix of the amoeba i s slightly acidic (pH approximately 6.8) but he makes no reference to the charges on the animal's surface.  Parameciums  have been observed to move toward the negative pole i f placed in an electrical f i e l d of direct current.  E l l i o t t (1952)  attributes this to the fact that externally the Paramecium i s positively charged but does not suggest the nature of this charge. Before the answer to the above observation can be found, i t w i l l no doubt be necessary to determine the  - 62 -  molecular structure of the c e l l membrane, generally, and of Paramecium multimicronucleatum and Pelomyxa carolinensis in particular.  Although several theories concerning the  c e l l membrane exist, Ponder ( l ° 6 l ) , i n an extensive treatise on the c e l l membrane, indicates that he i s not convinced about structure - or even the necessary existence - of the c e l l membrane as i t i s generally described.  He feels  that "many of the conclusions regarding the c e l l membrane are based on pre-existing ideas, on unallowable simplifications, as well as on a disregard of both physical chemistry and the results of experiments on the cells themselves." The observations regarding ingestion and formation of food vacuoles raised these questions: (1)  Why do Parameciums remain stationary while food vacuoles are formed about them; then commence very definite movement?  (2)  How does the amoeba sense the presence of the Paramecium and begin deliberate movements directed at surrounding the food with i t s pseudopodia? Regarding the f i r s t question, the reason for the  Paramecium remaining stationary while the food vacuole forms about i t i s not l i k e l y to be known until the explanation for the apparent attraction of Parameciums to amoebas i s found.  Regarding the second part of the f i r s t question, there i s ample evidence that a change i n pH occurs inside the food vacuole (Mast, 191*2).  This relatively sudden change i n  the environment could conceivably have a stimulatory effect on the Paramecium for i t i s known that protozoa are sensitive to pH changes i n the surrounding media. Parameciums tend to avoid an alkaline environment and to seek an acidic environment ,(Elliott, 1952). Mast (19U2) investigated the cause of death of living organisms after ingestion and concluded that i t was due to a decrease of oxygen i n the food vacuoles and not to the acidic environment.  He believes the oxygen decrease i s  due to respiration by the entrapped food organism and the diffusion of oxygen out into the cytoplasm as well as to decreased vacuolar fluid volume. Regarding the second question above, since ingestion (phagocytosis) i s a process very similar to pinocytosis, the essential features of which are similar i n a l l c e l l s , pseudopod formation can be considered analagous to the formation of a recess extending toward the inside of the c e l l by the sliding along of the membrane once a particle, molecule, or ion has been absorbed on the cell surface (Bennett, 1956).  However, the mechanisms underlying this  - 6h -  membrane s l i d i n g i s not well understood and u n t i l more knowledge i s acquired regarding the phenomena of pinocytosis and phagocytosis  t h i s question cannot be s a t i s f a c t o r i l y answered.  I t i s not yet known how the pinocytosed f l u i d or phagocytosed organisms become assimilated into the cytoplasm. Bennett, 1956, postulated that the vacuolar membrane disintegrates under the influence of cytoplasmic  enzymes  while others (Prescott, Chapman-Andresen and Holter) f e e l that the permeability properties of the vacuolar membrane are involved.  Regarding the d i s i n t e g r a t i o n of the membrane, i t has never a c t u a l l y been seen to disappear by the e l e c t r o n microscope (Holter,  1959 a, b; 196l).  Further evidence  against regarding membrane d i s i n t e g r a t i o n as the sole explanation of a s s i m i l a t i o n o f food into the cytoplasm i s found i n the work of Andresen and others  (1952) who  c a r r i e d out studies on the digestion o f P. carolinensis using Carbon"^ - l a b e l l e d food organisms.  They demonstrated  that the rapid spread of radio-active glucose throughout the cytoplasm was probably due to membrane permeability rather than to membrane d i s i n t e g r a t i o n because r a d i o - a c t i v i t y during subsequent feeding on unlabelled food revealed a period o f reduced a c t i v i t y but with persistence o f a c t i v i t y  - 65 -  as long as 700 to 800 hours.  Similar studies carried out  by Cohen recently (1959) on Amoeba proteus, but using c o l l o i d a l gold, confirmed Andresen's conclusions that food derivatives p e r s i s t i n vacuoles f o r some time after the o r i g i n a l feeding.  The plasmalemma of P. carolinensis i s  notoriously impermeable to many, especially high molecular substances.  Chapman-Andresen and Holter (1955) found the  c e l l membrane o f t h i s amoeba was almost completely impermeable to glucose.  Since the vacuolar membrane o r i g i n a l l y was  part of the c e l l surface and, according to Brandt (1958) and Marshall et a l (1959), preserves the morphological c h a r a c t e r i s t i c s of the plasmalemma, a permeability theory o f a s s i m i l a t i o n of phagocytosed  (or pinocytosed) material should  suggest a mechanism f o r a l t e r i n g the permeability o f the plasmalemma once i t has become the food vacuole w a l l . The small, dark granules which I noted streaming towards the point of ingestion (page 37) and l a t e r l i n i n g food vacuoles (pages 37 and 1*8) might conceivably be associated with assimilation of food vacuole contents.  I believe these  granules are i d e n t i c a l with those forming the compact l a y e r seen by Pappas (1959) and those studied by Mast and Doyle (1935 a, b ) .  My observations coincide with Horning's  (1933) who noted that the incorporated food i s brought intimate contact with some "chondriosomes".  into  He f u r t h e r  - 66 -  states that later a vacuole forms enclosing the food together with adjacent "chondriosomes".  However, since the  vacuolar membrane is i n fact what was plasmalemma prior to phagocytosis, a l l the granules would have to enter the vacuole at the same point through a narrow opening just before the two free ends join to form a vacuole.  I did not observe  this and I also found i t d i f f i c u l t to determine, from the photomicrographs  (Figure 25), whether these granules  are inside or on the outside of the membrane.  Horner  observed the diminishing size and eventual disappearance of the "chondriosomes" as digestion proceeds. observed to be so.  This, I also  deRobertis (1957) believes that the  events described above indicate a hydrolytic type of enzyme action i n connection with digestion.  The close relationship  of the "chondriosomes" to the food vacuoles, therefore, . would seem to support the permeability hypothesis of food assimilation.  Body Form Body form i s a reliable indicator of the physiological state of an amoeba at any given time. The spherical form seems to be the result of gradual withdrawal, i n a l l directions, from injury and therefore occurs when an unfavourable environment envelops the amoeba.  - 67 -  Club-shaped individuals, I believe, indicate that the organism i s concentrating i t s efforts i n one direction only in an attempt to reach food or more favourable environment existing some distance away.  This agrees with Kudo (195>1).  Formation of elongated pseudopodia i n a l l directions as seen i n star-shaped organisms where l i t t l e food remained probably indicates that the environment was not yet sufficiently unfavourable to cause withdrawal of pseudopodia and the anoebas were searching food i n a l l directions. Assumption of a star-shape when removed from old, barren cultures and placed i n fresh water might be explained by the fact that no inhibitory influence now prevented pseudopod formation (as when the sphere-shape occurs) hence the amoeba begin seeking food i n a l l directions. Regarding the more immediate cause of death of amoebas in old cultures, changes i n pH of the environment would be expected to have an immediate effect compared with starvation which i s a gradual process.  That i s , the organisms can  store food materials but when the internal milieu i s disrupted, degeneration occurs f a i r l y rapidly. The gradual fading away of "starving" amoebas appears to reflect a gradual lack of building materials for normal function and structure much as wasting i n multicellular  - 68 -  organisms possessing a negative nitrogen balance.  In contrast  to t h i s slowly acting (weeks) n u t r i t i o n a l e f f e c t , the d i s i n t e g r a t i o n of organisms exposed to d e l i t e r i o u s chemical agents follows rapid (minutes, hours) membrane disruption which can be observed as i t i s occurring.  Correlation of Body Form with M i t o t i c Events Body forms described i n Results (page 35)  indicated that  the n u c l e i were undergoing mitosis, a fact which was most useful i n studying -SH d i s t r i b u t i o n i n dividing amoebas since one could choose a desired stage of mitosis with great accuracy. Before d i v i d i n g , Amoeba proteus has also been observed to round up i n t o a spherical shape with short, blunt pseudopodia a l l over i t s surface (Schaeffer,  19U6).  Conditions which favour Reproduction and Frequency o f Mitosis The observations regarding p e r i o d i c i t y and frequency of m i t o s i s , as w e l l as a study of conditions favouring d i v i s i o n were of assistance i n obtaining material f o r l a t e r experimental work on -SH  groups.  The occurrence and frequency of nuclear d i v i s i o n and plasmotomy was correlated with food supply only.  Although  d i v i s i o n appeared to be r e l a t e d to size of the amoeba, t h i s can be interpreted as an aspect of adequate food.  Kudo (191*9)  - 69  -  believes that, as the number of nuclei in the parent increases, the number of resulting daughters also increases.  Perhaps this  explains why at times plasraotomy did not follow mitoses of the nuclei, and that often when plasmotomy was deferred until mitosis was repeated, the amoeba divided into more than 2 daughters. The fact that the variation i n size of newly divided amoebas was not great correlates with Prescott's (1959) findings concerning Amoeba proteus.  He weighed newly divided anoebas and  found that weight variations were insignificant except when division occurred i n strong light. The finding that distribution of nuclei amongst daughter amoebas i s random agrees with Kudo (19U9) who  carried out  careful statistical tests i n this connection and found that any equal distribution was not significant.  II.  HISTOLOGICAL STUDIES Vital Staining The formation and pinching off of vacuoles i s probably a means of eliminating toxic substances (in this case Neutral Red).  This is i n agreement with Kassel and Kopac (1953)  who exposed organisms to toxic materials.  It i s , therefore,  inferred that the " v i t a l " stain behaved as an injurious chemical material.  - 70 -  The red-stained vacuolar inclusions observed i n amoebas exposed to 1:100,000 concentrations of stain are believed analagous to the "neutral red bodies" described by Andresen (l9k2, 19U5 and 19k6) and others.  Regarding the  question as to whether these structures are normal cellular inclusions or whether they represent a response to the stain, I agree with Torch (1959) and others, who believe they are an artifact of v i t a l staining, since they were not seen i n any other material.  Torch (1959) offers a possible  explanation regarding the nature of these particles.  He  notes that identical structures are formed during staining with other v i t a l dyes (1959 paper refers to unpublished data).  Since these structures appear to be l i p i d i n  nature he believes they may represent a protective mechanism against the toxicity of the stain.  That i s , the amoeba  might u t i l i z e l i p i d to form an insoluble complex with the dye, thus removing the dye from the cytoplasm.  This could  explain why neutral red bodies occur onjy i n organisms exposed to dilute solutions, for higher concentrations k i l l the organisms before the bodies are formed. The bodies seen covering the contractile vacuoles i n organisms stained by Janus Green B are similar to those described by Mast and Doyle (1935, a, b) and Kassel and Kopac (1953).  I believe these particles are mitochondria  - 71 -  since Janus Green B i s a s t a i n for mitochondria.  Studies  c a r r i e d out by Torch (1955) and confirmed by recent electron microscope studies (Pappas, 1959) further support t h i s opinion.  Mitochondria i n P. carolinensis are further  discussed under permanent h i s t o l o g i c a l preparations.  Permanent H i s t o l o g i c a l Preparations That Osmium Tetroxide f i x a t i o n gave a picture closely resembling frozen material i s i n keeping with the findings of workers who have studied the action of various fixatives' on cultured c e l l s (Bang and Gey, 1959j Greider et a l , 1958 j  Palade, 1952j  Borysko and Roslansky, 1959).  f i x a t i v e s were evaluated by comparing the l i v i n g  The  cell  under phase contrast with the f i x e d c e l l under the electron microscope.  Best r e s u l t s were obtained with Osmium Tetroxide.  Morphology Plasmalemma Although the l i g h t microscope i s o f l i t t l e assistance i n revealing the structure o f the outer l i m i t i n g membrane, t h i s structure has been extensively studied by the electronmicroscope and i s described i n d e t a i l by the workers referred t o i n t h i s l i t e r a t u r e review.  Cytoplasmic Ground Substance Since material f i x e d i n Schaudinn's,  Osmium Tetroxide  - 72 -  and trichloroacetic acid closely resembles frozen preparations i t i s f e l t that the former three fixatives give a picture nearer the l i v i n g state than does Bouin's. Cytoplasmic Inclusions Mitochondria - Transformation of mitochondria from rod-shaped i n living organisms to round-shaped i n fixed material i s an occurrence sufficiently invariable to warrant Torch's (1955) regarding i t as a criterion of death of the amoeba.  Torch (1955) found that i n a l l cases Janus Green B  was concentrated i n eccentric granules or crescentic areas within the mitochondria.  This i s interesting i n view of  recent work on the chemistry of Janus Green B staining. Lazarow and Cooperstein (1953)J  Cooperstein, et a l (1953);  Cooperstein and Lazarow (1953) and Showacre (1953) have related colouration by Janus Green B to the presence of cytochrome oxidase within the mitochondria.  I f this i s so, the stained  granules or crescentic areas within the mitochondria of P . carolinensis possibly indicate localization of cytochrome oxidase. The characteristic accumulation of mitochondria around contractile vacuoles leads to speculations regarding the function of mitochondria i n P . carolinensis.  Most of the  knowledge concerning the function of mitochondria was derived from vertebrate studies.  Vertebrate mitochondria apparently  - 73 -  contain a l l the enzymes necessary for the respiration of the c e l l (Lindberg and Ernster, 195U).  Whilst the  biochemistry of protozoan mitochondria i s virtually unknown i t i s reasonable to assume that their enzymatic complement does not differ markedly from that of vertebrates. On the basis of morphological studies, Torch (1955) believes that mitochondria i n P. carolinensis possibly function i n one or more of the following processes: 1) accumulation and transport of fluids; 2) accumulation and transport of waste products; 3) ' digestion; k) respiration. No morphological evidence for the f i r s t tw> possibilities exists since there i s no difference between the granules surrounding contractile vacuoles and those freely circulating. Likewise, morphological evidence for a digestive function i s limited.  However, the meagre amount of work done concerning  function of protozoan mitochondria points to a probable function concerned with cellular respiration (Joyet-Lavergne, 1926, 1928, 1929, 1931*, 1935; Weiss, 1950).  Cowdry & Scott, 1928;  I f this i s so, their accumulation i n areas  of high energy expenditure such as contractile and food vacuoles i s understandable. Contractile Vacuoles - Although these vacuoles could be readily identified i n our histological preparations because  - 7k -  of the surrounding layer of mitochondria, l i t t l e else regarding structure or function could be determined. Electron microscopic studies of Pappas and Brandt (1958) and Pappas (1959) show that the contractile vacuole i s surrounded by a densely packed layer ( 2 u thick) of small vesicles which i n turn are surrounded by a layer o f mitochondria.  Pappas estimates the thickness of the o  contractile vacuolar membrane to be 7 0 A.. Food Vacuoles - The narrow rim of dense small particles probably represents the granular layer of material characteristically found around young food vacuoles observed by the electron microscope (Pappas, 1 9 5 9 ) .  The fact that they are seen  around newer food vacuoles, together with my own observations of their migration towards the site of ingestion, suggest they are concerned with digestion.  The empty vesicles  around older food vacuoles are identical with the vacuole refractive bodies described by Torch ( 1 9 5 9 ) .  Since they  surround older food vacuoles and contain crystals i t i s possible that they are concerned with waste products.  The  crystals contained within older food vacuoles, likewise are believed to represent wastes.  Because the latter crystals  were observed so clearly after Mercury Orange staining, they are further discussed under "Application of Mercury Orange Method to P. carolinensis (page 81*).  - 75 -  Crystals - In the past, several workers have speculated on the functional significance of these cytoplasmic crystals. Mast and Doyle (1935a) and Wilber (19U5) believe they originate within food vacuoles and hence represent a food reserve.  I found that the quantity of these crystals does  not decrease i n starving P. carolinensis.  This agrees  with the observation of Andresen and Holter (19U5). Further, both these workers and Torch (1955) maintain that the crystals arise i n the cytoplasm.  The fact that  expulsion'of crystals i s seen i n both well-fed and starving individuals suggests that they might be metabolic wastes. Torch (1955) believed they represent an accessory mechanism for excreting nitrogen.  At the time that I observed these  crystals, no decisive data could be found concerning their nature.  Recent data, however, shows conclusively that they  are new nitrogen excretion products (Griffin 1959, 1960j Grunbaum et a l , 1959).  Griffin has positively identified  the platelike crystals as carbonyldiurea on the basis of evidence from microanalysis, X-ray diffraction pattern, infrared absorption spectra and petrographic analysis. Grunbaum et a l suggest that the bipyramids are not carbonyldiurea but a related substance.  Allen (1961) feels  that both substances are probably breakdown products of purine metabolism through allantoic acid.  - 76 -  Nuclei - Although the number of nuclei i s usually described as "several hundred", I found fewer (75 to  150).  However, i t i s possible that more were present but not seen because of the difficulty i n observing a l l planes of the amoeba at once. I believe the granules found at the.periphery of the interphase nucleus represent nucleoli and are the same as those described i n the electron microscopic studies of Borysko and Roslansky (1959) and Brandt and Pappas (1959). Chalkley, as early as 1936,  found that these peripheral  granules were basophilic but contained no acid.  desoxyribonucleic  Their lack of DNA. i s revealed by the fact that they  are Feulgen negative. . Their basophilia i s evident by their staining reactions with acidified Methyl Green, Geimsa (ultra-marine blue), Grenacher's Borax Carmine, Crystal Violet and Heidenhain's Iron Haematoxylin (dense black granules with the latter stain.whilst chromatin stains paler). Electron micrographs of the nucleoli reveal them to be packed with tiny granules which resemble the ribosomes of the cytoplasm.  The nucleoli, i n fact are known to be rich i n  ribonucleic acid and appear to be active centres of protein and RNA  synthesis (Brachet, 1961).  Certain workers estimate  the protein content of nucleoli to be between 70$ to 85$ (Vincent and Huxley, 195U}  Nurnberger et a l , 1952).  Boltus (195U)  - 77 -  found a high content of nucleoside phosphorylase and the DPN-synthesizing enzyme i n isolated echinoderm nucleoli. Although nucleoli are not generally believed to contribute to cellular mitosis, the behaviour of these peripheral granules during mitosis suggests that they perform some function i n nuclear division, as discussed under mitotic nuclei below. Nuclear Division Synchronous mitotic nuclear division i n P. carolinensis was f i r s t reported by Schaeffer (1937).  Short (19U5, 19U6)  and Kudo (19U9) observed, as I did, that on rare occasions a few nuclei appeared i n more or less advanced stages. This difference, I found was very slight but significant. For example, Figure 30 shows most nuclei i n interphase with a few i n late prophase.  The differences i n distance  between anaphase plates as noted by Kudo probably does not constitute reliable evidence i n this respect since this could be an artifact - i.e. cytoplasm could have been stretched slightly during handling. Regarding the increase i n size of nucleus ( up to six times as noted by Schaeffer) which I did not observe, a possible suggestion might be accumulation of additional materials from the cytoplasm for energy or building  - 78 -  materials necessary to produce two nuclei from one. Electron microscopic studies definitely reveal pores i n the nuclear membrane of this organism (Pappas, 1959).  In  P. carolinensis the pores occupy 15 to 20 percent of the nuclear surface which i s approximately twice that estimated for mammalian cells (Watson, 1955). Observations of the behaviour of the peripheral granules during mitosis led me to agree with Kudo who believes that the peripheral granules contribute, i n part, to the formation of the mitotic apparatus.  Mitochondria i n  close proximity to the nuclear membrane and peripheral granules has been observed by Brandt and Pappas (1959). These workers feel that the outer membranes of mitochondria and nuclei are continuous during and shortly after nuclear division i n P. carolinensis.  This lends support to  Swanson's (i960) statement that, although the function of the nucleolus other than to manufacture proteins i s unknown, the fact that i t disappears during c e l l division leads him to suspect that i t may be involved i n passing genetic information and materials from nucleus to cytoplasm.  In view of the  intense staining with Mercury Orange of P. carolinensis' nucleoli, indicating a rich protein sulfhydryl content, i t i s possible that they represent a means of transfer i n the opposite direction as well (i.e. of materials from cytoplasm  - 79 -  to nucleolus to mitotic apparatus).  Thus, i f the'theory  that -SH groups contribute to spindle formation i s correct (i.e. by oxidation to -SS- or by some other as yet unknown bonding) then these peripheral granules might be the source of such -SH groups.  The movement of the peripheral  granules towards the central area of the nucleus as prophase beginsj  the spindle fibres appearing at f i r s t  to end i n the granules;  the decrease i n size and number  of granules concomitant with the appearance of spindle fibres during late prophase and early metaphase and their absence in metaphase and anaphase when the spindle apparatus i s f u l l y formed also lends support to this theory.  Schaeffer  (1937) noted small granules, perhaps from the disintegrating interplate fibres during telophase, coalescing to form the larger peripheral granules of the mature nucleus.  These  are also seen i n Figure 13 of this study. I believe that the change from oval (in prophase) to rectangular shape assumed by late metaphase i s related to the disappearance of the nuclear membrane. occur simultaneously.  The two seem to  The more indistinct the membrane becomes  the more nearly perfectly rectangular becomes the shape of the figure.  Perhaps a l l the spindle fibres can now attain the  same length since they are not restricted by the membrane. The significance of nuclear membrane disappearance i s  - 80 -  of interest but to date remains obscure.  The answer i s  unlikely to be found until the membrane's detailed structure i s known.  Connections between the nuclear membrane and  cytoplasmic membrane are evident and observations on the formation of the nuclear membrane at telophase suggest that the membrane i s a specialized cytoplasmic structure (Mirsky and Osawa, l°6l).  Yasuzumi (1959) i s of the  opinion that i t has i t s origin i n the cytoplasmic membrane system.  Another picture of nuclear membrane formation at  telophase i n Yoshida sarcoma cells i s provided by Barer et a l (1959).  It shows vesicles (indistinguishable from the  cytoplasmic membrane system) surrounding the chromosomes at telophase which eventually fuse to form a complete nuclear membrane.  Lafontaine (1958) also describes membranes around  the telophase chromosomes seen i n electron micrographs of other cells.  The close proximity of these vesicles  (apparently parts of the nuclear membrane) to the chromosomes suggests that nuclear membrane breakdown during mitosis could provide structural materials, enzymes, or energy for mitotic apparatus formation.  Schaeffer (1937) states that the  anaphase discs are pulled apart by protoplasmic streaming. However, since the plates i n P. carolinensis separate i n many directions simultaneously i t would appear that some other force i s involved.  Kudo (19U9) suggests that the interdisc  and polar fibres might be responsible for this movement.  - 81 -  Mazia (1961) feels that the poles are pushed apart by the growth of the interdisc fibres.  Although this may  be  descriptively correct, (and i s the most l i k e l y explanation of the observations i n this study) i t remains to be explained how the growth of the interdisc spindle i s translated into an actual movement of the discs.  The electron microscope  studies of Porter, Bernard and Dehaven (Mazia, 1961) show that the morphology of the interdisc and polar fibres i s similar - but - the l a t t e r must lengthen and the former must shorten when the poles move apart.  Thus, i t seems  reasonable that biochemical (and physical) alterations within the interplate fibres are involved.  This correlates  well with my hypothesis that there is a difference between polar and interplate fibres since Mercury Orange staining is exceedingly faint i n the latter compared with the former. In this study I made no attempt to count chromosomes and thus did not employ special methods for the purpose.  As  yet no satisfactory method seems to have been employed for this purpose and the results have been extremely variable. For example, Schaeffer (1937) quotes a figure of 2,£00j Short (19U5, 1956)  quotes a figure of 300 and Kudo (19U9)  believes the number of chromosomes i n this organism i s approximately 100. That doubling of the metaphase plate to form two anaphase  - 82 -  plates i s the result of fission of chromosomes seems plausible (Figure 37). Enhanced fusion of chromosomes during anaphase was also observed by other workers (Schaeffer, 1937).  It i s  not possible, by the methods used i n this study, to determine whether this enhanced fusion i s actual (i.e. chromosomes fused due to physical proximity or chemical alteration) or apparent ( i . e . failure of resolution of individual chromosomes due to fixation and staining techniques). Regarding the bluntly-ending polar fibres, a similar multi-polar appearance of the spindle fibres was observed by Dawson and co-workers (1935) who considered i t "due solely to fixation".  However, I (and Kudo, 19li9) have observed this  broad termination of the spindle apparatus i n late prophase, metaphase and early anaphase i n living organisms under phase contrast. Nowhere i n the literature have I found any  explanation  of why the polar fibres eventually terminate at a common point.  I believe the arching of the anaphase plates occurs  as the result of the convergence of polar fibres.  - 83 -  III. CYTOCHEMICAL STUDIES Preliminary Experiments with Mercury Orange on Toad and Mouse Muscle The reddish-orange colour of the untreated muscle fibres observed after Mercury Orange staining confirms Bennett s (l°5>l) 1  results which he attributes to the following reaction between sulfhydryl groups and the chlori-mercuri group of the Mercury Orange reagent:  R-SH  ,  + Cl-Hg-<^>.N  Protein containing -SH groups  X)H ^ ± R-S-Hg-O-lf  Mercury Orange Reagent  ^QH + HCl  (I)  Compound responsible for characteristic orange-red colour  The observation that muscle fibres which were treated with arsenic before Mercury Orange staining failed to become coloured i s believed to be due to the binding of the -SH groups by the arsenical according to the Dithiol Theory (Stacken and Thompson, l°li6, 19k&) as follows:  R-As=0  +  2 R -SH T  *  Trivalent Arsenical  »  S-R R-A/ '  Protein containing -SH groups  x  \  +  H0 o  2  (II)  x  Sulfhydryl groups bound to arsenical (Dithioarsenit e)  By thus blocking the -SH groups, reaction between the latter and the chlori-mercuri groups of the Mercury Orange reagent was prevented.  These experimental results indicate the specificity  of Mercury Orange for -SH groups.  - 8U -  Effects of Sodium Arsenic Tri-oxide on the Living Amoeba The immediate increase i n cytoplasmic streaming noted i n organisms which were immersed i n an 0.5  p$  solution of arsenic  trioxide i s likely a protective reaction, whereby the animal i s attempting to escape the effects of the toxic environment. However, i f one wishes to analyze this behaviour more closely, i t i s necessary to consider the animal's defence mechanisms i n the light of possible phenomena which are taking place at a molecular or ionic level. For example, the rate of cellular metabolism i s presumably increased i n order to produce the visible increase i n cytoplasmic movement.  The increase i n metabolism could, i n part, be a  reflection of increased rate of enzyme production. Since i t i s known that the toxic substance (namely arsenic) to which the amoeba was exposed combines with -SH groups, the postulated increase i n enzyme production would effectively replace those enzymes which are inSactivated as the result of binding by arsenic of -SH groups on the enzyme's active sites.  Application of the Mercury Orange Method to P. carolinensis There was a direct relationship between intensity of colour imparted by Mercury Orange and concentration of arsenic to which the amoeba was exposed (Table 2b).  That i s , the greater the  concentration of arsenic the less intense was the staining.  - 85 -  Since i t i s known that arsenic (in vitro studies) combines with -SH groups, the results suggest that arsenic combined with the -SH groups of P. carolinensis thus inhibiting the combination of Mercury Orange and -SH taking place.  This resulted i n less  intense staining with Mercury Orange i n the arsenic treated amoebas as compared with the controls. The fact that the experimental results of Mercury Orange staining are qualitative must be borne i n mind i n their interpretation (Table 3).  However, the differences i n  concentration noted i n the various areas are i n keeping with some experiments on -SH content i n A. proteus (Heller, 1959) and i t seems reasonable that a close similarity i n this respect exists between the two amoebas.  For example, the plasmalemma of  A. proteus contains mucopolysaccharides and proteins. probably contain -SH groups.  The latter  Cytoplasmic ground substance of  A. proteus contains -SH proteins that are low i n concentration compared with nucleolar concentration.  In Pelomyxa carolinensis  I also found that the ground substance contained less -SH (2 plus) than did the peripheral granules of the nucleus (k plus).  The  mitochondria of A. proteus contain sulfhydryl-containing proteins and the rims of contractile vacuoles i n Pelomyxa were 3 plus for -SH groups.  This, together with the fact that mitochondria  surround these contractile vacuoles would suggest that mitochondria i n P. carolinensis also contain -SH since the mitochondria  - 86 -  surrounding contractile vacuoles and those loose i n the cytoplasm seem comparable i n every way. That the number of crystals i n older food vacuoles i s greater than i n young food vacuoles suggests they may be wastes or storage material.  It i s interesting that these crystals show  up very distinctly i n the Mercury Orange stained amoebas.  This  seems to be due to the rims being coloured (indicating -SH superficially) thus outlining their structure clearly. Apart from the surface, these crystals probably do not contain -SH i n any appreciable anount since the inner portion i s colourless i n the -SH treated amoebas.  Although the size and shape of  the food vacuole crystals differed considerably from the cytoplasmic crystals of the amoeba i t was interesting that the cytoplasmic crystals, known to be nitrogen excretion products, were also coloured on their surface by the Mercury Orange reagent, (although the intensity of the colour reaction of the cytoplasmic crystals was much paler). Sulfhydryl distribution i n starved individuals differed from that i n normal amoebas i n that, i n the former, the cytoplasmic ground substance was more intensely stained, whereas the rim surrounding the contractile vacuoles was less intensely stained. These results correspond to Heller's (1959) findings i n A. proteus. He observed more densely staining cytoplasm and decreased numbers of mitochondria i n starving amoebas.  The marked decrease i n the  - 87 -  number of contractile vacuoles i s i n keeping with what one would expect as the result of a diminished ingestion of food organisms. The lack of structural detail, likewise, might conceivably be attributed to decreased "raw materials".  Perhaps the  increased intensity of staining of the nuclei i n the starved organisms i s due to increased staining of the nucleoplasm corresponding to the darker cytoplasm.  Mitotic Nuclei stained with Mercury Orange The intense staining of the interphase peripheral granules may be due to the fact that they represent material (containing -SH) which i s to be incorporated into the spindle apparatus during mitosis.  This i s i n line with the belief of other workers  that the nucleoli contribute to spindle fibre formation (Kudo, 1°1*6; Schaeffer, 1937). If the theory that oxidation of -SH groups to form -SS- bonds is i n part responsible for spindle formation, one would expect anaphase spindle fibres should be the least densely coloured with Mercury Orange staining since presumably the greatest quantity of -SH groups would be oxidized to form -SS- bonds.  This was found  to be the case with respect to interdisc fibres which were poorly stained.  However, polar fibres were very well stained and  metaphase fibres were faintly stained. (l)  These results suggest that:  the interdisc fibres contain many -SS- bonds (or, at any rate, are poor i n -SH groups)  - 88 -  (2)  polar fibres are i n fact rich i n -SH,  and  (3)  metaphase spindle fibres contain appreciable amounts of -SH groups.  The fact that the -SH content differs between polar fibres and interdisc fibres may be related to the fact that interdisc fibres are lengthening and polar fibres are shortening during anaphase (Taylor,  1959).  According to electron microscopic  studies, as the fibres become shorter or longer they do not become thicker nor thinner, nor do they become less straight. Therefore, i t i s postulated (Mazia, 1 9 6 l ) that the "elongation" might be actually growth i n one dimension (the addition of molecules) and "contraction" i s i n fact shortening due to removal of molecules. It i s quite l i k e l y that chemical groups (and/or bonds) other than -SH are also involved i n mitotic apparatus formation and although -SS- bonds i n fact appear to be significant i n spindle formation, they are not likely to be the sole factor. Other workers are also of this opinion.  For example, Mazia  (1959)  at  f i r s t stressed -SS- bonds but more recently (1961 a, b) attention has been turned to other (poorly understood) bonds involving protein sulfhydryl.  Gross  (i960)  provides convincing  evidence of a major role of hydrogen bonds. The following, highly speculative, explanation i s suggested as an explanation of the cytochemical investigations i n this study. Since peripheral granules are deeply stained with the  OBSERVATION  HYPOTHESIS Figure U9(a)  +  Mercury Orange -(-  ( l ) Interphase nucleus showing peripheral granules.  Inset of ( l ) - A hypothetical peripheral granule containing abundant sulfhydryl groups. Peripheral granules stain k plus, i n d i c a t i n g r i c h -SH content.  Figure 1+9 (b)  •  SH  l  l  l  l  - R . — R — R — R —%  -j- Mercury Orange -|-  ( 2 ) Majority of peripheral granules have migrated centrally. Faintly coloured spindle f i b r e s .  SH sH SH  Inset of ( 2 ) - A hypothetical single metaphase f i b r e . -SH groups from peripheral granules, which migrate c e n t r a l l y , contribute to mitotic apparatus. -SH attaches at "free" ends so that f i b r e grows i n length but not i n thickness. Fibres can shorten by reverse reaction.  Metaphase f i b r e s s t a i n 2 plus, indicating some -SH groups are present.  OBSERVATION  HYPOTHESIS Figure U9 (c)  s - s — s - •s-s I I I  -|-  Mercury Orange -f-  -R-R-R- •R-R-  I  I  s - s - s (3) Interdisc spindle f i b r e s poorly stained. Polar f i b r e s very intensely stained.  I  I  I  - s - s  Inset of (3) - A hypothetical single i n t e r d i s c anaphase f i b r e . -SH groups have been oxidized to form -SS- bonds (or some other form of high energy sulfur bond). No free ends on t h i s f i b r e , therefore i t must grow by cleavage of i n t e r n a l bonds with addition of chemical groups. In t h i s process of cleavage and r e formation o f bonds, chemical energy to push discs apart i s generated - f i b r e s elongate but do not thicken. Polar f i b r e s are actually the metaphase f i b r e s (2) above, therefore probably s t r u c t u r a l l y analagous - but contraction or increased a c c e s s i b i l i t y of -SH groups r e s u l t s i n more intense staining.  Polar f i b r e s s t a i n 3 plus. Interdisc f i b r e s s t a i n 1 plus  - 89 -  sulfhydryl reagent they are doubtlessly rich i n -SH groups (Figure h9 a).  Their close proximity to the nuclear membrane  during interphase might be related to synthetic processes taking place involving raw materials obtained from the cytoplasm. When these granules migrate centrally, sulfhydryl groups are made available to assist i n mitotic apparatus formation, but these -SH groups are not yet oxidized to -SS- bonds - therefore metaphase spindles stain faintly with Mercury Orange (Figure h9 b). A.s interdisc fibres are formed and elongate, -SH groups are oxidized to -SS- bonds and therefore Mercury Orange staining of these fibres is very faint (Figure k9 c).  On the other hand,  the polar fibres being structurally analagous to the metaphase fibres, contain -SH groups and therefore stain well with Mercury Orange.  The reason for the intense staining may be due to  increased accessibility of -SH groups (i.e. soon to become dissociated from the mitotic apparatus) or to the fact that the fibres are "contracting" hence -SH groups are closer together than they  were  i n metaphase.  It i s f e l t that the molecular structure of spindle fibres i s i n reality much more complicated than postulated i n Figure U9. For example, neither large protein molecules nor metal ions have been incorporated into the hypothetical structure of a spindle fibre postulated here.  - 90 -  SUMMARY  Although P. carolinensis i s a highly specialized structure, I believe i t i s a f a i r l y good representative of the living c e l l , and i t s use as research material for cellular studies i s justified. When one considers working with tissue cultures, whether fibroblasts or white blood cells, each i s a specialized cell and hence i t i s d i f f i c u l t to find a single organism or individual mammalian c e l l which represents a l l c e l l s . Feeding occurs only when the organism i s attached to the substratum. "healthy".  The latter must be clean and the environment Feeding habits are of a passive nature i n that  movement of the amoeba from place to place i s not the most significant factor i n obtaining food. advance towards the amoeba. i s as yet undetermined.  Rather, food organisms  The explanation of this behaviour  The Parameciums remain sluggish u n t i l the  food vacuole i s completely formed, then commence violent movement. Small dark granules surround newly formed food vacuoles but "empty" vesicles surround older food vacuoles.  Older food vacuoles  contain masses of crystals which are seen clearly with Mercury Orange staining. Environmental conditions (pH) appear to be of greater significance i n maintaining a healthy state i n P. carolinensis  - 91  than does the amount of food. Body form i s correlated with physiological state as well as mitotic events. The most satisfactory fixative for histological studies was Osmium Tetroxide but Schaudinn's and TCA, were also satisfactory. Results of this work confirmed that arsenic combines with sulfhydryl groups and therefore inhibits metabolic enzymes. Using activity (i.e.  cytoplasmic movement) as a criterion, low  concentrations had a stimulatory effect and this i s believed to indicate that metabolism i s temporarily stimulated at the molecular level, i n an attempt to cope with destruction of necessary substances.  Effects of arsenic i n these low  concentrations were reversible.  Arsenic i n concentrations  sufficient to cause death invariably damaged the membrane of P. carolinensis resulting i n complete dissemination of i t s contents into the surrounding environment. Frequency of mitosis i s apparently related primarily to food supply. A l l of the nuclei contained i n a given amoeba undergo mitosis synchronously except on rare occasions and then the difference i s slight.  r 92 -  Peripheral granules i n the nucleus stain intensely with Mercury Orange and appear to give rise to spindle fibres. 8.  Mitochondria characteristically surround contractile vacuoles and contain -SH groups.  The intensity of colour of  the mitochondria diminishes in starved individuals stained with Mercury Orange. 9.  The Mercury Orange method for the detection of -SH groups worked well for the material i n this study. Mercury Orange as used here i s believed to react with free, "masked" and "sluggish" -SH groups indiscriminately. Variation i n the distribution of -SH groups was observed i n the various structures of P.carolinensis.  Sulfhydryl distribution i n  starving individuals differed from that of normal, well-fed animals.  Crystals i n food vacuoles showed up very clearly  with Mercury Orange stain indicating considerable -SH content. 10.  Oxidation of -SH groups, the main source of which may  be  peripheral granules, to -SS- bonds i s believed to contribute, in part, to the formation of mitotic spindles i n P.carolinensis. Metaphase and polar (anaphase) fibres are believed to be structurally similar. They are, however, believed to differ structurally (chemically and/or physically) from interdisc anaphase fibres.  BIBLIOGRAPHY Adams, C.W.M. 1956 Allen, R.D. 1961  Histochem. Cytochem.  Us23.  Amoeboid movement. In: The C e l l . Biochem. Physiol. Morphol. 2:135. (Editors: Brachet and Mirsky). Academic Press, N.Y.  Andresen, N. 19U2 Cytoplasmic components i n the amoeba Chaos chaos Linne. Compt. Rend. trav. Lab Carlsberg, Ser. Chim. 2U:139. Andresen, N. 19U5 Coalescence between vacuoles during v i t a l staining with neutral red of Chaos chaos L. Compt. rend. trav. Lab Carlsberg, Ser. Chim. 25:lh7. Andresen, N. 19l*6 Cytoplasmic changes during neutral red staining of the amoeba Chaos diffluens (A. proteus. ibid) 25:169. Andresen, N. 1956 Cytological investigations on the giant amoeba (Chaos chaos). Compt. rend. trav. Lab Carsberg, Ser. Chim. 29_:h35. Andresen, N. 1957 Labile colloidal complexes of the cytoplasm. Comp. Physiol. h9}221.  J. Cellular  Andresen, N. and Holter, H. 19U5 Cytoplasmic changes during starvation of the amoeba Chaos chaos L. Compt. rend. trav. Lab Carlsberg, Ser. Chim. 25:107. Andresen, N., Chapman-Andresen, C. and Holter, H. 1952 Compt. rend. trav. Lab Carlsberg, Ser. Chim. 28:189. Armed Forces Instit. of Technology. I960 Manual of Histologic and Special Staining Techniques. 2nd Ed. Ashbel, R. and Seligman, A.M. I9I4.9 A new reagent for the histochemical demonstration of active carbonyl groups. Endocrinol, hit:565. Bachstrom, S. 1956 Experimental Cell Research 11:322. Bairati, A. and Lehman, F.E. 1956 Structural and chemical properties of the contractile vacuole of A. proteus. Protoplasraa U5*525.  Baker, J.R. 1958  Principles of biological microtechnique. and Sons, Inc., N.Y.  John Wiley  Bang, F.B. and Gey, G.O. et a l . 1951 The validity of cellular structures as seen with the electron microscope IX Meeting of E.M.S.A. Barer, R., Joseph, S., and Meek, G.A. 1959 Exptl. Cell Research 18:179. Barrnett, R.J. and Seligman, A.M. 1952 Histoehemical demonstration of protein-bound -SH groups. Science 116:323. Barrnett, R.J. 1953 Histoehemical demonstration of the sites of proteinbound -SH and disulfide groups. Abstract Anat. Record ll£s280. Barrnett, R.J. and Seligman, A.M. 1953a The histoehemical demonstration of protein-bound -SH groups. J. Nat. Cancer Inst. 13:905. Barrnett, R.J. and Seligman, A.M. 1953b Investigation of the histoehemical localization of disulfides (Abstract). J. Histo. and Cyto. 1:392. Barrnett, R.J. and Seligman, A.M. 1954a Histoehemical experiments on sulfhydryls and disulfides. In: A symposium on Glutathione. Academic Press, N.Y. page 89. Barrnett. R.J. and Seligman, A.M. 195Ub Histoehemical demonstration of -SH and -SS- groups of protein. J. Nat. Cancer Inst. 1^:769. Barrnett, R.J., Tsou, K.C. and Seligman, A.M. 1955 Further histoehemical characterization of proteinbound -SH groups; The use of naphthol-containing, mercaptide-forming and alkylating compounds as reagents. J. Cyto. and Histo. 3:U06. Barron, E.S.G. 1951 Thiol groups of biological importance. In: Recent Advances i n Enzyraology. Interscience Publishers, Inc. N.Y. 11:201. Barron, E.S.G. and Kalnitsky, G. 19U7 The inhibition of succinoxidase by heavy metals and i t s reactivation with dithiols. Biochem. J. Ul:3U6.  Barron, E.S.G. and Singer, T.P. 19h$ Sulfhydryl enzymes i n carbohydrate J . B i o l . Chem. 1^1?221. Barron, E.S.G., M i l l e r , Z.B.,  19l47a  metabolism.  and Kalnitsky, G.  The oxidation of d i t h i o l s .  Biochem. J . I4.lt62.  Barron, E.S.G., M i l l e r , Z.B., B a r t l e t t , G.R., Meyer, J . and Singer, T.P. 19U7b Reactivation by d i t h i o l s of enzymes, i n h i b i t e d by l e w i s i t e . Biochem. J . ltl:69. Barron, E.S.G., Nelson, L. and Ardao, M.I. 191*8 J . Gen. Physiol. 32:179. Baumberger, J.P. 19U1  Am. J . P h y s i o l . 133:206.  Beckett, E. and Bourne, G.H. 1958 Some histochemical observations on normal and diseased human skeletal muscle. J . Histo. and Cyto. 6:13. Bennett, H.S. 195l The demonstration of t h i o l groups i n c e r t a i n tissues by means of a new colored sulfhydryl reagent. Anat.  Rec. 110:231.  Bennett, H.S. 1956  J . Biophys. Biochem. C y t o l . 2(1*) part 2 (Suppl.):99.  Bennett, H.S. and Watts, R.M. 1958 The cytochemical demonstration and measurement of -SH groups by azo-aryl mercaptide coupling with s p e c i a l reference to Mercury Orange. In: General cytochemical methods. Edited by D a n i e l l i , J.F. 1:375. Academic Press Inc. N.Y. Bennett, H.S. and Yphantis, D.A. 19u8 l-(U-chloromercuriphenylazo)-naphthol-2.  Soc. 70:3522.  Binet, L. and Weller, G. 1936  B u l l . Soc. chim. B i o l . 18:358.  Binet, L., Joulmes, C. and Weller, G. 1937  Compt. rend. 20l*:176l.  J . Am. Chem.  Biology of the Amoeba June/59 Annals of N.Y. Academy of Sci. 78:Art. 2:h01-70lw Board, F.A. 1951  J. Cellular Comp. Physiol. 38:377.  Bolognari, A. 195U Aspetti dell' ovagenesi di Aplysia depilans L. ( Moll. Gast. Opis). Boll. Zool. 21:105. Bolognari, A. 1952 Arch. Sci. Biol. Italy. 36:UO. Boltus, E. 195U  Biochim. et Biophys.  Acta 15:263.  Borysko, E. and Roslansky, J. 1959 Methods for correlated optical and electron microscopic study of amoeba. In: Annals of N.Y. Acad. Sci.78(2):U32. Bourne, G. 1935 Brachet, J. 19u0  Mitochondria, Golgi apparatus and vitamins. Australian J. Exptl. Biol. Med. Sci. 13:238. La localization de l'acide thymonucleique pendant l'oogenese et l a maturation chez les amphibians. Arch. Biol. £1.15*1.  Brachet, J. 1950  Chemical Embryology.  Interscience Publishers Inc. N.Y.  Brachet, J. 1957  Biochemical Cytology. Academic Press Inc. N.Y.  Brachet, J. 1961  The Living C e l l .  Sci Amer.  205(3):5l.  Brandt, P.W. 1958 A study of the mechanism of pinocytosis. Research 15:300. Brandt, P.W. and Pappas, G.D. 1959 J. Biophys. Biochem. Cytol. 6:91. Brown, V.E. 1930 Buffa, E. 190U  The Golgi apparatus of A. proteus.  Exptl. Cell  Biol. Bull. 30:21*0.  Sur une combinaison sulfuree des tissues animaux. J. de Physiol, et de pathol. gen 6:6U5. Cafruny, E.J., DiStefano, H.S. and Farah, A, 195k Effects of mercural diuretics on the -SH content of kidney c e l l s . Fed. Proc. Amer. Soc. for Expl. Biol. 13:3H0.  Cafruny, E.J., DiStephano, H.S. and Farah, A. 1955 A cytometric determination of protein-bound -SH groups. J. Histochem. and Cyto. 3 0 5 U . Calkins, G.N. 19iil  Protozoa i n Biol. Research.  Columbia Univ. Press.  Cameron, G.R. 1951 The morphology of the normal c e l l . In: Path, of the Cell, pages 56-76. Charles C. Thomas, Springfield, 111. Casselman, W.G.B. 1959 Histochemical Technique. John Wiley and Sons, Inc., N.I. Chalkley, 1936  H.W. J. Morphol. 60:13.  Chapman-Andresen, C. and Holter, H. 1955 Exptl. Cell Research Suppl. 3:52. Chevremont, M. and Frederic, J. 19U3 Une nouvelle methode histochimique de mise en evidence des substances a fonction sulfhydryle. Arch. Biol. Liege 5U:589. Claude, A. 19U8  Cochran, W.G. 1957  Studies on Cells: Morphology, chemical constitution and distribution of biochemical functions. Harvey Lectures U3_:121. Springfield, 111. and Cox, G.M. Experimental design. 2nd Ed.  John Wiley and Sons, Inc., N.Y.  Cohen, A.., King, H. and Strangeways, W.I. 1931 . J. Chem. Soc.Pt 2:301+3 %  Cohen, A.I. 1957  Electron microscopic observations on A. proteus i n growth and inanition. J. Biophys. Biochem. Cytol. 3:859.  Cohen, A.I. 1959 Colien, F.E. 1938  Physiological and morphological observations on amoebae. Annals N.Y. Acad, of Sci. 78(2):609. J. Lab. Clin. Med. 2lj.:2U5.  Cooperstein, S.J.-, Lazarow, A. and Patterson, J.W. 1953 Studies on the mechanism of Janus Green B. Staining of mitochondria I I . Reactions and properties of Janus Green B and i t s derivatives. Exptl. Cell Res. 5?69.  Cooperstein, S.J., and Lazarow, A. 1953 Studies on the mechanism of Janus Green B staining of mitochondria III. Reduction of Janus Green B byisolated enzyme systems. Exp. Cell Research 5;82. Cowdry, C.V. and Scott, G.H. 1928 Etude cytologiques sur le paludisme III. Mitochondries, granules colorable au rouge neutre et appareil de Golgi. Arch. inst. Pasteur Afr. N. 17:233. Dalton, J.A. and Felix, M.D. 1956 A. comparative study of the Golgi complex. Biochem, Cytol. 2(Suppl):79. Danielli, J.F. 1957 Cytochemistry:  J. Biophys.  a c r i t i c a l approach. Academic Press,  Danielli, J.F., Danielli, M., Fraser, J.B., Mitchell, P.D., Owen, L.N., and Shaw, G. 19U7 BAL-Intrav: A new nontoxic thiol for intravenous injection i n arsenical poisoning. Bioch. J. 1+1:325. Davenport, H.A. I960 Histological and Histoehemical Techniques. Co., Phaladelphia (London.) Davson, H. 19U3  W.B.  Saunders  The permeability of natural membranes, page 25U. Cambridge and N.I.  deRey Pailhade, J.  1888  Compt. rend. 106:1683 and 107:1*3.  deRobertis, E.D.P., Nowinski, W.W., and Saez, F.A. 1957 General Cytology. W.B. Saunders, Philadelphia. Dulzetto, F. 1931 Arch. Biol. 1*1:221. Ecker, E.E. and Pillemer, L. 1938 Proc. Soc. Exptl. Biol. Med. 38:316. Eagle, H. and Doak, G.O. 1951 The biological activity of arsenobenzenes i n relation to their structure. Pharmacol. Rev. 3:107. Eagle, H, Hogan, R.B. and Doak, G.O. 19UU The therapeutic efficacy of phenyl arsenoxides i n mouse and rabbit trypanosomiasis. Pub. Health Rep. 59(?artl):765.  Ehrlich, P. 1909  Ber. 1*2:17.  E l l i o t t , A.M. 1952 Zoology.  Appelton Century Crofts, Inc. N.Y.  Engelhardt, V.A. 191*2 Yale J. Biol. Med. l £ : 2 1 . Faure-Fremiet, E. 1910 Etude sur les mitochondries des protozoaires et des cellules sexuelles. -Arch. Anat. Micr. 11:1*58. Findlay, G.H. 1955 J . Histo. and Cyto. 3:331. Ford, Peter 1958 19U9  Advanced Histological Technique.  Scholar's Library, N.Y.  * Arch. Biol. Liege 60:79.  Fugita, A. and Numata, I. 1939 Biochem. Z. 300:21*9. Gall, J.G. 1956  Small granules i n the amphibian oocyte and their relationship to RNA. J. Biophys. Biochem. Cytol. 2(Suppl.):393.  Gatenby, J.B. and Beams, H.W. (Editors) 1951 The Microtomists' Vade-Mecum (Bolles Lee). Blakiston Company, Pa.  The  Gatenby, J.B., Dalton, A*J. and Felix, M.D. 1955 The contractile vacuole of parazoa and protozoa and the Golgi apparatus. Nature 167:301. Ghosh, B. and Guba, B.C. 1937 Science and Culture 3:21*3. Giroud, A. 1931  Protoplasma 12:23.  Giroud, A., and Bulliard, H. 1933 Reaction des substances a fonctaon sulfhydryle. Protoplasma 19:281. Glahn, W.C. 1938  et a l . Effect of certain arsenates on the l i v e r . 21*: 1*88.  Arch. Path.  Glick, D. 19U9  Techniques of Histo. and Cyto-chemistry. New York.  Godeaux, J . 19hh  Bull. Soc. Roy. Sci. Liege 13:219.  Godeaux, J . 19U6  Corapt. rend. soc. b i o l . 11*0:678.  Gola, G. 1902  Lo zolfo e i suoi composti n e l l piante, Malpighia. 16:368.  1  Interscience,  economia delle  Goldacre, R.J. 1952 The folding and unfolding of protein molecules as a basis of osmotic work. Intern. Rev. Cytol. 1:135. Goldacre, R.J. and Larch, I.J. 1950 Folding and unfolding of protein molecules i n relation to cytoplasmic streaming, amoeboid movement and osmotic work. Nature 166:1*97. Goldetz, L. and Unna, P.G. 1909 Monotsschrift. prakt. Gomori, G. 19U9-50 Gomori, G. 1952 Gomori, G. 1956  Dermatol. 1*8:11*9.  P i t f a l l s i n histochemistry. In: Ann. New York Academy Sci. 50:968. Microscopic Histochemistry: Chicago Univ. Press.  Principles and Practice.  Histoehemical methods for protein-bound -SH and -SSgroups. J. of Microscopical Science. 97:1.  Goodman, L.S. and Gilman, A. 1956 The Pharmacological Basis of Therapeutics. The MacMillan Co. N.Y. Gray, P. 1958 Gray, P. 1951*  2nd Ed.  Handbook of Basic Microtechnique. 2nd Ed. McGraw-Hill Book Co. Inc. N.Y. (Toronto, London). The Microtomists Formulary and Guide. Press Co. Inc. N.Y. 1  The Blakeston  Greider, M.H., Kostir, W.J. and Frajola, W.J. 1956 Electron Microscopy of the nuclear membrane of A.proteus. J. Biophys. Biochem. Cytol. 2(Suppl.):l4lr5. Greider, M.H., Kostir, W.J. and Frajola, W.J. 1958 Electron microscopy of A. proteus. J. Protozool. 5(2):139. Gross, Paul, R. and Spindel, W. I960 Heavy water inhibition of c e l l division. An approach to mechanism. In: Annals of N.Y. Acad, of S c i .  90(Art.2):500.  Grunert, R.R. and Phillips, P.H. 1951 Arch. Biochem. 30:217. Gurr, E. 1956  Manual of Med. and Biolog. Staining Tech.  Guthwin, H. and Kopac, M.J. 1959 Microscopic enzyme chemistry of carboxylic esterases i n amoeba. Annals of N.Y. acad. of Sci. 78(2) :53s. Hall, R.P.  1953  Hall, R.P. 1930  Protozoology. Prentice-Hall, N.Y.^  Cytoplasmic inclusions of trichamoeba and their reaction to v i t a l dyes and to osmic and silver impregnation. J. Morphol. U9:139.  Hall, R.P. and Loefer, J . B . 1930 Studies on Euglypha I. Cytoplasmic inclusions of Euglypha alveolata. Arch. Protistenk. 72:365. Hammett, F.S. 1929 Protoplasma.  7:297.  Hammett, F.S. and Chapman, S.S. 1938 The quantitative unrealiability of the nitroprusside test for -SH and -SS-. J. Lab. Clin. Med. 2l±:293. Hardy, M.H.  1952  Am. J. Anat. 90:285.  Harris, L.J. 1923 Proc. Roy. Soc.  London. B9h:U26.  Heffter, A. 1908  Med. Naturwise. Arch. 1:81.  Heilbrunn, L.V. 1952 An outline of General Physiology, pages 367 to 380. Saunders, Philadelphia, Pa. Heilbrunn, L.V. 1956 The dynamics of living protoplasm. Academic Press, Inc. N.Y. Heller, I.M. and Kopak, M.J. 1955a Exptl. Cell Research 8:62. Heller, I.M. and Kopak, M.J. 1955b Exptl. Cell Research 8:563. Heller, I.M. and Kopak, M.J. 1956 Exptl. Cell Research 11:206. Hellerman, L., Perkins, M.E. and Clark, W.M. 1933 Proc. Natl. Acad. Sci. U.S. 19:855Hitchcock, P. 19U6 The effect of dithiols and other enzyme"inhibitors on blood vessels. J. Pharmacol, and Exper. Therap. 87:55. Hoffman, J.G. 1957 Life and death of cells. Garden City, N.Y. 'Holter, H. 1959a  Hanover House Books,  Problems of pinocytosis with special regard to amoebae. Annals N.Y. Acad, of Sci. 78(2):52U.  Holter, H. 1959b  Pinocytosis.  In: International Review of Cytology. 8:481  Holter, H. 1961 How things get into cells. S c i . Amer. 205(3):167. Holter, H. and Doyle, W.L. 1938 Uber die lokalisation der amylase i n amoeben. Compt. rend, trav Lab. Carlsberg, Ser. Chim. 22:219.  Hopkins, F.G. 1921 Biochem. J. 15:286. Hopkins, F.G. 1925 Glutathione, Its influence i n the oxidation of fats and proteins. Bioch. J. 19:787. Horning, E.S. 1926 Observations on mitochondria. Australian J. Exptl. Biol. Med. S c i . 3:lu9. Horning, E.S. 1928 Studies on the behavior of mitochondria within the living c e l l . Australian J. Exptl. Biol. Med. Sci._5_:lU3. Horning, E.S. 1933 Enzymatic function of mitochondria. Erg d. Enzymforsch 2:336. Houssay, B.A. 1950 Am. J. Med. S c i . 219:353. Hughes, W.L. 19U9  Protein mercaptides. Quant. Biol. l U : 7 9 .  Cold Spring Harbor Symposia on  Infantellina, F. and LaGrutta, G. 19U8 Arch. S c i . Biol. Italy. 32:85. Jacobs, M.H. and Carson, S.A. 193U Biol. Bull. 67:325. Jahn, T.L. 19 Jeener, R. 19U7  How to Know the Protozoa. Wm. E. Brown Co, Dubuque, Iowa. Experientia. 3:2u3.  Joyet-Lavergne, P. 1926 Reserches sur le cytoplasm des sporozoaires. Arch. d'Anat. Micr. 22:1. Joyet-Lavergne, P. 1928 Le pouvoir oxydo-reducteur du chondriome des Gregarines et les procedes de recherches du chondriome. Compt. Rend. Soc. Biol. 98:567.  Joyet-Lavergne, P. 1929 Glutathion et chondriome, Protoplasma 6 : 8 U . Joyet-Lavergne, P, 193U Une theorie nouvelle sur le mecanisme des oxydoreductions intracellulaires. Compt. Rend. Acad. Sci. Paris. 199:1159. Joyet-Lavergne, P. 1935 Recherches sur l a catalyse des oxydo-reductions dans l a cellule vivante. Protoplasma. 23'.$0. Joyet-Lavergne, P. 1938 Compt. Rend. Soc. Biol. Kassel, R. 1959  128;59.  Particulates of amoebae. In: Annals of N.Y, Acad. Sci. 78(2):U21.  Kassel, R. and Kopac, M.J. 1953 Experimental approaches to the evaluation of fractionation media. I The action of ions on the protoplasm of A. proteus and P. carolinensis. J. Exptl. Zool. 12li:279. Kawamura, N. and Dan, K. 1958 A cytochemical study of the -SH groups of Sea Urchin eggs during f i r s t cleavage. Jour. Biophys. Biochem. Cytol. U:6l5. Kawamura, N. 1960 Exptl. Cell Research. 20:127. Kaye, M. 192U Kirby, H. 1950  Biochem. J. 18:1289. Materials and methods for the study of the protozoa. University of Calif. Press., Berkeley and Los Angeles.  Knisely, M.H. 1936 A method of illuminating living structures for microscopic study. Anat. Rec. 6U:U99. Kopac, M.J. 1950 Kopac, M.J, 1951  The surface chemical properties of cytoplasmic proteins. Ann. N.Y. Acad. S c i . 50(8):870. Probable ultrastructure involved i n c e l l division. Ann. N.Y. Acad. S c i . 5 l ( 8 ) : l 5 l .  Kopac, M.J. 1959.  Research on the amoeba i n 2158 A.D. Acad. Sci. 78(2):696.  In: Annals  N.Y.  Kudo, R.R. 195U Kudo,R.R. 19U6 Kudo, R.R. 19U7 Kudo, R.R. 19U9  Protozoology. l*th Ed. G.C. Thomas, Springfield,  111.  Pelomyxa carolinensis Wilson. I. General observations on the I l l i n o i s stock. J. Morphol. 7 8 : 3 1 7 . Pelomyxa carolinensis Wilson. II. Nuclear division and plasmotomy. 3". Morphol. 80:93. Pelomyxa carolinensis Wilson. I I I . Further observations on plasmotomy. J . Morphol. 85:163.  Kudo, R.R. 1951  Observations on Pelomyxa carolinensis.  J. Morphol. 88:lU5.  Kudo, R.R. 1952 Kudo, R.R. 1959 Ruroiwa, Y. 1953 Labes, R. 1929  The genus Pelomyxa.  Trans. Am. Microscop. Soc. 71:108.  Pelomyxa and related organisms. In:. Annals N.Y. Acad. Sci.7bT2):U7U. J. Agr. Chem. Soc. Japan. 27:U73. Uber die pharmakologische bedeutung der chemischen reaktionen zwischen arseniger saure und thiol verbindungen. Arch Exp. Path. Pharm. I h l : l l i 8 .  Ladman, A.M. and Barrnett, R.J. 1951*  Endocrinology.  5k:35$.  Lafontaine, J.G. 1958 J. Biophys. Biochem. Cytol. 1*:777. Landau, J.V. 1959  Lazarow, A. 19U5  Sol-gel transformation i n amoebae. I I . Physical studies and c e l l division. Annals N.Y. Acad. Sci. 78(2):l+87. The chemical structure of cytoplasm as investigated i n Professor Bensley's laboratory during the past ten years. Biol. Symposia. 10:9.  Lazarow, A. 19U9  Physiol. Revs. 29:1*8.  Lazarow, A. and Cooperstein, S.T. 1953 Studies on the mechanism of Janus Green B staining of mitochondria - Review of the Literature. Expt. Cell Research 5:56. LeFevre, P.G. 19U8 J. Gen. Physiol. 31:505". Lehmann, F.E., Manni, E. and Bairati, A. 1956a Der feinbau von plasmalemma und kontraktiler vakuole bei A. proteus i n schnitt und fragment-proparaten. Rev. suisse Zool. 63s214.6. Lehmann, F.E., Manni, E. and Geiger, W. 1956b Der schichtenbau des plasmalemmas von A. proteus i n elektronenmikros-kopischen schnittbild~ Maturwiss 1*3:91. L i , C.H. and Evans, H.M. 19U8 Chemistry of anterior pituitary hormones. In: The Hormones. Academic Press, Inc. N.Y. (Editors: Pincus,G. and Thimann, K.V.). L i l l i e , R.D. and Burtner, H.J. 1953 J. Histochem. 1:87. Lindberg, 0 . and Ernster, L. 195U Chemistry and physiology of mitochondria and microsomes. Protoplasmatologia. 3 A , U : 1 . Linderstrom-Lang, K. and Jacobsen, C.F. 19U0 Compt. Rend. trav. Lab Carlsberg. Ser Chim. 23:289. Linneaus, K. 1758 ' Systema Naturae. London, England.  10th Ed. Brit. Museum, Nat. Hist.  Linneaus, K. 1767 Systema Naturae. London, England.  12th Ed. Brit. Museum, Nat. Hist.  Lison, L. 1953 Lyons, R.N. 19U5  Histochimie et cytochimie animales principes et methodes. '2nd Ed.. Gauthier-Villars, Paris. Nature. 155:633.  MacLennan, R.F. 19U0 A quantitative study of osmic acid impregnation i n protozoa. Trans. Am. Microscop. Soc. 59:149. MacLennan, R.F. 19Ul Cytoplasmic inclusions. In: Protozoa i n Biological Research, page 111. G.N. Calkins Editor. Columbia Univ. Press., N.Y. Mapson, L.M. and Moustafa, E.M. 1956 Biochem. J. 62:2U8. Marshal, J.M., Jr., Schumaker, V.N. and Brandt, P.W. 1959 Pinocytosis i n amoebae. Annals N.Y. Acad, of Sci. 78(2):5l5. Mason, H.L. 1930  J. Biol. Chem. 86:623.  Mast, S.O. 1938  Amoeba and Pelomyxa vs. Chaos. Turtox News. 16:56.  Mast, S.O. 19hZ  The hydrogen ion concentration of the content of the food vacuoles and the cytoplasm i n amoeba and other phenomena concerned with food vacuoles. Biol. Bull. Woods Hole. 83:173. Mast, S.O. and Doyle, W.L. 1935a Structure, origin and function of cytoplasmic constituents in A. proteus. I. Structure. Arch. Protistenk. 86:T55~. Mast, S.O. and Doyle, W.L. 1935b Structure, origin and function of cytoplasmic constituents in A. proteus with special reference to mitochondria and Golgi substance. I I . Origin and function based on experimental evidence. Effect of centrifuging on A. proteus.' Arch. Protistenk. 86:278. Mast, S.O. and Hahnert, W.F. 1935 Feeding, starvation and digestion i n A. proteus. Physiol. .Zool. 8:255.  Leidig  Mast, S.O. and Johnson, P.L. 1931 Concerning tine scientific name of the common large amoeba usually designated Amoeba proteus. Leidig. Arch. Protistenk. 75:lU.  Mazia, D. 195Ua Mazia, D. 195Ub Mazia, D. 1952  Mazia, D. 1955 Mazia, D. 1957 Mazia, D. 1958 Mazia, D. 1959  Mazia, D. I960 Mazia, D. 196la Mazia, D. 1961b  Glutathione, page 209.  Academic Press, N.Y. U0:$21.  Proc. Natl. Acad. Sci. U.S.  Physiol of the Cell Nucleus. Modern Trends i n Physiol, and Biochem. (E.G. Barron, Ed.). Academic Press, N.Y. 77:122. Symposia Soc. Exptl. Biol. 9:335. The Chemical Basis of Heredity, page 169. Hopkins Press, Baltimore. Exptl. Cell Research  The Johns  lU:U86.  The role of t h i o l groups i n the structure and function of the mitotic apparatus. In: Sulfur in Proteins:367. R. Benesch, et a l Editors. Academic Press, N.Y. The analysis of c e l l reproduction. N.Y. Acad, of Sci. 90(2):3U5.  In: Annals of  Mitosis and the physiology of c e l l division. 3:77. Academic Press, N.Y. How cells divide.  Sci. American.  Mazia, D. and Dan, K. 1952 Proc. Nat. Acad. Sci. U.S.  In: The Cell  205(3):100.  38:826.  McClung, C.E. 1950 Handbook of Microscopical Technique.  3rd Ed.  Hoeber, N.Y.  Mellors, R.C. (Editor). 1959 Analytical Cytology - Methods for studying cellular form and function. 2nd Ed. McGraw-Hill, Toronto. Mescon, H. and Flesch, P. 1952 J. Invest. Dermatol. 18:261.  Mir sky, A.E. and Anson, M.L. 1936 -SH and -SS- groups of proteins. I I . The relation between the number of -SH and -SS- groups and quantity of insoluble protein i n denaturation and i n reversal of denaturation. I I I . -SH groups of native proteins, hemoglobin and the proteins of the crystalline lens. The reducing groups of proteins. J. Gen. Physiol. 19:1*27, 1*39, 1*51. Mirsky, A.E. and Pauling, L. 1936 Proc. Natl. Acad. S c i . U.S. 22:1*39. Mirsky, A.E. and Osawa, S. 1961 The Interphase Nucleus. Inc.  In: The C e l l . 2:677 Acad. Press ~~  Morner, K.A.H. 1921 Biochem. J. 15:285. Neufeld, E.F. and Mazia, D. 1957 Exptl. Cell Research 13:622. Nickerson, W.J. and Falcone, G. 1956 Science 121*: 318. Nickerson, W.J. and Falcone, G. 1956 Science 121*: 722. N i g r e l l i , R.F. and Hall, R.P. 1930 Osmiophilic and Neutral Red stainable inclusions of Arcella. Trans. Am. Microscop. Soc. 1*9:18. Nurnberger, J., Engtrdm, A. and Lindstrom, B. 1952 J. Cellular Comp. Physiol. 39*215. Olcott, H.S. and Fraenket-Conrat, H. 191*7 Specific group reagents for proteins. Chem. Revs. I * l : l 5 l . Pace, D.M. and Belda, W.H. 19l*l* The effects of Potassium cyanide, potassium arsenite and ethyl urethane on respiration i n P. carolinensis. Biol. Bull. 87:138. Pace, D.M. and Kimura, T.E. 191*6 Relation between metabolic activity and cyanide inhibition i n P. carolinensis (Wilson) Proc. Soc. Expl. Biol. Med. 62:223. Pace, D.M. and McCashland, B.W. 1951 Effects of low concentrations of cyanide on growth and respiration i n P. carolinensis (Xvilson). Proc. Soc. Expl. Biol. Med. 76Tl6T^  Pace, D.M. 1952  and Frost, B.Li The effects of ethyl alcohol on growth and respiration i n P. carolinensis. Biol. Bull 103:97.  Palade, G.E. 19^2 Pappas, G.D. 195u Pappas, G.D. 1958 Pappas, G.Di 1959  A study of fixation for electron microscopy. J. Exp. Med. 95:285. Structural and Cytochemical Studies of the cytoplasm in the family Amoebidae.- Ohio J. Sci. 9±il9$. and Brandt, P.W. The Fine Structure of the contractile Vacuoles i n Amoeba. J. Biophys. Biochem. Cytol. 4:U85. Electron Microscope Studies on Amoebae. Annals of Acad, of Sci. 78(2):UU8.  Pearse, E.A.G. 1953 Histochemistry, Theoretical and - J.A. Churchill Ltd., London.  Applied.  Pearse, E.A.G. 1961 - Histochemistry, Theoretical and J.A. Churchill Ltd., London,  Applied.  N.Y.  Percival, G.A. and Stewart, C.P. 1930 Brit. J. Dermatol. Syphilis U 2 : 2 l 5 . Peters, R.A. 19ii9 -  The study of enzymes i n relation to selective toxicity in animal tissue. Soc. for Expl. Biol. Symp. 3:36.  Peters, R.A-., Sinclair, H.M. and Thompson, R.H.S. 19U0 An analysis of the inhibition of pyruvate oxidation by arsenicals in relation to the enzyme theory of - • vesication. Biochem. J. ijp_:5l6. P i l l a i , R.K. 1938  Action of Arsenate i n Glycolysis.  Prescott,-D.M. -. 1959 Microtechniques i n Amoebae studies. Sci. 78(2).655.•  Biochem. J. 32:1961. Ann. N.Y. Acad.  Pollister, A.W. and Pollister, P.F. 1957The structure of-the Golgi apparatus. cytol. 6:85.  Intern.  Rev.  Ponder, E. 1961  The C e l l Membrane and i t s Properties. In: The Cell 2:1 Ed. Braciet and Mirsky. Acad. Press, N.Y. and London.  Prosser, C.L. 1950 Comparative Animal Physiol, p. 630-639. Saunders, Philadelphia, P.A. Rao, H.S.M. 1928  Studies on Soil Protozoa. I I . The function of mitochondria i n some s o i l protozoa. J. Indian Inst. Sci. 11A:117.  Rapkine, L. 1931  Ann. Physiol. Physicochim Bio. J_:382.  Rapkine, L. 193U In: Langeron, Precis de Microscopie, Masson, Paris. Rapkine, L., Chatton, E., Lwoff, A. 1931 Compt. Eend. Soc. Biol. 106:626. Raven, C. P. 1958 Morphogenesis - The analysis of Molluscan Development. Pergamon Press, Los Angeles. Ravin, H.A., 1951 Rebkun, L.I. 1956  Tsow, K.C. and Seligman, A.M.J. Biol. Chem. 191:8U3. Electron Microscopy of Basophilic Structure of some invertebrate oocytes. I. Periodic lamellae and the nuclear envelope. J. Biophys. Biochem. Cytol. 2:93.  Robertson, J. D. I960 The Molecular Structure and Contact Relationships of Cell Membranes i n Progress i n Biophysics and Biophysical Chemistry. 1GT:3U8. Rollo, I.M., 19U9  Williamson, J. and Lourie, E.M. Studies on the Chemotherapy of Melaminyl Arsenicals and Antimonials i n Laboratory Trypanosome Infections. Ann. Trop. Med. U3:19U.  RBsel von Rosenhof, A.J. 1755 Der kliene Proteus. Monatlichhorausgegebenen Insectin-Belistigung 3:622. Rosenthal, 1932  S.M. Action of arsenic upon the fixed sulphydryl groups of proteins. Pub. Health Rep. U7:2Ul.  Roughton, F.J.W. and Clark, A.M. 1950 Carbonic anhydrase. In: The Enzymes. Vol. 1. pp. 1250-1265. Edited by Sumner, J.B. and Myrback, K. Acad. Press, N.Y. Rudall, K.M. 1952  The proteins of the mammalian epidermis. i n protein chem. J£:253.  Advances  Saiki, H. and Dai, K. 1959 Studies on sulfhydryl groups during c e l l division of sea urchin egg. Exper. Cell Res. 16:2)4.. Schaeffer, A.A. 1937 Morphology behaviour and reproduction in Type A and Type B of chaos chaos Linnaeus, the giant multinuclear amoeba of Rflsel, Biol. Bull. 73:355. Schaeffer, A.A. 1937a Rediscovery of the giant amoeba of RBsel, Chaos chaos Linnaeus, 1767. Turtox News 15:114.Schaeffer, A.A. 1938a Biol. Bull. Woods' Hole 91:221;. Schaeffer, A.A. 1938b Significance of 3-daughter divisions i n the giant amoeba. Turtox News 16:96. Schaeffer, A.A. 19U6 Formation of the nuclear membrane and other mitotic events i n Chaos spp. (observations during l i f e ) . Biol. Bull Woods' Hole 91:221;. Seligman, A.M. et a l . 19U9 Ann. Surg. 130:333. Seligman, A.M., Tsou, K.C. and Barrnett, R.J. 195U A new histoehemical method for demonstration of protein bound sulfhydryl. J. Histo and Cytochem. £:U8U. Serra, J.A. 1946  Histoehemical tests for proteins. Stain Tech. 21:(l):5.  Serra, J.A. and Lopes^ A. 19l*5 Queiroz Donnees pour une cytophysiologie du nucleole. I. L'activite nucleolaire pendant l a croisance de 1'oocyte chez les Helicidal. Portug. Acta Biol. 1:51. Shearer, C. 1922  Proc. Roy. Soc. London B93:213.  Short, R.B. 19hS  Spindle Twisting i n the giant amoeba.  Science 102; 1+81;.  Short, R.B. 19U6  Observations on the giant amoeba, Amoeba carolinensis (Wilson,1900). Biol. Bull. 9 0 : 8 . Showacre, J.L. 1953 A c r i t i c a l study of Janus Green B coloration as a tool for characterizing mitochondria. J. Natl. Ca.  Inst. 13:829.  Singer, T. 19U3 Smith E.L. 1951 Spector, W.S.  Enzyme systems containing active sulfhydryl groups. The role of glutathione. Science 97:356. Proteolytic enzymes. In iThe Enzymes Vol. 1. pp. 793-872. (Ed. Sumner, J.B. and Myrback, K.) Acad. Press, N.Y. Handbook of Toxicology. Philadelphia.  Vol. 1,1956.  Stern, H. 1956  Science 12U;1292.  Stem, H. 1957  J. Biophys. Biochem. Cytol. U:l57.  Vol.2,1957.  Stocken, L.A. and Thompson, R.H.S. 19U6 Arsenic and t h i o l excretion i n animals after treatment of lewisite burns. Biochem. J. ItO:51;8. Stocken, L.A. and,Thompson, R.H.S. 1925. Reactions of British antilewisite with arsenic and . other metals i n living systems. Physiol. Rev. 5:63. Straub, F.B. and Feuer, G. 1950 Biochim. et Biophys. Swanson, C.P.  I960  Swift, H. 1956  Acta U:U55.  The c e l l . Foundation of modern Biology Series. Prentice-Hall, Inc., New Jersey.. The Fine Structure of annulate lamellae. J. Biophys. Biochem. Cytol. 2_(Suppl):331.  Szent-Gyorgyi, A. 1930 The action of arsenite on tissue respiration. Biochem. J. 21;: 1723.  Tatum, A.L. 1937  Some relationships between structure and function of organic arsenicals i n experimental chemotherapy. Science 85:5U. No.2193.  Taylor, B.W. 1959 Dynamics of spindle formation and i t s inhibition by chemicals. J . Biophys. Biochem. Cytol. 6:193. Thompson, R.H.S. 19i|8 Biochem. Soc. Symposia. 2:28. Torch, R. 1955  Torch, R. 1959 Vincent, H. 1937  Cytological studies on Pelomyxa carolinensis with special reference to the mitochondria. J . Protozool. 2:(No.3):167. The cytology of Pelomyxa. Annals of N.Y. Acad, of Sci. 78:(2):U07. Compt. Rend. 20Utl693.  Vincent, W.S. and Huxley, A.H. 195U Biol. Bull. 107:290. Voegtlin, C , Dyer, H.A. and Leonard, C.S. 1923 On the mechanism of action of arsenic upon protoplasm. Pub. Health Rep. 38:1882. Part I I . Voegtlin, C. 1925 The Pharmacology of arsphenamine (Salvarsan) and related arsenicals. Physiol. Rev. 5:63. Voegtlin, C., and Dyer, H.A. and Leonard, C.S. 1925 On the specificity of the so-called arsenic receptor i n the higher animals. J. Pharm. exp. Ther. 2U:297. Walker, E. 1925  A colour reaction for disulfides.  Biochem. j . 19:1085.  Watkins, W.M. and Wormall, A. 19U8 Nature 162:535. Watson, M.L. 1955 The nuclear envelope. Its structure and relationship to cytoplasmic membranes. J . Biophys. Biochem. 1:257. Weather a l l , M. 195U Drugs and porphyrin metabolism. Pharmacological Reviews 6:lU5.  Weisz, P.B. 19^0  The mitochondrial nature of the pigmented granules i n stentor and blepharisma. J. Morphol. 86;177.  Whittaker, V.P. 19U7 An experimental investigation of the "ring hypothesis" of arsenical toxicity. Biochem. J. 1*1:56. Wilbur, C.G. 19l*2 Wilbur, C.G. 19U5  The cytology of Pelomyxa carolinensis. Micr. Soc. 61:2277  Trans. Am.  Origin and function of the protoplasmic constituents i n Pelomyxa carolinensis. B i o l . Bull. 8 8 : 2 0 7 .  Williams, R.R. 19U7 J . Immunol. 55:161. Wilson, E.G. 1952 An Introduction to Scientific Research. Publications. Wilson, 1900  Pure Science  H.V. Notes on a species of Pelomyxa. Am. Naturalist. 31*:535.  Yao, T. 191*9  Quart. J. Microscop. S c i . 90:1*01.  Yasuzumi, G. 1959 Z. Zellforch. u. mikroscop. Anat. 59:110. Zimmerman, A.M. 1959 Effects of selected chemical agents on amoebae. Annals of N.Y. Acad. Sci. 7 8 ( 2 ) : 6 3 1 .  


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