Studies of an Amoebo-Flagellate, Naegleria Griiberi

5*3

Studies of an amoebo-flagellate, Naegleria griiberi

By M. D. PITTAM (From the Lister Institute of Preventive Medicine, Chelsea Bridge Road, London, S.W. i)

Summary The amoeboid and flagellate phases of Naegleria griiberi were examined by phase- contrast microscopy, cytochemical techniques, and conventional staining methods. Some electron micrographs were taken. Results showed that lipid was confined to the cytoplasmic globules, cell membrane, and mitochondria. Glycogen was absent, but a polysaccharide, probably a protein-carbohydrate complex, was generally distributed throughout the cytoplasm and was particularly abundant in the food vacuoles. Particular attention was paid to the mitotic figure, with the result that stage I of mitosis, in which RNA-protein and DNA-protein particles were dispersed throughout the nuclear area, is claimed as being a constant and essential occurrence in the initiation of mitosis. After stage I, the RNA and DNA took up and remained in sharply demarcated areas of the mitotic figure. No lipid or carbohydrate was present in the mitotic figure. During the transformation from amoeba to flagellate, some of the mitochondria concentrated at the point on the periphery of the organism where the flagella later emerged, and in the fully formed flagellate appeared as a dense cap at the bases of the flagella. Electron micrographs showed that the mitochondria had a double limiting membrane and an internal system of tubules similar to those described in Acanth- amoeba. As the flagellate reverted to the amoeboid stage the flagella were resorbed by the endoplasm.

Introduction THE history of research on Naegleria griiberi (Schardinger) Wilson, 1916, is one of slow progress in the early stages and then stagnation. From Schardinger (1899) to Pietschmann (1929) a detailed knowledge of its cytology was accumulated. Nothing of any importance was added until, nearly 20 and 30 years later respectively, Rafalko (1947) described the distribution of deoxy- ribonucleic acid in the nucleus, and Willmer (1956) described physiological factors in the transformation of the amoeba to the flagellate. There are signs of renewed interest in this organism, and the present paper is an attempt to give a co-ordinated picture of it by phase-contrast microscopy, electron microscopy, and cytochemistry. The taxonomical treatment of the organism has caused confusion and argument. It cannot be dealt with here, but three well-known names that have been used are Amoeba Umax, Vahlkampfia tachypodia, and Dimastig-

In recent studies the organism was isolated from farm soils (Singh, 1952), and from rivers (Chang, 1958). It has not been shown to be pathogenic. [Quart. J. micr. Sci., Vol. 104, pt. 4, pp. 513-29, 1963.] 514 Pittam—Studies of an amoebo-flagellate Methods Cultivation The strain used was obtained from the Culture Collection of Algae and Protozoa, Botany School, Cambridge, with the identification code 15:18—3 Naegleria gruberi Gi Be 1510. It was most conveniently maintained by plate culture at room temperature (200 to 250). As a bacterial food supply is essential, Klebsiellapneumoniae was used. Many types of dilute nutrient agar are suitable as a basic medium. A successful one is: agar 1-5 g, yeast extract (Marmite brand) o-i g, peptone (Difco) o-i g, distilled water 100 ml. This is, in essentials, the medium used by Balamuth and Rowe (1955) for their studies of Tetramitus rostratus. Another medium, which yields a more luxuriant growth, is agar 1-5 g, beef extract (Oxoid Lab-Lemco brand) o-i g, glucose o-i g, distilled water 100 ml. Subcultures were made with a platinum loop. The bacteria carried over from the previous plate were usually sufficient for the new culture. If not, a drop of Klebsiella suspension in 0-25% saline was spread on a new plate before inoculation. Unsuccessful attempts were made to grow N. gruberi in axenic culture. Penicillin, streptomycin, and terramycin were used singly or in combination to suppress the bacteria, and the bacteria-free amoebae were tested on many types of nutrient agar or in nutrient solutions. Heat-killed Klebsiella was used as an additional nutrient. In all cases the amoebae survived for 2 or 3 days, but they dwindled in size and died without dividing or encysting. N. gruberi grows in liquid medium of the same composition as that used for the plate culture, with the omission of the agar. Overgrowth of the amoeba by the bacteria must be prevented, and good aeration is essential. 25 ml of medium in a 250 ml conical flask provided the conditions for a reasonable growth of amoebae.

Examination When the amoeboid form settled in a drop of medium on a glass slide, it flattened into a thin sheet of protoplasm, and was an almost perfect object for phase-contrast and dark-ground microscopy. For cytochemical or cytological staining procedures, amoebae were allowed to settle on slides and were washed free of bacteria with two or three changes of medium. The slides were then plunged into fixative.

General cytochemical survey of the amoeboid form Unna and Tielemann (1918) appear to have been the only workers who attempted a cytochemical study of A. Umax (= N. gruberi). They used staining and extraction techniques based on the classic methods of analytical chemistry. They concluded that the nucleolus consisted of an acid protein (a globulin), and an unidentified basic protein; that the nuclear sap contained Pittam—Studies of an amoebo-flagellate 515 the same type of basic protein as the nucleolus, with a protamine; and that the nucleus contained neither nucleic acid nor nucleoprotein. The morphology of the amoeboid form revealed by positive phase contrast may be described briefly. Organisms which settled on slides were approximately 15 to 80 /A long by 10 to 40 /x wide, according to the condition and type of culture. The largest organisms were usually multinucleate 'giants', and were probably abnormal forms produced by cultural conditions.

contractile mitochondrion lipid vacuole , , globule food J vacuole

advancing pseudopodium of clear ectoplasm

projection endoplasm of uroid

FIG. 1. N. gruberi: amoeba in characteristic Umax shape. Diagrammatic.

The ectoplasm (fig. 1), which was sharply separated from the endoplasm, had no inclusions. The most prominent feature of the cell was the large nucleus ranging in diameter from 6 to 10 /x, which contained a conspicuous central nucleolus. There was a well-defined nuclear membrane. Amoebae with two or more nuclei were not uncommon, especially in cultures where the amoebae were closely packed. Occasionally two nucleoli were present in one nucleus. There were usually a number of spherical globules, 0-4 to 1 -o )x in diameter, lying on the outside of the nuclear membrane, where (in optical section) they looked like a circle of beads of various sizes. Similar globules were distributed throughout the endoplasm. A contractile vacuole apparatus was always present, consisting of a large vacuole and small contributory vacuoles. Systole and diastole were readily observed. When the amoeba was in the typical limax shape, the contractile vacuole tended to lie at the 'posterior' end, i.e. the end at which the uroid forms (fig. 1). It must be emphasized, however, that none of the inclusions had a persistent location within the endoplasm. As the protoplasm streamed and surged in the course of normal locomotion, so the vacuoles, nucleus, and other cell inclusions were swept backwards and forwards, and rolled over and 516 Pittam—Studies of an amoebo-flagellate Numerous food vacuoles were present. The mitochondria were short pale-grey rods, about 0-5 p by 1-5 ft to 2 p; they were very numerous, and evenly distributed in the endoplasm. Amino-acids. With Baker's modificiation (1956) of Millon's test for tyrosine, and Baker's modification (1947) of Sakaguchi's test for arginine, the entire amoeba stained a pale pink. The colour was too faint for detailed observations, but it appeared that arginine and tyrosine were evenly distributed throughout the organism except in the nucleus, where the nucleolus was positive and the nuclear sap negative. The modification of Sakaguchi's test introduced by McLeish and others (1957) did not give a more intense colour. Nucleic acids. Desoxyribonucleic acid (DNA) was studied by the Feulgen reaction. The results were, on the whole, in agreement with all previous studies, such as those of Rafalko (1947) and Singh (1952). DNA was confined to the nucleus. In the non-dividing ('resting') nucleus it lay immediately beneath the nuclear membrane in the form of irregular granules. These were probably fixation artifacts produced by powerful precipitants of nucleic acids such as acetic acid. They did not occur after a non-precipitant fixative like formalin, nor were they visible by phase-contrast microscopy. In mitosis the movement of DNA can arbitrarily be divided into 4 phases, described below (p. 520). Ribonucleic acid (RNA) was studied by methyl green / pyronin (Jordan and Baker, 1955). Control preparations were incubated at 37° for 2 h in ribonuclease (Armour) solution, made up at o-ooi% in glass-distilled water. The solution was brought to boiling-point when first made up, to destroy non-specific proteolytic activity. In non-dividing amoebae the nucleolus, endoplasm, and ectoplasm were stained bright red by pyronin. The nuclear sap was tinged with green. In the amoeba undergoing mitosis the endoplasm and ectoplasm and the polar masses of the mitotic figure were stained red. When the DNA concentrated at the equator of the mitotic figure it stained bright green. The red-stained nucleolar material was present throughout mitosis. After prior treatment by ribonuclease the amoebae were completely unstained by pyronin, whereas the staining of their DNA by methyl green was unaffected. It was concluded that RNA distribution coincided with the pyronin staining. Lipids were studied by the following reagents: (1) Sudan III and IV in acetone/alcohol (Pearse, i960); (2) Fettrot 7B in propylene glycol (Pearse, i960); (3) Nile blue (Cain, 1947); (4) acid haematein (Baker, 1946); (5) osmium tetroxide / ethyl gallate (Wigglesworth, 1957); (6) acetic anhydride + sulphuric acid (Pearse, i960); (7) mercuric chloride / Schiff (Cain, 1949 a, b)\ (8) cold acetone followed by Sudan black (Pearse, i960). Pittam—Studies of an amoebo-flagellate 517 The reagents revealed two distinct types of cytoplasmic inclusions which contained or consisted of lipids: the cytoplasmic globules and the mitochondria

(%• *)• Tests 6, 7, and 8 respectively for cerebrosides, cholesterol, and acetal phosphatides were negative. The routine lipid stains, e.g. Sudan III and IV, Sudan black, and Fettrot 7B coloured the cytoplasmic globules intensely, while the rest of the cell remained practically colourless. The mitochondria were not coloured by any of these reagents. Positive results with Sudan III and IV and with Fettrot 7B may perhaps suggest 'neutral' lipid (Pearse, i960). Cain's (1947) Nile blue technique was used to check this. The cytoplasmic globules in N. gruberi were immediately and intensely coloured blue with both the 1% and 0-02% solutions of Nile blue; all that can be inferred is the presence of free fatty acids and/or glycerophosphatides, but triglycerides might also be present. To detect glycerophosphatides, Baker's acid haematein with pyridine- extracted controls was used. A true positive result was obtained only with the mitochondria. The nucleolus occasionally stained black both in the test material and in the pyridine-extracted controls: this might be expected, since nucleoprotein stains with acid haematein (Baker, 1946). However, in the control material, many nucleoli had a peculiar washed-out appearance. In some cases they had one or two clear areas, which presumably might have arisen as the result of extraction of material. In a few cases the 'vacuolation' was so extreme that the remaining material looked like a deeply stained reticu- lum. It is uncertain whether such a result indicates the presence of glycero- phosphatide in the nucleolus. Pyridine is a strongly basic substance, and it is possible that it might react with the RNA of the nucleolus and extract it, thus producing the washed-out appearance. With osmium tetroxide / ethyl gallate the whole amoeba was coloured in shades of grey. The cytoplasmic globules were nearly black. The mitochondria and the nucleolus were well shown in pale grey. This result also raises the question of lipid in the nucleolus. Wigglesworth (1957) states that in tissues, reaction with protein can be ignored, and the technique used as a test for unsaturated fatty acids. Support for this conclusion comes from Bahr (1954). In a study of the reactions of osmium tetroxide with solutions of biological materials, Bahr found that carbohydrate and nucleic acids are inert towards it; that the reaction with the ethylenic linkages of lipids is exceptionally strong; and that amino- acids that contain •—SH or —S— react vigorously, as also do amino- acids with basic groups which are in a terminal position of a peptide chain and are not salt-linked. Bearing in mind that in Naegleria the mitochondria and the nucleolus are blackened to the same extent, there are at least three possible interpretations. (1) Lipid is present in the mitochondria and the nucleolus and is responsible for the binding of osmium. (2) Lipid is present in the 518 Pittam—Studies of an amoebo-fiagellate mitochondria only. The staining seen in the nucleolus is caused by basic groups (or groups containing sulphur in the requisite form) in the poly- peptide chains. (3) Lipid, terminal basic groups of amino-acids, —SH and —S—, are present in the mitochondria and the nucleolus. As it proved impossible to demonstrate lipid in the nucleolus by any of the standard stains, the second interpretation is perhaps the most likely. Carbohydrate. For histochemical purposes Pearse (i960) divides the carbohydrates into polysaccharides (the glycogen group), acid mucopolysaccharides, neutral mucopolysaccharides, mucoprotein and glycoprotein, and glycolipid. The periodic acid / Schiff (PAS) reaction coloured the food vacuoles bright red and the cytoplasm pale pink. A similar result was obtained when preparations were incubated in a solution made by diluting saliva to twice its volume with sterile glass-distilled water, centrifuging to clear of mucus, and then staining with PAS. The failure of the salivary enzymes to remove the red- staining material indicated that the glycogen group of polysaccharides was absent; similarly the preparations treated with iodine became pale lemon- yellow with no sign of the deep reddish-brown characteristic of glycogen. The PAS reaction was unchanged in amoebae from which lipid had been extracted. Three tests were used for the acid mucopolysaccharides: metachromasia of toluidine blue (standard method) (Pearse, i960); methylene blue extinction (MBE), (Dempsey and Singer, 1946; Pearse, i960); alcian blue (Steedman, 1950). With toluidine blue the amoebae stained purple. No red metachromatic colour was seen. In the cysts, however, a layer of the wall was coloured red, indicating acid mucopolysaccharide in that structure. The methylene blue extinction (MBE) test was used to check the results of the toluidine blue staining. Solutions at pH from 2-62 to 4-66 were used for untreated preparations of amoebae and for preparations after incubation in ribonuclease solution (p. 516) and subsequent washing. Ribonuclease was applied because RNA considerably increases basiphilia and so affects the MBE. In both sets, amoebae at pH 4-66 were stained pale blue. At pH 3-62 the amoebae extracted with RNase were unstained, whereas in the unex- tracted amoebae there were traces of blue staining, particularly in the nucleolus. As the nucleolus is known to contain RNA (p. 516), it was concluded that the staining in the amoebae at pH 3 -62 was due to RNA, and that as the MBE of Naegleria was not below pH 4 when RNA was removed it was unlikely that acid mucopolysaccharide was present. Finally, alcian blue (Steedman, 1950) stained the entire amoeba a uniform blue colour, and not the bright green or blue-green indicative of acid mucopolysaccharide. The position in the amoeboid form of Naegleria as regards carbohydrates, therefore, was as follows. In the cytoplasm (including the food vacuoles) there was a substance, or group of substances, which gave characteristic staining reactions. These were the orthochromatic purple-blue of toluidine blue; the pale pink (cytoplasm) or the bright red (food vacuoles) of the PAS Pittam—Studies of an amoebo-flagellate 519 test; and the failure to bind methylene blue below pH 4 if RNA is removed. These reactions persisted when glycogen, RNA, and lipid were extracted. Acid mucopolysaccharide was absent. The substance or substances giving these reactions can, therefore, only be neutral mucopolysaccharide, mucoprotein, or glycoprotein. These, unfortunately, cannot be distinguished by cytochemical means. Vital dyeing Some of the early work (1778 to 1900) on protozoa with vital dyes is sum- marized by Baker (1958). The most recent research, apart from the special case of enzyme studies, is that of Morisita (1939), who used 71 dyes on Tricho- monas foetus. In the present work attempts were made to colour the nucleolus of Nae- gleria and watch its behaviour during mitosis (brilliant cresyl blue); to gain further information on the food vacuoles and lipid globules (neutral red and brilliant cresyl blue); and to determine the distribution of mitochondria during the transformation from amoeba to flagellate (Janus green B and triphenyl- tetrazolium chloride). The dyes were used at strengths of o-oi% to o-oooi% (w/v) in solutions approximately isotonic with the culture medium. With tryphenyl-tetrazolium chloride, a succinate substrate was used. The living organisms were placed in a drop of the dye solution on a slide, covered, and examined at intervals of about 15 min, 2 to 4 h, and 24 h. Brilliant cresyl blue stained neither the nucleus nor any of the cytoplasmic components. Neutral red stained the lipid globules and the food vacuoles, but not the contractile vacuole system. The globules and the food vacuoles were red, indicating that the colourable matter had an acid pH. According to Marston (1923), proteolytic enzymes within the cell can be demonstrated by azine dyes. Accordingly, the transformation from flagellate to amoeba was studied in organisms immersed in o-ooi % neutral red. It was thought that when the flagella were absorbed into the cytoplasm in the final stage of the transformation, any proteolytic activity might result in a con- centration of the dye. Although the transformation was quite normal, no neutral red staining occurred in the area of writhing cytoplasm (p. 526) where the flagella had been withdrawn. The lipid globules and the food vacuoles in both flagellate and amoeboid phases were coloured red. Janus green B, used at o-oooi%, gave variable results. Sometimes the mitochondria were tinged with pale green, at other times they were colourless. The results with triphenyl-tetrazolium chloride were similar: sometimes the mitochondria were coloured pale pink, sometimes they remained un- coloured. No coloration of the mitochondria took place in less than 18 h. In every case, however, the picture was confused by the readiness with which the red formazan was produced in the lipid globules. As the smallest of these were of about the same size as mitochondria, critical examination was neces- sary to distinguish them. 520 Pittam—Studies of an amoebo-flagellate

Cytology and cytochemistry of mitosis The nuclear division of Naegleria has attracted much attention, probably because of the conspicuous nature of the nucleus and the mode of division of the nucleolus. As a result, the stages of mitosis are fairly well known, though far from understood. The following account is based on phase-contrast microscopy of living amoebae, conventional cytological fixation and staining, and standard cytochemical techniques. These observations were correlated as closely as possible. Amoebae were watched under phase until the nucleus of an individual amoeba was seen in the first stage of mitosis; the position of the amoeba was established by stage Verniers, and the preparation fixed. Amoebae in the second, third, fourth, and fifth stages of the division were similarly treated. In this way comparisons between phase-contrast, cytochemical, and cytological preparations of the same mitotic phase were made. Several preparations of each stage of mitosis were made. One set was stained by Jordan and Baker's (1955) methyl green / pyronin technique for RNA. A control set was incubated in a solution of crystalline ribonuclease (Armour) (o-ooi % in distilled water) for 1 h at 370, and then stained in methyl green / pyronin. Another set was carried through the Feulgen technique, and control preparations were used in which acid hydrolysis was omitted. These sets gave the distribution of nucleic acids throughout mitosis. A third set was treated as follows. (1) Fixative washed out. (2) Mann's stain; dehydrated, mounted in xylene. (3) Nucleus of the selected amoeba on each slide drawn in colour. (4) Preparation rehydrated and washed in running water to remove stain. (5) Incubated in a solution of RNase at 370 for 1 h; controls were incubated in the solvent alone. (6) Washed, stained in Mann's stain, dehydrated, and mounted in xylene. (7) Nucleus of the selected amoeba on each slide drawn in colour. (8) Stages 4 to 7 repeated, except that in (5) the preparations were not incubated in ribonuclease, but for 15 min at 20° in trypsin (Armour) made up at o-i% in Sorensen phosphate buffer pH 8. (9) Stages 4 to 7 repeated except that in (5) the preparations were incubated in pepsin (Armour) for 30 min at 200 in 0-02 N HC1 at pH i-6. Controls were run in (8) and (9) as in (5). These preparations gave information on the protein matrix of the mitotic figure (pp. 522, 523). A fourth set was carried through Alfert and Geschwind's (1953) technique for the demonstration of basic protein, a fifth through the PAS reaction for carbohydrate, and a sixth through the Sudan black method for lipid. The results are best described by relating them to 4 arbitrary divisions in the mitotic sequence. Most writers give these the conventional metazoan names of prophase, anaphase, metaphase, and telophase. As these names are linked with chromosome configurations and movements, of which little is known in Naegleria, the terms stage I, II, III, IV, are substituted for them in this paper (fig. 2). Pittam—Studies of an amoebo-flagellate 521 Before stage I, the nucleus is in its non-dividing or 'resting' state. The nucleolus contains RNA, some basic protein, and an unidentified 'residual' protein which has a strong affinity for basic dyes. Stage I is characterized by an enlargement of the nucleus and disintegration of the nucleolus. When the living nucleus is examined by phase contrast it appears as a disk of about the same refractive index as the surrounding cytoplasm. It is exceedingly difficult to see, and unless one is familiar with the

non- stage stage stage dividing I II (= resting) nucleus

• sites occupied by RNA EU sites occupied by DNA ' FIG. 2. Diagram of the nucleus of N. gruberi in mitosis. phases of mitosis in the living amoeba it is easy to imagine that the nucleus has disappeared entirely. The nucleolar RNA is intermingled with the DNA of the nuclear sap throughout the nuclear area. There is a well-defined nuclear membrane. Stage II. Spindle fibres appear among the mixed nuclear material. What appears to be a re-aggregation of nucleolar RNA and nucleolar protein produces the typical squat dumbell (fig. 2, stage II). Sometimes the mass of the nucleolar material obscures most of the spindle fibres. During this stage the DNA migrates to the equatorial region of the dumbell figure, forming a band which sometimes has the appearance of a considerable number of irregular elongated bodies. These stain intensely with the methyl green of Jordan and Baker's (1955) method, and with the SchifFs reagent in Feulgen's reaction. Stage III. The mitotic figure elongates and the spindle fibres are stretched out. Two large masses now form, one at each pole of the mitotic figure. The masses are composed of RNA and protein, and were called 'polar masses' by Rafalko (1947). Sometimes the polar masses are sharply separated from each other; sometimes they are connected by an irregular wisp of material, and sometimes by a thick column (fig. 2). The material connecting the polar masses is composed of RNA and protein. The DNA bodies separate into two groups. One group moves on (or is moved by) the spindle fibres towards one of the polar masses, whilst the other group moves in a similar manner to the opposite mass. The nuclear membrane breaks down in the equatorial region but remains intact round the polar masses. 522 Pittam—Studies of an amoebo-flagellate Stage IV. The nucleus now enters the final stage of mitosis. In this stage it reaches its greatest elongation. The main elements of the mitotic figure consist of polar masses of RNA, basic protein, and acidic protein; a compact group of bodies containing DNA adjacent to each polar mass; and a long slender strand of nucleolar RNA and protein stretching between the DNA bodies. The nuclear membrane persists round the polar areas. The slender strand parts in the middle and appears to retract, forming a compact body next to the DNA bodies. Separate daughter nuclei are now present. Division of the cytoplasm follows within seconds or, at the most, within a minute or two. For a few minutes the daughter nuclei remain with DNA in the centre, and RNA at the periphery, of the nucleus, i.e. a reversal of the normal condition. It is possible that this corresponds to what happens in mammalian nerve cells (Hyde'n, 1943), where the foundation of a nucleolus is preceded by the aggregation of DNA-protein particles in a ground substance rich in basic protein. On the other hand, Alfert and Geschwind's test shows very little basic protein at this stage in the Naegleria nucleus. About 10 min after separation of the daughter nuclei, and after division of the amoeba, the nuclei in the daughter amoebae have assumed their normal non-dividing ('resting') appearance, i.e. there is a large nucleolus lying centrally in the clear nuclear sap, and the entire nucleus is surrounded by a well-defined membrane. It cannot be emphasized too strongly that mitosis as seen by phase contrast conveys quite a different impression from that studied in fixed and stained material. The absence of dyes tends to draw attention to the dynamic nature of this system. As these nuclear changes are taking place the body of the amoeba follows a regular pattern of movement. In the early stages (I and II) of mitosis, locomotion is normal, and the dividing nucleus is rolled backwards and forwards in the surging and streaming cytoplasm. As stage III is ap- proached, amoeboid movement slows down, and the elongated mitotic figure tends to become fixed in the long axis of the amoeba. In stage IV the change in the amoeba is dramatic, and events proceed in rapid succession. All amoeboid movement ceases; the amoeba flattens into a thin, delicate sheet of protoplasm; for a moment it is motionless; then tiny pseudopodia are rapidly protruded and withdrawn at each end of the organism; a waist appears at the centre of the organism; the nuclear figure parts to give daughter nuclei; the waist constricts, and the two halves of the amoeba draw apart, usually pulling out a long slender strand of cytoplasm between them. The preceding description, and the facts recorded above, indicate that RNA and DNA remain clearly demarcated throughout mitosis. Carbohydrate and lipids are absent from the nucleus (except the lipid of the nuclear membrane) during mitosis. Basic protein is present in the nucleolar figure, and also in the nuclear sap, if an exceedingly pale green stain can be taken as a positive result with Alfert and Geschwind's test. One would expect both RNA and DNA to be associated with basic protein, but some of the protein may be affected by the Pittam—Studies of an amoebo-flagellate 523 drastic extraction with trichloracetic acid which Alfert and Geschwind's test entails. A residual 'acid' protein is demonstrable by basic dyes when basic protein has been removed by trypsin or by mild acid hydrolysis. The validity of such demonstrations rests on the assumption that short hydrolysis with trypsin, or mild acid hydrolysis, will remove basic protein before the remaining protein is affected. It is obvious that the cytochemical analysis of a complex protein body such as the nucleolus is unsatisfactory. A satisfactory analysis must await either new cytochemical methods or the separation of the nucleoli from large num- bers of amoebae and their biochemical examination. Little has been written about stage I of mitosis. This is not equivalent to the 'prophase' of the majority of writers on Naegleria, who have either missed or completely misunderstood stage I. Glaser (1912) figured it but made no comment on it. Zuluetta (1917) gave good figures of it but was so mystified by it, and by the seemingly endless variety of mitotic figures which Naegleria can produce, that he made this stage I the starting point for part (the 'proto- dieresis') of his complicated double system of mitosis. There was no evidence for such a system of mitosis in the strain of Naegleria examined. The place, though not the explanation, of stage I in mitosis is most evident from phase- contrast observations. It is clearly a constant occurrence in the initiation of mitosis, and follows a constant course in which the nucleolus becomes fainter in appearance and blends with the nuclear sap, while the nucleus as a whole increases in size. In one case where measurement was possible, the resting nucleus was about 6 fi in diameter, whereas the swollen nucleus was about 10 ix in diameter. Stage I may be connected with spindle formation. The protein of the nucleus, if utilized for this, would have to be in solution; hence, presumably, the disintegration of the nucleolus. This disintegration might be a reversible dissociation (Haurowitz, 1950) which would account for the observed decrease in viscosity (p. 521) and swelling of the nucleus. It is therefore possible that in the semi-fluid content of the nucleus there now follows a process analogous to the formation of fibrin in the blood (Heilbrunn, 1956) and to the end-to-end linkage of certain of the peptide chains by enzymes (Ferry, 1949). These linked chains might then aggregate by lateral association to produce the spindle fibres (Mazia, 1955). However, spindle fibres are not visible in the majority of preparations. This raises the question, are spindle fibres artifacts? Mazia (1955) dealt with this in detail. He extracted the mitotic spindles from the eggs of Strongylo- centrotus purpuratus and, after critical tests, concluded that the fibres were not artifacts. In phase-contrast studies of mitosis in Naegleria a well- developed fibrous spindle (though not of the metazoan type) may appear in one amoeba, and not in an adjacent amoeba. That spindle fibres are not visible in every case does not necessarily mean that they are not formed. For example, they may form at the end of stage I, and then in stage II become 524 Pittam—Studies of an amoebo-flagellate fused with the nucleolar mass. My belief is that they always form, but, like every other part of the mitotic figure, they are subject to considerable variation in mass and in duration. Three other morphological features, the 'polar caps', interzonal body, and centrioles, are controversial. I regard 'polar caps' as spurious. Ford (1912) was the first person to name and describe these structures. He claimed that they were stained only by Dobell's alcoholic iron-haematein. More recently Rafalko (1947), Singh (1952), and Chang (1958) described them after staining with aqueous iron- haematoxylin, or with light green. I have seen them in aqueous iron-haematoxylin preparations, and do not believe that they are separate structures worthy of a special name, but that they are, as Pietschmann (1929) observed, the ends of the spindle protruding beyond the polar mass. The term 'interzonal body' was coined by Rafalko (1947), though Glaser (1912) had called what was evidently the same structure der Zwischenkorper. It applies to the nucleolar material which frequently occupies the centre of the mitotic figure in stages III and IV. Rafalko states that 'as anaphase progresses, particles of the polar masses appear to migrate along the spindle fibres to the middle to form a so-called interzonal body often mistaken for true chromatin'. I found no evidence of this migration. It seems that the interzonal body is a normal consequence of mitosis in a nucleus where there is a large amount of nucleolar material. Sometimes the nucleolus divides cleanly, producing large polar masses, easily visible spindle fibres, and no interzonal body. At other times the division of the nucleolar material is not clear-cut, and, as the nucleus elongates, nucleolar material, often in coarsely granular form in fixed preparations, stretches between the polar masses to produce the inter-zonal body. Perhaps the most inadequate exposition of the origin of the interzonal body comes from Chang (1958). He says 'When the karyosome divides into two in the prophase, a piece drops out . . .'. This piece then becomes the interzonal body. The reversible transformation from amoeba to flagellate Pietschmann's (1929) account of this transformation is particularly good. The present account adds some new facts on the resorption of the flagella and the movement of the mitochondria. As in the study of mitosis, observations on living organisms were correlated with those on fixed and stained specimens. When amoebae were placed in a drop of distilled water as a stimulant to transformation, the following events were observed by phase-contrast microscopy. An amoeba which had been moving in the usual manner gradually came to a standstill and assumed a spherical shape. A few small pseudopodia were occasionally thrust out, but pseudopodial activity soon ceased. The amoeba, though spherical, was still attached to the substratum, and the nucleus occupied a central position within the amoeba. The contractile Pittam—Studies of an amoebo-fiagellate 525 vacuole, food vacuoles, lipid globules, and mitochondria were present. They did not occupy fixed positions in the cell, though many of the mitochondria came to do so. A pair of flagella suddenly appeared, though sometimes one flagellum was extruded before the other. When both flagella had newly emerged they beat slowly, but with rapidly increasing tempo, causing the amoeba to vibrate. This vibration soon changed to a slow rotation: a half- turn clockwise, then a half-turn counterclockwise. Since the amoebo- flagellate was still attached to the slide, its protoplasm twisted round the point of attachment. Finally, the half-turns gave place to a rapid spinning, which was clockwise or anticlockwise, accompanied by an increase in the rate of the flagellar beat. The spinning, lasting anything from a few seconds to a minute or two, broke the attachment to the slide, and the organism swam away as a spindle- or torpedo-shaped flagellate. Usually it had one nucleus and two flagella; this form was produced by a uninucleate amoeba. Multi- nucleate amoebae, usually produced in old or very crowded cultures, were also capable of the transformation, forming flagellates with 2, 3,4, or even 5 nuclei. In these, each nucleus was not necessarily associated with a pair of flagella; a flagellate with two nuclei may have either 3 or 4 flagella, and one with 5 nuclei may have 8 flagella. The free-swimming flagellate stage lasted for 30 min to 24 h; then the flagellate lost its rigidity and settled on the slide again, rotating in a clockwise or anticlockwise direction as it did so. As it settled, pseudopodia were thrust out at random. Occasionally pseudopodia were protruded a few seconds before the amoeba-flagellate settled. A striking feature of the nearly settled amoeba-flagellate was the immobility of the nucleus. Whereas in the normal amoeboid phase the nucleus was moved about by the surging of the cytoplasm (p. 515), now it was held stationary, close to the point where the flagella emerged from the periphery of the organism. The flagella did not change in length, or in rate of beat. As the random extrusion of pseudopodia gradually changed into the typical Umax action of a single broad pseudopodium, the amoebo-flagellate flattened on to the slide, and the flagellaan d nucleus became clearly visible. The thickened bases of the flagella appeared to be attached to the nuclear membrane, which was drawn out towards the point of emergence of the flagelia. The existence of a physical connexion between nucleus and flagella was indicated by the vibration of the nucleus within the cytoplasm in rhythm with the lashing of the flagella. The amoeba-flagellate stage may be quite protracted, though it usually took less than 1 h. The last phase of the transformation back to the amoeba was marked by a faltering in the rapid beat of the flagella. For a fraction of a second a flagellum remained quite motionless, then resumed its beating; alternating in this manner for several minutes, until finally the flagellum became motionless, bent, and was rapidly withdrawn into the organism. The flagellum was invisible within the cytoplasm, but its presence was marked by a snake-like writhing. While this was happening the second flagellum was still beating normally, but soon stopped; for a second it was held out 526 Pittam—Studies of an amoebo-flagellate motionless, then bent and was withdrawn into the organism. The writhing in the cytoplasm was now particularly clear. As the flagellawer e withdrawn, the nucleus moved away from the periphery and down the long axis of the organism. As it moved it was jerked from side to side by the writhing of the flagella—yet another point in favour of a physical connexion between nucleus and flagella. During the period of resorption of the flagella the amoeba was motionless. Gradually the writhing in the cytoplasm ceased; the nucleus moved freely as the cytoplasm resumed its normal streaming; and the organism reverted to a characteristic limax amoeba, moving on its course by a single bulging pseudopodium. The nucleolus underwent no detectable change during any of the events concerned with the reversible transformation, and it did not seem to be involved in the production of flagella. This description is in disagreement with that of Willmer (1956) in the following respects. Flagella are not produced at a 'posterior' end, nor is their production associated with the uroid. In most cases the amoeba is spherical, or nearly so, when the flagella are produced. The nucleus and contractile vacuole are not in any fixed spatial relationship to each other, so that the terms 'anterior' and 'posterior' cannot be applied. Willmer states that 'More often than not this amoeba does not produce a single flagellum but a cluster of three or four, most commonly the latter'. Flagellates with more than two flagella do occur (p. 525), but not usually; the biflagellate is the typical form. Further, if the above statement implies that flagellates with a single flagellum occur, then that implication must be denied. Willmer also states that first the flagella appear from the 'posterior' end, then the amoeba rounds up, then the spinning commences. The sequence is that the amoeba becomes stationary, rounds up, produces flagella and then spins. Willmer's description seems to confuse stages in the secondary change from flagellate to amoeba with the primary change from amoeba to flagellate.

The relationship between flagella and mitochondria In phase-contrast preparations a dark patch appeared at the periphery of the rounded amoeba as the amoeba changed to the flagellate form (fig. 3). When the flagella emerged, this dark patch surrounded the point of their emergence, and remained at the base of the flagella in the fully formed flagellate. The rounded form, and rapid movement of the living flagellate, made it impossible to resolve the individual mitochondria; but they can be resolved in stained preparations. All stages of the transformation of the amoeba to the flagellate were studied with Baker's acid haematein. In the stained flagellate the mitochondrial cap at the base of the flagella (fig. 3) was the characteristic feature. When the flagellate settled down and reverted to the amoeboid form, the mitochondria dispersed throughout the endoplasm. Pittam—Studies of an amoebo-flagellate 527 In one instance, where the change from flagellate to amoeba was being watched by phase contrast, it was noticed that the mitochondria in the cytoplasm occupied by the writhing withdrawn flagella were larger than normal, and were globular instead of rod-shaped. It is possible that the mitochondrial movements are connected with the synthesis of flagellar protein, and with the energy requirements of the flagella.

mitochondrion

Normal amoeba nucleus

Amoeboid form unchanged, but mitochondria concentrating at periphery

Rounded immediate pre- flagellate form. Mitochondrial "cap" present; flagella protruding

Normal flagellate form.Nucleus near flagellate end of organism; dense "cap" of mitochondria round bases of flagella

FlG. 3. Schematic representation of the movement of mitochondria in the transformation from amoeba to flagellate.

The time relationship of the reversible transformation The variability of the time-course of transformation is evident from the fact that some amoebae transformed to flagellates in a matter of minutes; others in the same preparation took hours. The age and condition of the culture were important factors in this metamorphosis. However, given a healthy young culture not more than 3 days old, and given that the events proceeded undisturbed in distilled water, most of the amoebae responded to this stimulus within 3 h. Once the amoeboid movement ceased and the organism rounded up, it took 15 to 30 min to attain the full flagellate state. 528 Pittatn—Studies of an amoebo-flagellate The free-swimming flagellate phase did not usually last longer than 6 h. The transformation back to the amoeboid phase took 10 to 20 min. The relationship between nucleus and flagella This relationship was well investigated by Pietschmann (1929), and the studies recorded here attest the accuracy of her work. The direct physical connexion between the nucleus and flagella is obvious. The nature of the connexion is still unknown. The light microscope showed, particularly at the beginning and at the ends of the flagellate phase, the bulbous bases of the flagella attached, or closely apposed, to the nuclear membrane. These bases appeared as black dots in iron-haematoxylin preparations. There is little doubt that these objects are the basal granules (blepharoplasts) of earlier workers. However, in view of what the electron microscope has revealed about the nature and size of the base of the flagellum and the basal granule complex in many protozoa, judgement must be reserved on this point in Naegleria. The problem of the relationship between nucleus and flagella, indeed of the whole transformation from amoeba to flagellate, requires a thorough study with the electron microscope. Some electron micrographs were taken by personnel of the Wheatstone Laboratory, King's College, London. One micrograph showed an elongate body (part of a flagellum ?) stretching from the nuclear membrane to a point near the periphery of the cell. There was an indentation of the cell membrane and adjacent cytoplasm opposite this point. There was also an indentation of the nuclear membrane where the base of the elongate body rested. It is likely that this elongate body is the same structure as the cylindrical object which is seen in iron-haematoxyl preparations on, or even within, the nuclear membrane, and which is the rudiment of a flagellum. Another micrograph of the series showed the mitochondria concentrating near the periphery in the immediate pre-flagellate stage. The mitochondria appeared to have a double membrane and an internal system of tubules, similar to those figured for Amoeba proteus by Mercer (1959) and for Acanthamoeba by Vickerman (i960). Cytochemistry of the flagellate stage All the tests listed on pp. 516-20 were also carried out on the flagellate stage. The results were essentially similar to those for the amoeboid form. All the components of the amoeboid form were present-in the flagellate. No nucleic acid was detected in the rudiments of the flagella.

I have pleasure in acknowledging my debt to Dr. Muriel Robertson, my supervisor, who brought the problems associated with Naegleria to my attention, and was always ready to draw from her immense experience in order to advise and discuss. The work, which is part of a thesis submitted for the degree of doctor of philosophy of the University of London, was financed by an Agricultural Pittam—Studies of an amoebo-flagellate 529 Research Council grant, and was carried out at the Lister Institute of Pre- ventive Medicine, London, S.W.i. The electron microscopy was carried out at the Wheatstone Laboratory, King's College, London, by kind permission of Prof. Sir John T. Randall, F.R.S.

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