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)

SummaryThe 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 thecytoplasmic globules, cell membrane, and mitochondria. Glycogen was absent, but apolysaccharide, probably a protein-carbohydrate complex, was generally distributedthroughout the cytoplasm and was particularly abundant in the food vacuoles.Particular attention was paid to the mitotic figure, with the result that stage I ofmitosis, in which RNA-protein and DNA-protein particles were dispersed throughoutthe nuclear area, is claimed as being a constant and essential occurrence in the initia-tion of mitosis. After stage I, the RNA and DNA took up and remained in sharplydemarcated areas of the mitotic figure. No lipid or carbohydrate was present in themitotic figure.

During the transformation from amoeba to flagellate, some of the mitochondriaconcentrated at the point on the periphery of the organism where the flagella lateremerged, and in the fully formed flagellate appeared as a dense cap at the bases of theflagella. Electron micrographs showed that the mitochondria had a double limitingmembrane 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 theendoplasm.

Introduction

T H E history of research on Naegleria griiberi (Schardinger) Wilson, 1916, isone of slow progress in the early stages and then stagnation. From Schardinger(1899) to Pietschmann (1929) a detailed knowledge of its cytology wasaccumulated. Nothing of any importance was added until, nearly 20 and 30years later respectively, Rafalko (1947) described the distribution of deoxy-ribonucleic acid in the nucleus, and Willmer (1956) described physiologicalfactors in the transformation of the amoeba to the flagellate.

There are signs of renewed interest in this organism, and the present paperis an attempt to give a co-ordinated picture of it by phase-contrast micro-scopy, electron microscopy, and cytochemistry.

The taxonomical treatment of the organism has caused confusion andargument. It cannot be dealt with here, but three well-known names thathave 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

MethodsCultivation

The strain used was obtained from the Culture Collection of Algae andProtozoa, Botany School, Cambridge, with the identification code 15:18—3Naegleria 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 wasused. Many types of dilute nutrient agar are suitable as a basic medium. Asuccessful 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 usedby Balamuth and Rowe (1955) for their studies of Tetramitus rostratus.

Another medium, which yields a more luxuriant growth, is agar 1-5 g, beefextract (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 overfrom 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 platebefore inoculation.

Unsuccessful attempts were made to grow N. gruberi in axenic culture.Penicillin, streptomycin, and terramycin were used singly or in combinationto suppress the bacteria, and the bacteria-free amoebae were tested on manytypes of nutrient agar or in nutrient solutions. Heat-killed Klebsiella was usedas 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 usedfor the plate culture, with the omission of the agar. Overgrowth of theamoeba 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 areasonable growth of amoebae.

ExaminationWhen 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 forphase-contrast and dark-ground microscopy. For cytochemical or cytologicalstaining procedures, amoebae were allowed to settle on slides and werewashed free of bacteria with two or three changes of medium. The slideswere then plunged into fixative.

General cytochemical survey of the amoeboid formUnna and Tielemann (1918) appear to have been the only workers who

attempted a cytochemical study of A. Umax ( = N. gruberi). They usedstaining and extraction techniques based on the classic methods of analyticalchemistry. They concluded that the nucleolus consisted of an acid protein (aglobulin), 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 thenucleus contained neither nucleic acid nor nucleoprotein.

The morphology of the amoeboid form revealed by positive phase contrastmay be described briefly.

Organisms which settled on slides were approximately 15 to 80 /A long by10 to 40 /x wide, according to the condition and type of culture. The largestorganisms were usually multinucleate 'giants', and were probably abnormalforms produced by cultural conditions.

contractile mitochondrion lipidvacuole , , globule

food J

vacuole

advancingpseudopodiumof clearectoplasm

projection endoplasmof 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 indiameter from 6 to 10 /x, which contained a conspicuous central nucleolus.There was a well-defined nuclear membrane. Amoebae with two or morenuclei were not uncommon, especially in cultures where the amoebae wereclosely packed. Occasionally two nucleoli were present in one nucleus. Therewere usually a number of spherical globules, 0-4 to 1 -o )x in diameter, lying onthe outside of the nuclear membrane, where (in optical section) they lookedlike a circle of beads of various sizes. Similar globules were distributedthroughout the endoplasm.

A contractile vacuole apparatus was always present, consisting of a largevacuole and small contributory vacuoles. Systole and diastole were readilyobserved. When the amoeba was in the typical limax shape, the contractilevacuole tended to lie at the 'posterior' end, i.e. the end at which the uroidforms (fig. 1). It must be emphasized, however, that none of the inclusionshad a persistent location within the endoplasm. As the protoplasm streamedand surged in the course of normal locomotion, so the vacuoles, nucleus, andother cell inclusions were swept backwards and forwards, and rolled over and


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 tyro-

sine, and Baker's modification (1947) of Sakaguchi's test for arginine, theentire amoeba stained a pale pink. The colour was too faint for detailedobservations, but it appeared that arginine and tyrosine were evenly distributedthroughout the organism except in the nucleus, where the nucleolus waspositive 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 Feulgenreaction. The results were, on the whole, in agreement with all previousstudies, such as those of Rafalko (1947) and Singh (1952). DNA was confinedto the nucleus. In the non-dividing ('resting') nucleus it lay immediatelybeneath the nuclear membrane in the form of irregular granules. These wereprobably fixation artifacts produced by powerful precipitants of nucleic acidssuch as acetic acid. They did not occur after a non-precipitant fixative likeformalin, 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 (Jordanand Baker, 1955). Control preparations were incubated at 37° for 2 h inribonuclease (Armour) solution, made up at o-ooi% in glass-distilled water.The solution was brought to boiling-point when first made up, to destroynon-specific proteolytic activity.

In non-dividing amoebae the nucleolus, endoplasm, and ectoplasm werestained bright red by pyronin. The nuclear sap was tinged with green. In theamoeba undergoing mitosis the endoplasm and ectoplasm and the polarmasses of the mitotic figure were stained red. When the DNA concentratedat 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 un-

stained by pyronin, whereas the staining of their DNA by methyl green wasunaffected. It was concluded that RNA distribution coincided with the pyroninstaining.

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).


The reagents revealed two distinct types of cytoplasmic inclusions whichcontained or consisted of lipids: the cytoplasmic globules and the mitochondria(%• *)•

Tests 6, 7, and 8 respectively for cerebrosides, cholesterol, and acetalphosphatides were negative.

The routine lipid stains, e.g. Sudan III and IV, Sudan black, and Fettrot 7Bcoloured the cytoplasmic globules intensely, while the rest of the cell remainedpractically colourless. The mitochondria were not coloured by any of thesereagents.

Positive results with Sudan III and IV and with Fettrot 7B may perhapssuggest 'neutral' lipid (Pearse, i960).

Cain's (1947) Nile blue technique was used to check this. The cytoplasmicglobules in N. gruberi were immediately and intensely coloured blue withboth the 1% and 0-02% solutions of Nile blue; all that can be inferred is thepresence of free fatty acids and/or glycerophosphatides, but triglyceridesmight also be present.

To detect glycerophosphatides, Baker's acid haematein with pyridine-extracted controls was used. A true positive result was obtained only with themitochondria. The nucleolus occasionally stained black both in the testmaterial and in the pyridine-extracted controls: this might be expected, sincenucleoprotein stains with acid haematein (Baker, 1946). However, in thecontrol material, many nucleoli had a peculiar washed-out appearance. Insome cases they had one or two clear areas, which presumably might havearisen 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 ispossible that it might react with the RNA of the nucleolus and extract it, thusproducing the washed-out appearance.

With osmium tetroxide / ethyl gallate the whole amoeba was coloured inshades of grey. The cytoplasmic globules were nearly black. The mito-chondria and the nucleolus were well shown in pale grey. This result alsoraises the question of lipid in the nucleolus. Wigglesworth (1957) states thatin tissues, reaction with protein can be ignored, and the technique used as atest for unsaturated fatty acids.

Support for this conclusion comes from Bahr (1954). In a study of thereactions of osmium tetroxide with solutions of biological materials, Bahrfound that carbohydrate and nucleic acids are inert towards it; that the reactionwith the ethylenic linkages of lipids is exceptionally strong; and that amino-acids that contain •—SH or —S— react vigorously, as also do amino- acidswith basic groups which are in a terminal position of a peptide chain and arenot salt-linked. Bearing in mind that in Naegleria the mitochondria and thenucleolus are blackened to the same extent, there are at least three possibleinterpretations. (1) Lipid is present in the mitochondria and the nucleolusand 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 basicgroups (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 thestandard stains, the second interpretation is perhaps the most likely.

Carbohydrate. For histochemical purposes Pearse (i960) divides the carbo-hydrates into polysaccharides (the glycogen group), acid mucopolysaccharides,neutral mucopolysaccharides, mucoprotein and glycoprotein, and glycolipid.

The periodic acid / Schiff (PAS) reaction coloured the food vacuoles brightred and the cytoplasm pale pink. A similar result was obtained when prepara-tions were incubated in a solution made by diluting saliva to twice its volumewith sterile glass-distilled water, centrifuging to clear of mucus, and thenstaining with PAS. The failure of the salivary enzymes to remove the red-staining material indicated that the glycogen group of polysaccharides wasabsent; similarly the preparations treated with iodine became pale lemon-yellow with no sign of the deep reddish-brown characteristic of glycogen. ThePAS reaction was unchanged in amoebae from which lipid had been extracted.

Three tests were used for the acid mucopolysaccharides: metachromasia oftoluidine 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 metachromaticcolour 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 resultsof the toluidine blue staining. Solutions at pH from 2-62 to 4-66 were used foruntreated preparations of amoebae and for preparations after incubation inribonuclease solution (p. 516) and subsequent washing. Ribonuclease wasapplied because RNA considerably increases basiphilia and so affects theMBE. In both sets, amoebae at pH 4-66 were stained pale blue. At pH 3-62the amoebae extracted with RNase were unstained, whereas in the unex-tracted amoebae there were traces of blue staining, particularly in the nucleo-lus. As the nucleolus is known to contain RNA (p. 516), it was concluded thatthe staining in the amoebae at pH 3 -62 was due to RNA, and that as the MBEof Naegleria was not below pH 4 when RNA was removed it was unlikely thatacid mucopolysaccharide was present.

Finally, alcian blue (Steedman, 1950) stained the entire amoeba a uniformblue colour, and not the bright green or blue-green indicative of acid muco-polysaccharide.

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 characteristicstaining reactions. These were the orthochromatic purple-blue of toluidineblue; the pale pink (cytoplasm) or the bright red (food vacuoles) of the PAS


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 givingthese reactions can, therefore, only be neutral mucopolysaccharide, muco-protein, or glycoprotein. These, unfortunately, cannot be distinguished bycytochemical means.

Vital dyeingSome 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 specialcase 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 gainfurther information on the food vacuoles and lipid globules (neutral red andbrilliant cresyl blue); and to determine the distribution of mitochondria duringthe 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 solutionsapproximately isotonic with the culture medium. With tryphenyl-tetra-zolium chloride, a succinate substrate was used. The living organisms wereplaced in a drop of the dye solution on a slide, covered, and examined atintervals of about 15 min, 2 to 4 h, and 24 h.

Brilliant cresyl blue stained neither the nucleus nor any of the cytoplasmiccomponents. Neutral red stained the lipid globules and the food vacuoles,but not the contractile vacuole system. The globules and the food vacuoleswere red, indicating that the colourable matter had an acid pH.

According to Marston (1923), proteolytic enzymes within the cell can bedemonstrated by azine dyes. Accordingly, the transformation from flagellateto amoeba was studied in organisms immersed in o-ooi % neutral red. It wasthought that when the flagella were absorbed into the cytoplasm in the finalstage of the transformation, any proteolytic activity might result in a con-centration of the dye. Although the transformation was quite normal, noneutral red staining occurred in the area of writhing cytoplasm (p. 526) wherethe flagella had been withdrawn. The lipid globules and the food vacuoles inboth flagellate and amoeboid phases were coloured red.

Janus green B, used at o-oooi%, gave variable results. Sometimes themitochondria were tinged with pale green, at other times they were colourless.

The results with triphenyl-tetrazolium chloride were similar: sometimesthe 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 whichthe red formazan was produced in the lipid globules. As the smallest of thesewere of about the same size as mitochondria, critical examination was neces-sary to distinguish them.


Cytology and cytochemistry of mitosis

The nuclear division of Naegleria has attracted much attention, probablybecause of the conspicuous nature of the nucleus and the mode of division ofthe nucleolus. As a result, the stages of mitosis are fairly well known, thoughfar from understood.

The following account is based on phase-contrast microscopy of livingamoebae, conventional cytological fixation and staining, and standard cyto-chemical techniques. These observations were correlated as closely as possible.Amoebae were watched under phase until the nucleus of an individual amoebawas seen in the first stage of mitosis; the position of the amoeba was establishedby stage Verniers, and the preparation fixed. Amoebae in the second, third,fourth, and fifth stages of the division were similarly treated. In this waycomparisons between phase-contrast, cytochemical, and cytological prepara-tions of the same mitotic phase were made.

Several preparations of each stage of mitosis were made. One set wasstained by Jordan and Baker's (1955) methyl green / pyronin technique forRNA. 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 methylgreen / 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'sstain; dehydrated, mounted in xylene. (3) Nucleus of the selected amoeba oneach slide drawn in colour. (4) Preparation rehydrated and washed in runningwater 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'sstain, dehydrated, and mounted in xylene. (7) Nucleus of the selected amoebaon each slide drawn in colour. (8) Stages 4 to 7 repeated, except that in (5) thepreparations were not incubated in ribonuclease, but for 15 min at 20° intrypsin (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 inpepsin (Armour) for 30 min at 200 in 0-02 N HC1 at pH i-6. Controls wererun in (8) and (9) as in (5).

These preparations gave information on the protein matrix of the mitoticfigure (pp. 522, 523).

A fourth set was carried through Alfert and Geschwind's (1953) techniquefor the demonstration of basic protein, a fifth through the PAS reaction forcarbohydrate, and a sixth through the Sudan black method for lipid.

The results are best described by relating them to 4 arbitrary divisions inthe mitotic sequence. Most writers give these the conventional metazoannames of prophase, anaphase, metaphase, and telophase. As these names arelinked with chromosome configurations and movements, of which little isknown in Naegleria, the terms stage I, II, III, IV, are substituted for themin this paper (fig. 2).


Before stage I, the nucleus is in its non-dividing or 'resting' state. Thenucleolus 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 disintegra-tion of the nucleolus. When the living nucleus is examined by phase contrastit appears as a disk of about the same refractive index as the surroundingcytoplasm. It is exceedingly difficult to see, and unless one is familiar with the

non- stage stage stagedividing 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 nucleushas disappeared entirely. The nucleolar RNA is intermingled with the DNAof the nuclear sap throughout the nuclear area. There is a well-definednuclear membrane.

Stage II. Spindle fibres appear among the mixed nuclear material. Whatappears to be a re-aggregation of nucleolar RNA and nucleolar proteinproduces the typical squat dumbell (fig. 2, stage II). Sometimes the mass ofthe nucleolar material obscures most of the spindle fibres. During this stagethe DNA migrates to the equatorial region of the dumbell figure, forming aband which sometimes has the appearance of a considerable number ofirregular elongated bodies. These stain intensely with the methyl green ofJordan and Baker's (1955) method, and with the SchifFs reagent in Feulgen'sreaction.

Stage III. The mitotic figure elongates and the spindle fibres are stretchedout. Two large masses now form, one at each pole of the mitotic figure. Themasses are composed of RNA and protein, and were called 'polar masses' byRafalko (1947). Sometimes the polar masses are sharply separated from eachother; sometimes they are connected by an irregular wisp of material, andsometimes by a thick column (fig. 2). The material connecting the polarmasses is composed of RNA and protein. The DNA bodies separate intotwo groups. One group moves on (or is moved by) the spindle fibres towardsone of the polar masses, whilst the other group moves in a similar manner tothe opposite mass. The nuclear membrane breaks down in the equatorialregion but remains intact round the polar masses.


Stage IV. The nucleus now enters the final stage of mitosis. In this stageit reaches its greatest elongation. The main elements of the mitotic figureconsist of polar masses of RNA, basic protein, and acidic protein; a compactgroup of bodies containing DNA adjacent to each polar mass; and a longslender strand of nucleolar RNA and protein stretching between the DNAbodies. The nuclear membrane persists round the polar areas.

The slender strand parts in the middle and appears to retract, forming acompact body next to the DNA bodies. Separate daughter nuclei are nowpresent. Division of the cytoplasm follows within seconds or, at the most,within a minute or two. For a few minutes the daughter nuclei remain withDNA in the centre, and RNA at the periphery, of the nucleus, i.e. a reversalof the normal condition. It is possible that this corresponds to what happensin mammalian nerve cells (Hyde'n, 1943), where the foundation of a nucleolusis preceded by the aggregation of DNA-protein particles in a ground sub-stance rich in basic protein. On the other hand, Alfert and Geschwind's testshows very little basic protein at this stage in the Naegleria nucleus.

About 10 min after separation of the daughter nuclei, and after division ofthe amoeba, the nuclei in the daughter amoebae have assumed their normalnon-dividing ('resting') appearance, i.e. there is a large nucleolus lyingcentrally in the clear nuclear sap, and the entire nucleus is surrounded by awell-defined membrane.

It cannot be emphasized too strongly that mitosis as seen by phase contrastconveys quite a different impression from that studied in fixed and stainedmaterial. The absence of dyes tends to draw attention to the dynamic natureof this system. As these nuclear changes are taking place the body of theamoeba follows a regular pattern of movement. In the early stages (I and II)of mitosis, locomotion is normal, and the dividing nucleus is rolled backwardsand forwards in the surging and streaming cytoplasm. As stage III is ap-proached, amoeboid movement slows down, and the elongated mitotic figuretends to become fixed in the long axis of the amoeba. In stage IV the changein the amoeba is dramatic, and events proceed in rapid succession. Allamoeboid movement ceases; the amoeba flattens into a thin, delicate sheet ofprotoplasm; for a moment it is motionless; then tiny pseudopodia are rapidlyprotruded and withdrawn at each end of the organism; a waist appears at thecentre of the organism; the nuclear figure parts to give daughter nuclei; thewaist constricts, and the two halves of the amoeba draw apart, usually pullingout a long slender strand of cytoplasm between them.

The preceding description, and the facts recorded above, indicate thatRNA and DNA remain clearly demarcated throughout mitosis.

Carbohydrate and lipids are absent from the nucleus (except the lipid ofthe 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 Alfertand Geschwind's test. One would expect both RNA and DNA to be associ-ated with basic protein, but some of the protein may be affected by the


drastic extraction with trichloracetic acid which Alfert and Geschwind's testentails.

A residual 'acid' protein is demonstrable by basic dyes when basic proteinhas been removed by trypsin or by mild acid hydrolysis. The validity of suchdemonstrations rests on the assumption that short hydrolysis with trypsin, ormild acid hydrolysis, will remove basic protein before the remaining proteinis affected.

It is obvious that the cytochemical analysis of a complex protein body suchas the nucleolus is unsatisfactory. A satisfactory analysis must await eithernew 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 tothe 'prophase' of the majority of writers on Naegleria, who have either missedor completely misunderstood stage I. Glaser (1912) figured it but made nocomment on it. Zuluetta (1917) gave good figures of it but was so mystifiedby it, and by the seemingly endless variety of mitotic figures which Naegleriacan 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 evidencefor 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 ofmitosis, and follows a constant course in which the nucleolus becomes fainterin appearance and blends with the nuclear sap, while the nucleus as a wholeincreases in size. In one case where measurement was possible, the restingnucleus was about 6 fi in diameter, whereas the swollen nucleus was about10 ix in diameter.

Stage I may be connected with spindle formation. The protein of thenucleus, if utilized for this, would have to be in solution; hence, presumably,the disintegration of the nucleolus. This disintegration might be a reversibledissociation (Haurowitz, 1950) which would account for the observed decreasein viscosity (p. 521) and swelling of the nucleus. It is therefore possible that inthe semi-fluid content of the nucleus there now follows a process analogousto the formation of fibrin in the blood (Heilbrunn, 1956) and to the end-to-endlinkage of certain of the peptide chains by enzymes (Ferry, 1949). Theselinked chains might then aggregate by lateral association to produce thespindle 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 withthis in detail. He extracted the mitotic spindles from the eggs of Strongylo-centrotus purpuratus and, after critical tests, concluded that the fibres werenot artifacts. In phase-contrast studies of mitosis in Naegleria a well-developed fibrous spindle (though not of the metazoan type) may appear inone amoeba, and not in an adjacent amoeba. That spindle fibres are notvisible in every case does not necessarily mean that they are not formed. Forexample, they may form at the end of stage I, and then in stage II become


fused with the nucleolar mass. My belief is that they always form, but, likeevery other part of the mitotic figure, they are subject to considerable variationin mass and in duration.

Three other morphological features, the 'polar caps', interzonal body, andcentrioles, are controversial.

I regard 'polar caps' as spurious. Ford (1912) was the first person to nameand describe these structures. He claimed that they were stained only byDobell'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-haema-toxylin preparations, and do not believe that they are separate structures worthyof a special name, but that they are, as Pietschmann (1929) observed, the endsof 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 ofthe mitotic figure in stages III and IV. Rafalko states that 'as anaphaseprogresses, particles of the polar masses appear to migrate along the spindlefibres to the middle to form a so-called interzonal body often mistaken fortrue chromatin'. I found no evidence of this migration. It seems that theinterzonal body is a normal consequence of mitosis in a nucleus where thereis a large amount of nucleolar material. Sometimes the nucleolus dividescleanly, producing large polar masses, easily visible spindle fibres, and nointerzonal body. At other times the division of the nucleolar material is notclear-cut, and, as the nucleus elongates, nucleolar material, often in coarselygranular form in fixed preparations, stretches between the polar masses toproduce the inter-zonal body.

Perhaps the most inadequate exposition of the origin of the interzonalbody comes from Chang (1958). He says 'When the karyosome divides intotwo in the prophase, a piece drops out . . .'. This piece then becomes theinterzonal body.

The reversible transformation from amoeba to flagellatePietschmann's (1929) account of this transformation is particularly good.

The present account adds some new facts on the resorption of the flagella andthe movement of the mitochondria.

As in the study of mitosis, observations on living organisms were correlatedwith those on fixed and stained specimens.

When amoebae were placed in a drop of distilled water as a stimulant totransformation, the following events were observed by phase-contrastmicroscopy. An amoeba which had been moving in the usual manner gradu-ally came to a standstill and assumed a spherical shape. A few small pseudo-podia were occasionally thrust out, but pseudopodial activity soon ceased.The amoeba, though spherical, was still attached to the substratum, and thenucleus 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. Theydid not occupy fixed positions in the cell, though many of the mitochondriacame to do so. A pair of flagella suddenly appeared, though sometimes oneflagellum was extruded before the other. When both flagella had newlyemerged they beat slowly, but with rapidly increasing tempo, causing theamoeba 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 pointof attachment. Finally, the half-turns gave place to a rapid spinning, whichwas clockwise or anticlockwise, accompanied by an increase in the rate ofthe flagellar beat. The spinning, lasting anything from a few seconds to aminute or two, broke the attachment to the slide, and the organism swamaway as a spindle- or torpedo-shaped flagellate. Usually it had one nucleusand two flagella; this form was produced by a uninucleate amoeba. Multi-nucleate amoebae, usually produced in old or very crowded cultures, werealso capable of the transformation, forming flagellates with 2, 3,4, or even 5nuclei. In these, each nucleus was not necessarily associated with a pair offlagella; a flagellate with two nuclei may have either 3 or 4 flagella, and onewith 5 nuclei may have 8 flagella.

The free-swimming flagellate stage lasted for 30 min to 24 h; then theflagellate lost its rigidity and settled on the slide again, rotating in a clockwiseor anticlockwise direction as it did so. As it settled, pseudopodia were thrustout at random. Occasionally pseudopodia were protruded a few secondsbefore the amoeba-flagellate settled. A striking feature of the nearly settledamoeba-flagellate was the immobility of the nucleus. Whereas in the normalamoeboid phase the nucleus was moved about by the surging of the cyto-plasm (p. 515), now it was held stationary, close to the point where the flagellaemerged from the periphery of the organism. The flagella did not change inlength, or in rate of beat. As the random extrusion of pseudopodia graduallychanged into the typical Umax action of a single broad pseudopodium, theamoebo-flagellate flattened on to the slide, and the flagella and nucleus becameclearly visible. The thickened bases of the flagella appeared to be attached tothe nuclear membrane, which was drawn out towards the point of emergenceof the flagelia. The existence of a physical connexion between nucleus andflagella was indicated by the vibration of the nucleus within the cytoplasm inrhythm with the lashing of the flagella.

The amoeba-flagellate stage may be quite protracted, though it usuallytook less than 1 h. The last phase of the transformation back to the amoebawas marked by a faltering in the rapid beat of the flagella. For a fraction of asecond a flagellum remained quite motionless, then resumed its beating;alternating in this manner for several minutes, until finally the flagellumbecame motionless, bent, and was rapidly withdrawn into the organism. Theflagellum was invisible within the cytoplasm, but its presence was marked bya snake-like writhing. While this was happening the second flagellum wasstill beating normally, but soon stopped; for a second it was held out


motionless, then bent and was withdrawn into the organism. The writhingin the cytoplasm was now particularly clear.

As the flagella were withdrawn, the nucleus moved away from the peripheryand down the long axis of the organism. As it moved it was jerked from side toside by the writhing of the flagella—yet another point in favour of a physicalconnexion 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 asthe cytoplasm resumed its normal streaming; and the organism reverted to acharacteristic limax amoeba, moving on its course by a single bulging pseudo-podium.

The nucleolus underwent no detectable change during any of the eventsconcerned with the reversible transformation, and it did not seem to be involvedin the production of flagella.

This description is in disagreement with that of Willmer (1956) in thefollowing respects.

Flagella are not produced at a 'posterior' end, nor is their productionassociated with the uroid. In most cases the amoeba is spherical, or nearly so,when the flagella are produced. The nucleus and contractile vacuole are notin 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 asingle 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; thebiflagellate is the typical form. Further, if the above statement implies thatflagellates with a single flagellum occur, then that implication must be denied.

Willmer also states that first the flagella appear from the 'posterior' end, thenthe amoeba rounds up, then the spinning commences. The sequence is thatthe amoeba becomes stationary, rounds up, produces flagella and then spins.

Willmer's description seems to confuse stages in the secondary changefrom 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 ofthe rounded amoeba as the amoeba changed to the flagellate form (fig. 3).When the flagella emerged, this dark patch surrounded the point of theiremergence, and remained at the base of the flagella in the fully formed flagel-late. The rounded form, and rapid movement of the living flagellate, made itimpossible to resolve the individual mitochondria; but they can be resolved instained preparations. All stages of the transformation of the amoeba to theflagellate were studied with Baker's acid haematein. In the stained flagellatethe mitochondrial cap at the base of the flagella (fig. 3) was the characteristicfeature. When the flagellate settled down and reverted to the amoeboid form,the mitochondria dispersed throughout the endoplasm.


In one instance, where the change from flagellate to amoeba was beingwatched by phase contrast, it was noticed that the mitochondria in the cyto-plasm occupied by the writhing withdrawn flagella were larger than normal,and were globular instead of rod-shaped. It is possible that the mitochondrialmovements are connected with the synthesis of flagellar protein, and with theenergy requirements of the flagella.

mitochondrion

nucleusNormal amoeba

Amoeboid form unchanged,but mitochondria concentratingat periphery

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

Normal flagellate form.Nucleusnear flagellate end of organism;dense "cap" of mitochondriaround bases of flagella

FlG. 3. Schematic representation of the movement of mito-chondria 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 thefact that some amoebae transformed to flagellates in a matter of minutes;others in the same preparation took hours. The age and condition of theculture were important factors in this metamorphosis. However, given ahealthy young culture not more than 3 days old, and given that the eventsproceeded undisturbed in distilled water, most of the amoebae respondedto this stimulus within 3 h. Once the amoeboid movement ceased and theorganism 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 thestudies recorded here attest the accuracy of her work. The direct physicalconnexion between the nucleus and flagella is obvious. The nature of theconnexion is still unknown. The light microscope showed, particularly at thebeginning and at the ends of the flagellate phase, the bulbous bases of theflagella attached, or closely apposed, to the nuclear membrane. These basesappeared as black dots in iron-haematoxylin preparations. There is littledoubt that these objects are the basal granules (blepharoplasts) of earlierworkers. However, in view of what the electron microscope has revealedabout the nature and size of the base of the flagellum and the basal granulecomplex in many protozoa, judgement must be reserved on this point inNaegleria.

The problem of the relationship between nucleus and flagella, indeed of thewhole transformation from amoeba to flagellate, requires a thorough studywith the electron microscope. Some electron micrographs were taken bypersonnel of the Wheatstone Laboratory, King's College, London. Onemicrograph showed an elongate body (part of a flagellum ?) stretching fromthe nuclear membrane to a point near the periphery of the cell. There was anindentation of the cell membrane and adjacent cytoplasm opposite this point.There was also an indentation of the nuclear membrane where the base of theelongate body rested. It is likely that this elongate body is the same structureas the cylindrical object which is seen in iron-haematoxyl preparations on, oreven within, the nuclear membrane, and which is the rudiment of a flagellum.Another micrograph of the series showed the mitochondria concentrating nearthe periphery in the immediate pre-flagellate stage. The mitochondria ap-peared to have a double membrane and an internal system of tubules, similarto those figured for Amoeba proteus by Mercer (1959) and for Acanthamoebaby Vickerman (i960).

Cytochemistry of the flagellate stage

All the tests listed on pp. 516-20 were also carried out on the flagellatestage. 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, mysupervisor, who brought the problems associated with Naegleria to myattention, and was always ready to draw from her immense experience in orderto advise and discuss.

The work, which is part of a thesis submitted for the degree of doctor ofphilosophy of the University of London, was financed by an Agricultural


Research Council grant, and was carried out at the Lister Institute of Pre-ventive Medicine, London, S.W.i. The electron microscopy was carried outat the Wheatstone Laboratory, King's College, London, by kind permissionof Prof. Sir John T. Randall, F.R.S.

ReferencesALFERT, M., and GESCHWIND, I. I., 1953. Proc. nat. Acad. Sci. Wash., 39, 991,BAHR, G. F., 1954. Exp. Cell Res., 7, 441.BAKER, J. R., 1946. Quart. J. micr. Sci., 87, 441.

1947. Ibid., 88, 115.1956. Ibid., 97, 161.1958. Principles of biological microtechnique. London (Methuen).

BALAMUTH, W., and ROWE, M. B., 1955. J. Protozool., 2, Supplement. Abstract No. 57.CAIN, A. J., 1947, Quart. J. micr. Sci., 88, 383.

I949«. Ibid., 90, 75.1949*. Ibid., 90, 411.

CHANG, S. L., 1958. J. gen. Microbiol., 18, 565.DEMPSEY, E. W., and SINGER, M., 1946. Endocrinology, 38, 270.FERRY, J. D., 1949, in Blood clotting and allied problems. Transactions of the Second Confer-

ence, New York. (Josiah Macy, Jr., Foundation).FORD, E., 1912. Arch. Protistenk., 34, 190.GLASER, H., 1912. Ibid., 25, 27.HAUROWITZ, F., 1950. The Chemistry and biology of proteins. New York (Academic Press).HEILBRUNN, L. V., 1956. The dynamics of living protoplasm. New York (Academic Press).HYDJSN, H., 1943. Acta physiol. Scand., 6, Supplement 17, 1.JORDAN, B. M., and BAKER, J. R., 1955. Quart. J. micr. Sci., 96, 177.MARSTON, H. R., 1923. Biochem. J., 17, 851.MAZIA, D., 1955. Symp. Soc. exp. Biol., 9, 335.MCLEISH, J., BELL, L. G. E., LA COUR, L. F., and CHAYEN, J., 1957. Exp. Cell Res., 12, 120.MERCER, E. H., 1959. Proc. roy. Soc. B, 150, 216.MORISITA, T., 1939. Jap. J. exp. Med., 17, 1.PEARSE, A. G. E., i960. Histochemistry, theoretical and applied. London (Churchill).PIETSCHMANN, K., 1929. Arch. Protistenk., 65, 379.RAFALKO, J. S., 1947. J. Morph., 81, 1.SCHARDINGER, F., 1899. S. K. Akad. Wiss. Wien, 108, 713.STEEDMAN, H. F., 1950. Quart. J. micr. Sci., 91, 477.SINGH, B. N., 1952. Phil. Trans. B, 236, 405.UNNA, P. G., and TIELEMANN, E. T., 1918. Zbl. Bakt., Originale Pt. I, 80, 66.VICKERMAN, K., i960. Nature, London, 188, 248.WIGGLESWORTH, V. B., 1957. Proc. roy. Soc. B, 147, 185.WILLMER, E. N., 1956. J. exp. Biol., 33, 583.WILSON, C. W., 1916. Univ. Calif. Publ. Zool., 16, 241.ZULUETTA, A., 1917. Trab. Mus. Cienc. nat., Madrid, Ser. Zool., 33.

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Studies of an amoebo-flagellate, Naegleria griiberi

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