Cytology. - An electron~microscopical study of bull sperm I. By L. H. BRETSCHNEIDER and WOUTERA VAN ITERSON. (From theZoological Laboratory at Utrecht and the Netherla'll!ds Institute for Electron Microscopy at Delft.) (Communicated by Prof. CHR. P. RAVEN.)

(Communicated at the meeting of November 30, 1946.)

1. Introduction.

The Research Group for Endocrinology at Utrecht, under the auspices of the organization for Applied Scientific Research (T.N.a.), wanted for their studies on al'tificial insemination a thorough iflivestigation of the development and structure of bult sperm.

Wi,rh the Hght microscope we candistinguish as main features of the spermatozoon a flat head with a tender cap, a neck, the body and a long tail. In tohese, finer structures are hardly visibl'e, a's they !ie beyond the possibihties of resolutionof rthe i,nstrument. To reveal such details it seem~ cd necessary to use the 1 OO~fold improwment in resolution given by the electron microscope. A review of the recent American literature taught us that some submicroscopical investigations of spermatozoa had alread'y been undertaken by various workers (2, 6, 8). The present communication bears only a preliminary charader.

Apart from the aim with which this study was undertaken, it may gvie a first impressio:l of the possibilities of the Dutch electron microscope with a view to the solution of problems of morphology and genesis of the healthy and abnormal sperm. This microscope, which will be described elsewhere, was designed and built by Mr. J. B. LE POOLE in the Ph,ysical Institute of the Technical University at Delft during the la~er years of the war. It is characterized amongst others by a th ree-lens system which. without changing the pole pieces and without image distortion, enables a continuous va<riation of the magni­fication from 1000 to 60.000 times. The accelerating voltage cao be va ried from 50-120 kv, opening the possibility to work with higher tensions, if the density of the object makes this desirabie.

The image formation with the electron microscope depends on the electron scattering power of the structure of the specimen. The magnitude of this scattering is dependent on the thickness and density of the object. In general an obj ect thickness of 0.1 fl is considered suitable for electron microscope research. The relatively lal'ge bull sperm (thickness -+- 1 fl-400mfl) haroly answers this requiremeIlJt, but owing to the heterogenity of its structure it still proved possible to destect same interesting details. An electron acceleraHng voltage of 90 kv provedl !Jo give a suitable contrast. MoreOver, we succeeded in increasing this heterogenity with the aid of the so~called shadow~casting method of WILLIAMS and WVCKOFF (10).

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2. Technique.

The sperm use.d was taken from a normal bull. Colloidal material. when present in an electron microscope preparation, will dry up against objects aud spon their ima9le. For this reason the sperm was c1eaned by repeated centrifugatiÏon. After this procedure a part of the sperm was mounted fresh in a drop of distiUed water on the film of t:he specimen holder, another part was fixed in chloramine. The chloramine treatment appeared to be favour~ able for the contrast in the tender struCitures which wiU be due to a shrink~ age of the protein structures. In particular the so~called axial filament showed better in the fixed preparations.

As object holders for the apparatus at Delft na screens are used, but small silver cones with a very tiny hole of 0.3 mm diameter in the top to allow for the passage of the electron beam. The film is prepared by bringing with a glass capil!ary one drop of a solution of "Geisselthallack" on thesurface of distil!ed water in a 10 cm Petri dish. Af ter the film has settled, which takes same minutes, the specimen holder m~unted on a metal rad is brought into touch with the film, either in the center or in any suitable area. Then the holder is removed by turning the rod, sa as to roll the surplus of the film around the conical si des. On top of this film the objects in distilled water are now mounted with the aid of a platinum loop. After the drop has dried in the air, the specimens are locked in the vacuum of the electron microscope or subjected to the procedure of shadow-casting.

The procedure of drying, fresh sperm in ,the air on the object film for electron microscope examination is comparable to the light~microscopical technique in which air~dried smears are used. The drying has the ad~ vantage of flatteIlling the sperm, thus increasing its eIectron~per:meability. The w~dth of the head increases aboUit 20%.

The influence of the vacuum on ~he sperm is unlknown to us; heat damage as a 'consequence of e'Iec,tron absorption was not observed.

To increase the contrast of tender structures two methods are in use. The influence of seleCitive staining will be consi:deredi in a subsequent report. In these experim~nts a part of the preparations were submi,ued to a second' technique, a sha,dow~casting with g:old.

WILLIAMS and WVCKOFF (10) developed a method of increasing contrast for electron microscopy by evaporating particles of a piece of metal (gold or chromium for instance) heated in vacuum. When the object holders are placed in au oblique position to th is metal piece in an auxilia~ vacuum chamber, the evaporated metal particles wil! condense all over the specimen, but as in the case of a snow storm, in the shelter of a higher structure no particles will settle, thus causing a blank "shadow" in proportion to the height of the structure. This procedure of coating objects with a few atoms of metal has the double advantage of increasing the density of hardly perceptible structures and making it possible to estimate their relative heights (compare the micrographs 1 and 2. or 3 and 7).

When considering thephotographs · one should realize that in favour of the eHect It!he prints of shadow~castings are made in negative. In ourcase

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the shadowing with gold sometimes showed the disadvantage of a rough coagulation of this metal when the object was too intensely "illuminated" by the electron beam which spoiled the details of fin er structures (Cf. Plates, fig. 10). But in g:eneral the micrographs obtained in this way, in contrast to what holds for the ordinary pictures, greatly facilitate the mental recon­struc-tion of the three..-dimensional structure.

3. Spermiogenesis.

For a better understanding of the disdosed facts we give a brief account of the growing process of the spermatozoon.

A. The spermatid (Text fig. la). From this primitive stage the so complicately built spermatozoon develops by differentiation. In spermio­genesis we notice first aglobular cell body in which we distinguish: in the center a round nucleus, the diplosomes, the acrobl'ast originating from a Golg:i-body, numerous mitochondria and some separate Golgi-bodies.

B. The meta-spermatozoon. After this first development has come to an -end, the cell strdches longitudinally. The nucleus moves .to the apical cd} pole and the acroblast assumes a caplike position by covering the apical part of the nu deus (Text fig. lb). The diplosomes move ,antapically to­wards the axis of the cello An axis fibril. the axial filament, grows from the peripheral centrosome; it can be seen to continue outside ithe cell body in the direction of the longitudina} axis. The Golgi-bodi-es and mitochondria arrange themselves in a vortex around this axial f.ilament.

C. The immature spermatozoon stage. A sheathlike lamella, the collar, differentiates at the equator of the nuclear membrane and grows in a caudal direction round the cell-axis, until a ,length of 165 mfi -Î'S reached. It encloses the axial filament, the diplosomes, GoIgi-bodies, mitochondria an.d the central protoplasm, the so-called involucrum. Where this collar ends, the axial filament surrounded by the involucrum cont:inues and reaches a lerugth of 70fi. The cell plasm not used for construction contmats to a drop and is separated as residual protoplasm. By dehydration and 10ss of protopl'asm all structures a're still more condensed. by which process the nucleus flattens.

As a consequence of this. the structures of the mature sperm are packed into a space of less than 1 fi thickness, making a further analysis with the light microscope impossible, thus necessitating the electron-microscopical investigations.

4. The structure of tlle mature bull sperm.

The electron-microscopical image shows:

A. The structure of the sperm as also visible with the ordinary micros­cope.

B. Submicroscopical structures leading down to the macromolecular protein components.

Spermiogenesis

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10. head. 11. neck. 12. mld die piece. 13. residual plasm. 14. tail.

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Schematized resumption of the electron-microscopical observations.

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Fig. 2. 1. head fibriIs. 2. collar. 3. articular strands. 4. cortical helix (middle piece). 5. splitting up into thinner threads. 6. cortical helix ( tail) . 7. chromosome shadow. 8. centrosome. 9. articular strands.

10. Golgi-bodys. 11. axial filament. 12. braad helix. 13. corticaJ plasm. 14. involucrum. 15. protofibrils (axial filament) .

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A. Description of the more conspieuous structural elements.

a. The head (Cf. Plates, fig. 1. 2, 3). lts shape is slightly asymmetri­cal. both at the concave base and alt the top. On one side the top is a little rounder than on the other side. The apex has shifted over to one side; how­ever, the rdeviation in the axis is so small Ithat it is not detectable with the light microscope.

The apical part is covered by a teilider cap, ca~led protoplasmic cap by BAYLOR, NALBANDOV and CLARK 1934 (2), whïch shows in thephotographs as a lighter zone surrounding the outside. Evidently the cap is lifted a little from the head as indicated by the broader zone at the top. (Cf. Plates, fig. 3.)

The distal part of the cap is marked by a line or a nOitch in the profile. Here in the equator of the head a double dark shadow ba1nd becomes visible. This appal1ent doubling is caused bydiffer,ent extension of the cap at front and back side, both margins coveringeach other incompletely. These shadows are often lined by a lighter zone, the "sub-equatorial zone".

Both zones can be made visible for the light microscope by staining with iron haematoxylin. They can be understood as differences in thickness of the nucleus or nuclear structures' in this area.

In spermiogenesis the chromosomes are completely condensed in the nucleus- or head-base. In our eleotIron microscope pictures we can see them indicated as a vague "chromosome shadow" (Cf. Plates, fig. 3, 4).

b. The neck (Cf. Plates, fig. 4, 5). The tail articulates with the broad ( 1,5-2 fh) base of the head by 2 lateral columns, the "articular strands". These strands are connected with ithe collar, a ring>-shaped membrane fit­ting closely to the head-base (Cf. Plates, fig. 4). As an artifact the collar sometimes detaches itself, by which the articulation poiIllts are ddsplayed (Cf. Plates, fig. 5) . In extreme cases protoplasmic threads are seen to stretch between headbase and collar. The proximal centrosome is situated in the center of the neck, against the base of the nucleus. It is the origin of the axial filament, and is of ten covered by the collar. The remaining space is found to be empty both on examination in the light and in the electron microscope, and in vivo is probably filled with a liquid (Cf. Plates, fig.4,5).

c. The middle piece (Cf. Plates, fig. 6, 7). The neck passes into the cylin­drical middle piece (leng th 16-18fh' width 750 mfh-lfh). Cytogenetically it is distinguished by containing several cell S1tructures which Ior a large part envelop each other. As a consequence their images overlap in electron microscope pictures, thus complicating their interpretation.

In the proximal part the axial filam·eIllt runs through the middle piece. This is seen as a lighter medial zone in ordinary pictures (Cf. Plates, fig. 4).

In the micrographs of lthis part, immediately behind the neck a more dense structure of irregular shape can be distinguished, and in some cases more-

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over vacuoles and largIel' granu~es. In cytogenesis bhe m1tochondria are de* posited here. These irregular structures are transformed into a broad helix. The regular coils of this helix run obliquely backwaIlds, and encircle the axial filament until ,the end of the middle pieee. As a proof of ,this anti~ clockwise spiral nature, both the upper and lower parts of the coil ean be seen as a diagonally crossing system in shadow~cast pictures (Cf. Plates, fig. 6). The thickness of the spiral filament is 200 mft and the numher of coils in the middle piece amounts '10 -+- 25.

Tthis spiral ori'ginat'es from mitochondria linked tog'ether and af,ter inse* mination of the ovulum it d'isperses again into mitochondria. Excess mito~ chondria are of ten found in the separated residual protoplasm (Cf. Plates. fig. 9). Perhaps they are identical with the granules measuring also 200 m,u which we can distinguish vaguelyon some of theelectron micrographs. Axial filament and helix are embedded in the endoplasm. the so~called in~ volucrum. while this is surrounded by the ectoplasm being richer in vacuoles.

d. The tail (Cf. Plates. fig. 4.7.8. 10). The middle piece is continued into the long tail (45 ft). lts width diminishes from beginning to end. from 800 IDft to 400 mft. As the large helix is lacking here one ean of ten, notice that the axial filament is composed of a number of subf.ibrils. Especially Ï'I1

the chloramine~fixed preparations this is 'evident. In the upper pal.'lt of the tail the axial filament is still enclosed by the involucrum. Inthe proximaJ part of the taH the involucrum diminishes quickly in widbh. so Ithat at the end the axial filament is only wrapped in ectoplasm. In such a construction the tail of the sperm resembles a flagellum or cilium of animal cells. It is known that both of these consist of a fibrillar axis enveloped by ectoplasm.

It seemsevident fhat in vivo also the termina'l point of the tail is sur* rounded by ectoplasm. When the specimen is washed and dried for electron~ microscopical examination, the protoplasmic sheath probably breaks up so as iOO free the axial filaments, Wlhich now 'Split up into a brush of 9 sub* fibrils (Cf. Plates. fig. 10). Each of the fibrils has a width of -+- 30 mft. their length varying with the location .of the ectoplasm rupture. They can have a length of several ft.

E. The more delicate electron~microscopical st'TUctures. As is Ithe case in every animal cello alsothe sperm is bord'ered by a thin

exterior limiting layer. the so~called cortical plasm. In accordance with the great mecha,nical requirements of the motile sperm, there are to be found in this outer layer appropriately arranged meèhanical fibrils. the so~called tender helix.

On examining the head in shadow~cast pictures (Cf. Plates. fig. 1. 2). one i':J struck by a regular structure of crossing fibrils on its surface. The same is revealed in ordinary pictures (Cf. Plates. fig. 3. 4). where - although with some difficulty - a mosaiclike structure is unmistakably observed. In this connection we want to emphasize the difference noticed in chlora* mine and non*chloramine fixed preparations. Noticeably the latter pictures

of the head reg ion show (Cf. Plates, fig. 5) on a rather dark background still darker marbIed indications of the spiral systems, while the chloramine­fixed heacls are much more penetrabIe to the electron beam, but show this structure less clear (Cf. Plates, fig. 3, 4). They, however, have the advantage of giving a better "chromosome shadow".

In the neck we observe as a continuation of the head fibril structures 3-4 knots in ItJhe articular strands, corresponding on both sides, which might represent ,transverse bands. From the collar to the end of the tail an anti­clockwise spiral is wound, enwrapping, first with wide coils the involucrum, the broad helix and the axial filament, bUit more backwards (wi~h smaller coils) it seems only to hold the fibrils of the axial filament together in a string~like way (Cf. Plates, fig. 10).

It seems reasonable to consid'er all of this spiral Sitructure as a whoIe. This woulid mean that we aredealing with onecontinuous fibril system from head to tail, with some kind of transition in the articular strands. In the base of the head the fibrils çross at an angle of -t- 90°, near the top this angle is enlarged to -t- 120°. The significance of this value should not, bowever, be overrated without knowledge of the real shape of the head, as: tlle angle only represents some projection in ' the p}ane of the photograph. About 120 fibrils in each direction cover the whole head.

The diameter of the fibrils in the head is -t- 25 - 40 mJ-l. The diameter of the first part of the cortical helix is -t- 75 mJ-l, more caudally it is diminished to -t- 25 - 40 mJ-l, but greater magnifications vaguely indicate that this pro~ bably results from a splitting' up into 2-3 thinner threads.

At the broken end the subfibrils of the axial filament of -t- 25-40 mJ-l

show a sub~division into a tuft of finer strands, the protofibrils. As we are suffering from lack of contrast in our micrographs of these very tender structures, it is difficult to count these strands, but there may be at least some 5 of these subunits. Their diameter will fall in some tens of A units. This means that the electron microscope reveals here structures of only a small number of protein molecules, united to larger fibrillar units, these being bundled again to form the axial filament.

When considering the pictures we are tempted Ito describe a banded structure both in the axial and in the helical fibrils of the cortex. However, we give this observation in this preliminary report only with great reserve, as more evidence is needed to eiclud:e influences of underl'ying structure of the supporting film, while in shadow~cast pictures a disturbance by the coarseness of the gold grains has to be elimin<lJted.

As an unexpected fact we observed on the head somewhat below the equatorial zone aporus, shaped like a cmter, i.e., consisting of a wall and a pit (Cf. Plates, fig. 1). We are unable to give any interpretation of this unknown formation.

5. Discussion. (Cf. Textfig. 2.)

The preceding shows clearly that electron~microscopical investigation

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greatly adds to our knowledge of the structure of the bull sperm. After these first attempts many questions arise. We expect that an increase in contrast by applying selective stains will be of value.

In agreement with HARVEY and ANDERSON'S observations (6) on the sperm of Arbacia, and with those of BAYLOR, NALBANI)OV and CLARK (2) on the bull sperm, we a150 found the axial filament to consist of + 1 0 sub~ fibrils. Our analysis, however, went further, since we were able to detect in our micrographs (not printed here) a secondary splitting up of these sub~ fibrils into protofibrils, having the dimeIlJSÎOn of only a relatively small number of protein molecules.

Surpass'ing the observations of BAYLOR, NALBANI)OV and CL ARK (2) we were able to demonstrate in the middle piece the broad helix, formed out of mitochondria, the centrosome, and the axial filament originating from the latter. We also stressed the existence of acortical structure, consisting pro~ bably of 2 crossing fibril systems in the head region, and one anti~clockwise helix in the middle piece and tail, whereas both are connected in a rather uncertain way in the neck region.

A photograph of a part of an assumedly comparable helix from an ultra~ sonically fragmented human sperm tail is ,given by F. O. SCHMITI (8).

HARVEY and ANDERSON (6) noticed regularly cross striations in the tail of their Arbacia sperm. They hesitated, however, to compare these with the phenomenon reported for collagen Eibers by SCIjMITI (see bel'ow), and feared that it might he produc'ed in the washing and drying process. We suppose, however, that also in the case of Arbacia this oross striation may be attributoo to the presence of such a helical fibril in the sheath of .the tail. The process of drying for electron~microscopical purposes is as a de~ hydration one of the least radical preparation techniques. Already B. M. KLEIN (7) noticed in 1928 the favourable influence of drying as compared to fixation with chemical ag1ents on the structure of ithe protoplasm.

Even if the banded structure we noticed in the axial as weIl as in the helical fibrils should have to be considered as an artifa'Ct due Ito drying, it still would reveal somethingl of a structural regulafi,ty. However, when we succeed in excluding disturbing influences from the molecular structure of the supporting film and the gold coagu13lte, we expect some cross striation

to remain as ademonstration of some underlying fundamentallaw in nature. In this respect we want to reEer to the recentl:y published eleatIon micro­graphs of ten don (F. O. SCHMITT e.a. (8)) and muscle (RICHARD ANDER~ SON and HANCE 1942 (5) ) and mos,t striking of all, to ASTBURY' S trystalline

protein fibre. From the fact Ithat in these cases the value of the regular ~pacing between the successive bands is in fair agreement with x~ray dif~

fraction measurements, it is inferred that the cross striations should be

designed as subperiods in itlhe polypeptrde chains. We hope to bring in a

future publication moreevIdence that the same holds for the protein fibrils observed by us.

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The most striking result of our invesbgation seems to be the continuous fibril syst!em enwrapping head and tail of the sperm cello Spiral structures in cell waHs are by no means an unknown feature in nature. We want to remind of this phenomenon in the pellicula of the Ciliata (Ophryoscolecid~ es) (3,4). An inspection of our pictures of the head region strongly recalls in mind the spiral sllructures in ,the cell walls of the green aIgae Valonia and Cladophora as described amongst others by ASTBURY (9 and 1). One ean see an ,analogy betw~en the continuation of the spiml structures from the head over the tai! in Ithe sperm cell and the situation occurring in the branchingi of Cladophora celIs. In the latter case the spiral striations indi~ cating the di'rection of the celluIose chains, clearly pass over on the waH of the newly formed cello In our case Ibhe protein chains from the tenrder helix might split up at the base of the head into two spiral systems running in opposite directions. When considering the pictures we should realize that we have on the head at least two or three layers on the upper and lower side coveringeach other. Apart from the lower side - which in shadow~ cast photographs does not 50 muoh effect the whol:e picture as in ordinary micrographs - we have nuclear membrane and the cortical sheath and in the upper par,t moreover the cap as three structures complicating each other.

With regard to the motility of the sperm we expect that there will be an antagonism between axial filament and cortical helix. Which of the two will play the active and which the passive part, we do not venture to d~cide.

Summary.

The underlying study is a first application of the Delft electron micros~ cope in the investigation of spermstruoture.

Normal bull sperm, fresh and af ter ohloramine fixation, in same cases ~ubmitted to a procedure of shadow~casting, with 'glold, has been studied and photographed at an eleotron emission voltage of 90 kv. It is conc1U'ded that the electron microscope can be of great use in elucidating the morphology of the sperm cello We were ab Ie to observe:

A. As structures also visible with the ordinary microscope:

1. The asymmetricaI head, covered by thecap and containing ,the chro~ mosomes showing as a vague shadow.

2. The neck, where the tail is seen to articulate by the articular strands wi,th the collar, a ring~S!haped membrane. In the neck the centrosome is sÏltuated from which the axial filament originates.

3. The middle piece with braad helix, involucrum and several other elements.

4. The tail with cortical sheath and axial filament consisting of 9 sub~ fibrils.

L. H. BRETSCHNEIDER and WOUTERA VAN ITERSON: An electron-microscapical study af bull sperm I.

-----:1 Int1,c <1 tcs 1 ." in all picturrs.

Fig . I. Head of bull sperm. Shadow-cast 18.000 X 9J kv. Showing: crossing fibril systcm; in thc rquatcr the edge of the cap from front and back side; below these a por us.

Fig . 10. Braken end of tai!. Chlora mine-fixed. Shadow-cast 36.000 X 90 kv. Showing: tuft of subfibrils.

Fil]. 2. Head of bull sperm. Shadow-cast 18.000 X 90 kv. Showing: crossing fibril system: edg~ of cap and porus vag uely in the e4uator: surroundi'lg top : escapcd proto·

plasmic contents.

Fig. 8. Fragments of tail. Chloramine-fi:~ed . Shadow-cast 36.000 X 90 kv. Showing : tender helix and axial filament (at the left: part of head and ncck) .

Fig.9. Fragment with residual protoplasm. Chloraminc-fixed. Shadow-célst 20.000 X 90 kv. Notice: mitochondria.

Fig. 3. Head of udll öpcrril. Chlcrauine-fixed 20.000 >.: 90 kv. Notice : a: the outsiue lighter zone of the cap: its edg : a5 ij line in the equator: less den se the sub-equatorial

ZO!le: chro:nos:Jll1~ s>liI:1ow: porus ver,: indistinct .

Fig. 6. Middle piece. Shadow-cast 20.000 X 90 kv. Showing: tender helix enwrapping broad helix.

Fig . 7. Transition of rniddle piece to tail. Shadow-cast 20.000 X 90 kv.

Fig. 4. Head. neck and proximal part of middle piece. Chloramine-fixed 42.000 X 90 kv. Showing: chromosome shadow; articular strands with knots. attached to collar; axial filament attached to centroscme. To the right: part of tail w;th tender helix and axial

filament.

Fig. 5. Displayed articular strands. 42.000 X 90 kv. Notice: detacheci centrosome.

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B. Suhmicroscopical structures:

1. The cortical plasmexists of a crossing fibril system in the head reg ion which continu es af ter an uncertain transition in the neck into an anti­clockwise winding spiraI. the "tender helix" enveloping middle piece and tail as the sheath.

2. The subfibrils of the axial filament showed to consist of protofibrils with a diameter of only some tens of A units.

3. Tentatively we have described in helical and axial filaments a cross striation structure, but further 'evidence is needed in order to exclude disturbing influences of object film andgold coagulate.

4. As a new structural element a porus was observed near the equatorial zone of the head.

With regard to sperm motility an antagonism between axial' filament and cortical helix isdeemed probable.

1. ASTBURV, W . T. and R. D. PRESTON, Proc. Roy. Soc. B. 129, 54 (1940) . 2. BAVLOR, M. R. B., A. NALBANDOV and G. L. CL ARK, Proc. Soc. Expt. Biol. Meel.

54, 229 (1943). 3. BRETSCHNEIDER, L. H., Verbandl. D. Zool. Ges. 324 (1931). 4. BRETSCHNEIDER, L. H., Arcb. f. Protistenk. 82, 298 (1934) . 5. BURTON, E . F. and W. H. KOHL, Tbe Electron Microscope, 268 (1946) . 6. HARVEV, E. B. and TH. F. ANDERSON, Biol. Bull. 85, 151 (1943). 7. KLEIN, B. M., Arcb. f. Protistenk. 62, 177 (1928). 8. SCHMITT, F. 0 .. Advances in Protein Cbemistry, Vol. I. 37 (1946). 9. PRESTON, R. D. and W. T. ASTBURV, Proc. Roy. Soc. B. 122, 76 (1937).

10. WILUAMS, R. C. and R. W. G. WVCKOFF, J. Appl. Pbys. 17, 23 (1946).

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