The Electron Microscopy of Trichomonas muris

114 ELECTRON h”1cRoscoPY OF Tricho.monas

The criteria and their application in the opalinid-anuran com- plex (-4bstr.). J . Parasitol., 27, (Suppl.), 27 .

59, hlugard, H. (1948). Contribution i 1’6tude des infusoires hymenostomes histiophages. Ann . sci. nut. 2001. et b i d . ani- nzak (ser. 11). 10, 171-268.

60. Neresheimer, E. (1907). Die Fortpflanzung der Opalinen. Arch. Prolistenk., Suppl. 1. 1-42.

61. Potts. B. P. & Tomlin, S. G . (1955). The structure ol cilia. Biochinz. c t Riophys. Acta, 16, 66-71.

62. Purliinje, J. E. 8- \‘alentin, G. (1835). De phenomeno generali et fundamentali motus vihratorii . . . \’ratislaviae.

63. Raabe. 2. (1948). Pr6ba rewizji systemu pierwotniakhw. An attempt of a revision of the system of Protozoa. A n n . Univ. Marine Curie-Sklodowska, Ltrblin-Polonia, 3, 259-276.

61. Reynolds, M. E. C. (1932). Regeneration in a n amicro- nucleate iniu5orian. J . E x p t l . Zool., 62, 327-361.

65. Scheniakoff, W . (1896). The organization and system- atics oi the Iniusoria kpirotr icha (Holotvicha aitctorunz ) .

M t m . acad. imp&. sci. St. Petersbourg (Gr . 8 ) , 4, 1-395. j In Russian.]

66. Stein, F. (1860). Uher die Eintheilung der holotric1ie.i Infusionsthiere und ciniae neue Gattung und .4rten aus diepry Ordnung. Sits ber. biihin. Ges. Wiss., iVaturw.-Math., Prarue, 1860. 56-62.

67. van Overheek de Meyer, G. A. W. (1929). Beitrage zu Wachstums- und Plasmadifferenzierungs-Erscheinungen an Opalina ranariim. Arch. Protistenk., 66, 207-284.

68. Villeneuve-Brachon, S. (1940). Recherches sur les cilii.; hCtCrotriches. Arch. zool . e x p t l . e t g i n . , 82, 1-180.

69. Weisz, P . B. (1954). Morphogenesis in protozoa. Quart . Rev. Bid . , 29, 207-229.

70. Wenrich, D. H., editor (1954). Sex in Microorganisins. A.4.4S Pub., Washington, D. C.

71. Woodruff, L. L. (1921). Micronucleate and aniirronu- cleate races of infusoria. J . E x p f l . Zool., 31, 329-337.

J. PR~lTOZO(lL., 2, 114-124 (1955).

The Electron Microscopy of Trichomonas mzcl-is*+

E V E R E T T A N D E R S O N Department o j Zoology, State Zfniversity o j Iowa, Iowa City, Io.ula

SUMMARY. Thin sections of Trichoinonas muris, from the caecum of Mesocricetits auratus, were observed with the aid of the electron microscope in order to determine structural details of organelles of this species.

The blepharoplas,t appears to he limited by a mem’brane. What seem to be basal granules occur in the hlepharoplastic area for the axostyle and the lamellae of the undulating membrane and meas- urc approximately 500 A in diameter.

In cross section the anterior flagella and posterior flagellum are composed of eleven fibrils, two centrally and nine peripherally located. Each fibril measures about 380 2% in diameter.

The so-called “accessory filament” of the undulating membrane is devoid of any filamentous structure and a p p e a s to be composed of two differentiated mesh-a.ork areas. The undulating membrane is composed of a series of lamellae ranging from 300 to 400 in thickness. This organelle is attached t o the external body surface by fine fibers ranging from 167 to 300 -c in thickness.

The costa consists of a series of disks embedded in a matrix. The costa is attached to the internal body surface by what appear to he extensions of the costal disks. These disks are approsi- matelv 370 A in thickness and spaced 490

The axostyle is limited by a double, corrugated membrane. The corrugations are about 150 A in thi’cknes and spaced 110 .k apart. The so-called chromatic ring is composed of a series of rods approximately 640 A in thickness.

The parahasal body consists of a series n l filaments approximately 190 A in thickness. Mitochondria, spheroidal in shape, are limited by a membrane about 1SO A in thickness. Thc

internal structure consists of a varyinz number of projections which make this organelle appear in section as a series of compartments.

Chromatic granules (paracostal, para-asostylar, endo-asostylar and those sca,ttered in the cytoplasm) are irregular in shape and limited bv a membrane approximately 200 -1 in thickncss. These granules typically display a vacuolated internal structure.

apart.

richomonas nrzrsis (Grassi. 1879) has been found T to inhabit the caeca of many rodents(20,39). In the genus Trichomonas there exists a variety of different kinds of organelles of which the structure, division and reorganization have attracted the attention of protozoologists and cytologists. (For a detailed

* . A revision of a dissertation in zoology presented to the Faculty oi the Graduate School of the State University of Iowa in partial fulfillment of the requirement for the degree of Doctor of Philosophy (1955).

t Grants to the Radiation Research Laboratory from the Iowa Division o i the iZmerican Cancer Society have made pos- sible the purchase and maintenance of the electron microscope.

description of Trichomonas mzrris see Kirby and Hon- igberg( 20) .)

Differences of opinion regarding the details of structure of the organelles of trichomonads exist in the liter- ature. For a Letter understanding of the morphology of these organelles, a detailed picture of their fine structure would appear to be essential. The electron microscope, with its resolving power and magnification far beyond the limits of the light microscope, and the technique of ultra-thin sectioning provide a new ap- proach to the study of these structures.

ELECTRON ~IICROSCOPY OF Trichonzonas 115

X4TERIALS AKD hlETHODS Trirhoiiio7ias nzuris, used in this investigation, was

obtained from the caecum of the golden hamster (Blcsocric etirs aziratus). For the purpose of diagnosis, cover slip smears of the caecal contents diluted with saline were fixed in Schaudinn’s solution with 570 acetic acid. and stained with iron-haematoxylin. Trirkonzonas 11iiii.i.~ was also examined for mitochondria after vital staining with a solution of Janus green B ( 1 : 15 .OOO prepared in saline solution).

Portions of the caecal contents, about the size of a large pea. were suspended in centrifuge tubes that con- tained about 1 cc. of 1 % osmium tetroxide solution (fixative). buffered a t pH 7.4 with acetate veronal buffer according to the method of Palade(28). The material was fixed for 10-15 minutes and subsequently washed. dehydrated and infiltrated with methacrylate monomer. The methacrylate monomer consists of 727% K-butyl methacrylate and 28% methyl methacrylate with a catalyst added (Luperco, 0.2 gm./ 10 cc.). The material to be embedded was placed in fresh monomer in Number 4 gelatin capsules. Each capsule was centrifuged in order to concentrate the organisms a t one end and polymerized in an oven a t 47°C. for eight hours.

The polymerized block was sectioned with an Tnter- national Minot Rotary Ultra Thin Sectioning hlicro- tome using a ‘5 inch plate glass knife. Sections were cut approximately 0.025 p thick and ribbons so obtained were floated directly into a trough containing 25% diosan. The sections were mounted on grids previously covered with a supporting film prepared from a 2‘3 solution of celloidin in amyl acetate. After the sections were dried, the embedding mixture was removed: all qrids were stored in a desiccator until esamined.

Observations were made using a Model EMU-2B- RCA electron microscope. The electron micrographs were taken at an original magnification of 4600 or 640.0 s and thereafter enlarged photographically as desired.

Diagnosis of the stained slides revealed that Tricho- 17IOI iUS minictcr and T . microti in very small numbers were also present in the caecal contents. Since T . iiiziris is the largest of the three, electron micrographs were taken only of the largest fragments or cross sections found. Sections small enough to have been possibly those of species other than T . T ~ Z U ~ ~ S were ignored.

DESCRIPTION O F RESCLTS Rlcpharoplast. The blepharoplast of Trichoiiionas

inuris is located a t the anterior end of the major axis of the body (LB, Fig. 7 ) and is the point of origin of the flagella and a number of other structures (undulating membrane, costa, axostyle and parabasal

body). It seems to be limited by a membrane, inside of which may be seen cross sections of the anterior flagella (AF) and posterior flagellum ( P F ) . IVithin the blepharoplast, a t the lower right (Fig. 7 ) , is seen a portion of a membrane in the form of a broken cir- cle with no visible internal structure. This membrane is possibly related to a third anterior flagellum in the region of its origin. At the left, attached to the periphery of the blepharoplastic membrane, is seen a part of the undulating membrane (UM).

=interior flagella. Figs. 2 and 3 are tangential sections of the anterior end of the organism showinq longitudinal arrangement of the fibrils of the anterior flagella (AF). In cross section the anterior flagella appear to consist of eleven fibrils (AF, Figs. 6 and 7 ) . Nine of these fibrils are peripherally located, whereas the other two occur as a pair in the center (Fig. 1 ) . The central fibrils of a flagellum are not always resolvable into two distinct fibrils. In this situation the central structure measures approximately 630 LJ in diameter, which is about twice the diameter of a single fibril. The fibrils of the flagella measure about 380 ri in diameter and are surrounded by a relatively structureless matrix. Fibrils and matrix are enclosed by a limiting membrane approximately 270 .\ in thickness.

Posterior f lagellum, “,-Iccessory filament” and Urr- dulating membrane. Two structures, the posterior flagellum and “accessory filament,” are described as forming the outer limits of the undulating membrane. The posterior flagellum (PF, Fig. 6 ) appears to originate in the blepharoplastic area (BA, Fig. 6 ) and con- stitutes the major marginal filament of the undulating membrane (UM, Figs. 4, 14, 15 and 16 ) . This flagellum has the same submicroscopic structure as described for the anterior flagella.

The so-called “accessory filament.“ located parallel to the posterior flagellum, may be seen a t ACF in Figs. 4, 15, and 16. I t appears to be composed of two differentiated mesh-work areas which occupy opposite sides of a zone between the posterior flagellum and the laniellae of the undulating membrane. In many cases (e .g . Fig. 16), there is little indication that these two areas are intimately connected. The entire zone is wider than the diameter of the posterior flagellum (Fig. 16) , but its radial depth is small.

The undulating membrane, (UM, Figs. 1, 4, 8, 1 5 and 16) which in light microscope observations takes a spiral course along the surface of the organism. seems to originate from the blepharoplastic area ( B A . Fig. 8 ) . This organelle appears to consist internally of a series of lamellae, arranged parallel to the longitudinal axis of the undulating membrane (LUAI, Figs. 4. 7 and S ) , ranging from 300 to 470 -4 in thickness. The lamellae of this organelle seem to have their origin

116 ELECTRON MICROSCOPY OF Trichonzonas

Figs. 1-19. Tlir clcctroii iiiicrograplis slioiv seetioiis of Triclwtrio/icis ? t i i / r i s that, mere oht:iiiicd f rom the caoc:i of three dif-

Fig. 1. Tanpciitinl section slioiviiig niidu1:ttiiig mriiibraiie ( U M ) aiitl anterior flagello ( A F ) origiii:itiiig in tlic hlrpli- :irol)lastic area ( B A ) . Note oiie of the anterior flagella sliows two filbrils iu i ts wiitrr. iiig longitudiiial arrangeiiient of tihrils of the anterior fl:tgella (AF ) , origiiiatiiig iii tlir b1epli:iropl:istic aw:t ( B h ) . Nito- c.lioiitlri:t shoi~iiig coinpa~tiiinits are seen a t (M). Fig. 3, Tniigeiiti;rl section showing loiigitudiiinl arr:iiigtwiriit~ of tlic filjrils of the :interior flagella ( A F ) . The aiitcrior flagella aiid costa (C) origiii:itc? from the l)lepli:i~ol,l:istic :ire:i (ELL). 1\Iitocl1oiitlrin (M) may hc seeii a t the peripliery of the orgniiisiir ant1 tlrc corrugated ii:itiirr of tlir :i~ostylr. :it (AX) . d chroniatic gr:inulc niny be swii at CG :ind a food vacuole at FV. Pnrwcostal gr:iiiiilrs (PCCi) iii:iy IJ? st'(w along tlic costa. Also the niidulntiug niciiil~r:iilc i x y IJC sreii xitli its 1:imell:ic (LUM), so-c:~lle~l " acccssorg filmiieiit " (ACF) and posterior Aagrllui~i (PF). Fig. 5. T;riig~~iitinl section slion.iiig the asostgle (AX') diffcreiiti:ited iii the blepharoplastic :rwa ( R A ) :is :I biilb-like sirtxlliiig arising froiii u-lint ap- ~ J W ~ S to be n basal grniiiile ((;A ).

frrri i t Iiaiiisters. A inag1iific:itioii scale represcriting oiie microii is d r a ~ v i i 0 1 1 c ~ i c . l i fipur~~.

Fig. 2. T:iiig:mtial

Fig. 4. Tangenti:il sectioii showing nucleus ( N ) and costa (C).

ELECTRON ~UICROSCOPY OF Trichontonas 117

Figs. 6-9. Electron iiiicrogr:ilrlis of tliiii sectioiis of Tviclro?iiontrs n ~ f t r i s . Fig. (i. T;iiig(wti:il section showing cross srctioii of tlic :iiitcrior fl;igrlln ( A F ) ancl posterior flagelluiii (PF). The nxo-

style [-IS I i - st’c’ii iii tlic I~lcplinroplnstic area (BA) as a hxll)-likc swelling. Endonxostylar grniiulev :It EAG, niicl tlic so- callrd “c~- t i i~ tc~i i i r ’ : :it, CY. Fig. 7. Loiigitudiiial sertion showing tlir liiiiit of tlip hlrpharoplast (LE) with its iiitrrior eniitaiiiiiig c i u \ s srctioi i of the anter ior flagella ( A F ) and iiostcsrior fi:igclluiti (PF). Note uiiclulatiiig iiicnibmie (UJ1) rritli 1 : i i i i t ~ l I : i t ~ (I ,L->t) :it,tacliecl to tlie periphery of t h e b1epli:irolil:istic nieiiihrnnc. The nucleus (N), endosome (ED) an& the, s o - ~ : i I l ~ ~ d ” ~ . ~ . t i i s t o i i i r ~ ’ ’ ( C Y ) i i i ~ y he seen. Fig. 8. T:ingeiiti:il sectioii shoiviiig uiitlulnting iiiemliraiie (UM), costa (C) a i d p:tr:ib:i?;:il (PE ) :irjsiiig from the blepharoplastic area. A grniiiile for the lninellae of tlie uiidul:itiiig nienibrane iiiny be wen nt C;L-Jl. Sotr fil:nnmit, of tlir parabasal body (PB) :iii11 liitnellac of tlie iiiiiliilnting ~ncnibrniic (LUM). Fig. 9. ~~oi lg i tud i i i : i~ e w t j o i i slio\ring the costa (C) originntiiig froin the hlrplinroplnstie awn (EA’J. The attacliiiieiit of the 1111- ilu1:itiiig 1iiciiilIr:i t i t > (1 ’AT) iiiny lir srcii a t AUAf. The nxostylcx ( A S ), c~iit1o~isostyl:iI grmiulrs (EAC;), eliromatic graiiiile (CC;i, iiiiclvus i S 1 :lii(l tlic so-called “c~-toatoine” (CY) niny l ~ , serii.

118 ELECTRON MICROSCOPY OF Trichomonas

from a basal granule ( G C N , Fig. 8) that measures approximately 500 A in diameter. In most electron micrographs this internal mass of aggregated lamellae does not have uniform width. The width of this mass is greatest in the region adjacent to the so-called “accessory filament” and least where it joins the base of the undulating membrane. Consequently the undulating membrane is wedge-shaped when seen in cross section (Figs. 1.3, 15 and 16). The limiting boundary of the undulating membrane surrounds the posterior flagellum and the so-called “accessory filament” as well as the lamellae. In some of the electron micrographs the limiting boundary is lifted away from the lamellae; this “blistering” perhaps occurs during the preparation of the material.

At AURS in Figs. 9 and 1 5 the undulating membrane appears to be connected to the body surface by a series of fine fibrils that range from 167 to 300 W in thickness.

Costa. The costa ( C , Figs. 3, 8 and 9 ) takes origin in the blepharoplastic area and passes posteriorly in the cytoplasm in a curved path (Figs. 3, 7 and 13). This organelle is narrow a t the anterior end (Fig. 3 ) , where it leaves the blepharoplastic area and becomes thicker in its niidregion (Figs. 13 and 14). The costa consists of a linear array of areas of relatively high electron density alternating with others of low density. In Fig. 14, these regions of high density, cut a t their surfaces may be seen to have a curved contour. In Fig. 13 the costa is cut longitudinally, and a cross section of these areas is seen. This suggests that the regions of high electron density are disks and that the regions of low density constitute a matrix. Measure- ments indicate that the costal disks are approximately 370 A in thickness and spaced 490 A apart.

Where the costa is opposite the body surface (C, Figs. 12 and 14), orientated cytoplasmic strands are seen. These strands approximate the costal disks in thickness and can be traced from the body surface to the costal disks. This arrangement suggests that the cytoplasmic strands are extensions of the costal disks and make the costa appear to resemble a series of table tennis paddles placed adjacent to one another with their handles oriented toward and attached to the body surface.

In Fig. 9, filaments may be seen attached to the costa a t its origin. These filaments could not be iden- titied with any previously described structure.

The avostyle is an elongated tube originating in the blepharoplastic area perhaps from what appears as a basal granule (G.4. Fie. 5 ) . It proceeds posteriorly in its course. passing around to one side of the nucleus (AS. Fig. 6 ) and then through the major axis of the body to project at the posterior end, where it tapers sharply to a point

.IxostyZe and Chromatic ring.

(Fig. 18). This point does not penetrate the body surface but is surrounded by the outer membrane of the organism.

The membrane of the axostyle appears to be double (AX, Fig. 1 5 ) and corrugated (Figs. 3. 10 and 13). ’The axostyle is differentiated in the blepharoplastic area where it displays a bulb-like swelling (Figs. 5 and 6 ) . h1easurements show that the corrugations of the limiting boundary of the axostyle are approximately 150 A in thickness, with spacings measiirinc abnut 110 A.

Surrounding the axostyle in the cytoplasm of the posterior end of the body is the so-called chromatic ring (CR, Figs. 17 and 18). I t seems to consist of a series of electron dense rods, located in a vesiculated area. These rods appear to project from the outer limits of the vesicle toward the surface of the axostyle, and a few can be seen attached to the asostyle (Fig. 17). Measurements show that the individual rods have thicknesses of approximately 640 A.

The parabasal body orieinates in the blepharoplastic area, and proceeds posteriorly where it curves around the nucleus (PB. Fies. 10 and 11 ) . The fine structure of this organelle as revealed by this study consists of a series of parallel filaments approximately 190 A4 in thickness.

Aiitochondria. Certain spheroidal bodies which are interpreted as mitochondria, are evident at >I in Figs. 2 and 3. Their diameter is 2630 to 4220 A and they have a limiting membrane 180 ‘4 in thickness. The internal structure of the mitochondria is composed of a varyiny number of projections from the external limiting membrane that come in contact with an internal membrane. This arrangement of the projections makes the mitochondria appear to consist of a series of compartments (31, Figs. 2 and 6 ) . However. this internal structure could not be seen clearlj- in all mitochondria and some appear as vesiculated spheres pos- sessing an electron dense outer area (Fic. 3 ) . A region devoid of ohvious structure is present in the center of each mitochondrion and in each ”cninpartment.” These regions probably constitute the matrix of this organelle.

For the most part, the mitochondria are located a t the periphery of the organism and a few are scattered around in the cytoplasm (31. Fie>. 3 and 6 ) . This same spatial arrangement was als3 observed in animals that were prepared by the Janus green 8 technique for mitochondria.

Chromatic- granules. In trichomonads. cytoplasmic bodies which stain densely with basic d j es have been reported and named chromatic granules. Certain of these granules in Trichoinonus nzirris ha\ye a definite spatial arrangement in the cytoplasm. A few are located parallel to the costa. the paracostal zranules

Parabasal body.

ELECTRON MICROSCOPY OF Trichonionas 119

(PCG. Figs. 3 and 13). and along the asostyle. the paraasostylar granules (PAG, Figs. 15 and 19). A number of these granules are found inside the asostyle, the endoaxostylar granules (EAG, Figs. 6 and 9 ) . A few chromatic granules are scattered in the cytoplasm without apparent orderly arrangement (CG, Fig. 9 ) .

The chromatic granules are generally spherical in shape and very irregular in size, ranging from 3 160 to 4210 A in diameter. Typically they display a vacuolated, internal, submicroscopic structure limited by a membrane about 200 A in thickness (Fig. 9).

“Cytostoirw.” The so-called “cytostome” seems to be a cytoplasmic region of low electron density adjacent to the axostyle a t the anterior end of the organism (CY, Figs. 6, 7 and 9). It is limited on one side by the membranes of the axostyle but on the opposite side no demonstrable membrane is evident.

DISCUSSION I t has long been evident that in protozoan and meta-

zoan cells there exists morphological complexity which cannot be resolved by the light microscope. Knowl- edge of this organization is important, since it con- tributes not only to protozoan morphology, but also aids in the understanding of physiological processes and problems dealing with morphogenesis.

Very little is known with certainty regarding the details of internal structure of the blepharoplast of trichonionads. Kofoid and Swezy( 2 1 ) reported that the blepharoplast of Trichomonas ttzurz‘s consists of two parts, one of which is a centrosome and the other a basal granule for the flagella. Wenyon(48) and b‘enrich( 43) also described the blepharoplast of Tric-honzonas nuiris as double, but with the posterior flagellum attached to one granule and the chromatic basal rod (costa) attached to the other.

-4 number of investigators have described the blepharoplast of other species of Trichomonas to be a composite structure with the number of granules either approximating or equalling the number of flagella (4, 7,13.15,16.34,45). Ludvik( 24 ) published electron micrographs of whole mounts of Trirlionzonos foetus and reported the blepharoplast to consist of six granules surrounded by a denser opaque cytoplasm.

The blepharoplastic region is widely recognized as a center for attachment of organelles (e.g. 13,19.26,44) and some writers imply that each organelle is associ- ated with a granule. The electron micrographs here presented support this idea and are not in agreement with the interpretation advanced by Kofoid and Swezy ( 2 1 ) and Wenrich(43) for Trichomonas muris.

Since the advent of the electron microscope, flagella have been the subject of investigation more than any other single protozoan organelle. A number of observers have described electron micro-

Blcpharoplast.

Fln,yclla.

graphs of whole mounted specimens that had been either dried, hydrolyzed or shadowed with chromium. Depending upon the technique employed. workers have demonstrated that the axoneme either is a dense core or is resolvable into nine or eleven parallel fibrils surrounded by a sheath with or without a helical coil (5.6,9,12,1 S.22.23,24,25,32.38,41).

Woken and Palade(49) studied thin sections of the flaqella of Euglena gracilis and Poferiochromonas stipitczta and reported that in cross section the flagella consists of eleven fibrils embedded in a matrix. Nine of these fibrils were found to be peripherally located while the other two occurred as a pair in the center. The electron micrographs here presented of the flagella of Trichomonas muris, demonstrate essentially the same internal structure as that found by I\’olken and Palade (49 ) .

Posterior flagellum, “ A rressory filanzent” and Un- dulating membrane. A number of investigators are of the opinion that the margin of the undulating niem- brane consists of two filaments: the marginal filament. which continues as the trailing flagellum. equal in diameter to the anterior flagella: and an “accessory filament” with a diameter greater than that of the marginal filament ( 13,20,2 6,2 7.43,44,45,46). \\’enrich (44) reported that sometimes the “accessory filament” appeared double. Buttrey ( 7 ) described three filaments embedded in the undulating membrane of Tritricho- inonas azrgusfa: the marginal, “accessory” and “subaccessory” filaments. Buttrey reported that with silver impregnation the “accessory” and “subaccessory” filaments are thicker than the marginal filament. which is the posterior flagellum. Gabel( 13) described three filaments as parts of the undulating membrane of Tritrichomolzas acnrichi: (1 ) a primary filament, the trailing flagellum ; ( 2 ) a secondary filament, appar- ently the “accessory filament” of other authors, a t the edge of the primary filament; and (3) a fine supra- costal filament embedded in the undulating membrane.

As has been stated, the posterior flagellum appears to have the same internal structure as the anterior flagella. This flagellum as far as morphology is con- cerned, is the marginal filament. Since the submicroscopic structure of the “accessory filament” is mesh- like, the author prefers to call this area the iwtramar- ginal mesh-work area. Perhaps the two differentiated areas are what Wenrich(44) observed when he men- tioned that the “accessory filament” is sometimes double. Also one of these areas may be the “subaccessory filament” as reported by Buttrey( 7 ) for Tritricho- 7nonas augzrsta.

Wenrich (44) observed that after Trickornonus honzinis was subjected to several different fixation procedures the “accessory filament” in some cases ex- panded considerably. A number of investigators have

120 ELECTRON MICROSCOPY OF Trichonzonas

ELECTRON ~IICROSCOPY OF Trichomonas 121

122 ELECTRON MICROSCOPY OF Trichomoiias

reported a difference in staining reaction of the marginal and “accessory” filaments. When the organisms are subjected to the silver-protein technique, the “accessory filament” is often more heavily impregnated than the marginal filament or posterior flagellum : however, this is not usually the case with iron-haematoxylin stain( 7,20,27.43,46). The submicroscopic structure of the marginal filament and the intramar- ginal mesh-work area substantiates the occurrence of a fundamental difference in the organization of these structures as was suggested by observations of Wen- rich and others on fixation and staining.

Jlost observations upon trichomonads have described the undulating membrane as a protoplasmic sheet originating from the blepharoplast and taking a spiral course along the surface of the organism. The evidence from the electron micropraphs seems to indicate that the undulating membrane is attached to the surface by fine fibers and is composed internally of a number of parallel lamellae.

Costa. The costa is usually described as a slender homogenous rod located beneath the undulating membrane( 17,27,37,43,44,45). Ludvik(24) from an electron microscope study of whole mounts of Tricho- m o m s joetus found the costa to consist of a “series of striations.” Evidence here presented indicates that the costa is made up of a series of disk-like structures embedded in a homogeneous matrix.

The costa is assumed by numerous investigators to be a supporting structure. In light microscope studies it has been observed that changes in the position of the costa are usually accompanied by corresponding changes in the form of the body. The present study supports this observation. The structure of the costa, as herein described, could provide both rigidity and f’iexibjlity.

hfoskowitz (26) reported the axostyle of several reptilian trichomonads to be a hyaline tube formed from a spirally rolled membrane. Saxe( 38) described electron micrographs of Trichomonas wcnyoni ? and found the axostyle to be composed of three spirally arranged ridges which he believed to be fibrils. There is no evidence in the present study of Trichomonas mzrris to indicate a spiral membrane or fibrillar internal structure of the axostyle. Ludvik( 24) in his study of Trichomonas foetus described the base (posterior termination) of the axostyle as forming a “collar.” Ludvik’s interpretation does not appear to be applicable to Trichomonas muris.

Trichomonas muris, when stained with iron-haema- tosylin, shows a dark staining cytoplasmic area (chromatic ring or rings) surrounding the posterior end of the axostyle(3,4,49). Other workers have reported this area in muris-type trichomonads to consist of one

:I.xostyle and Chromatic ring.

or more rings(7,13.27). Ludvik(24) from an electron microscope study of Trichoinonas Joetzrs, reported the chromatic ring to consist of granules.

This study demonstrates that the “chromatic ring” is composed of a series of electron dense rods. Since the dimensions of these rods lie below the limit of resolution of the light microscope the staining of the “chromatic ring or rings” presumably results from an absorption of dye on the rods or in the surrounding cytoplasm.

ParabasaZ body . Nearly every investigator has described the parabasal body of trichomonads of the muris-type as a sausage-shaped structure oriqinating from the blepharoplast. GrassC( 14) believed that the organelle typically consists of two parts. a chromo- phobic interior and a chromophilic exterior. A similar organization for the parabasal body has been described by other workers( 13,20.27,43). Ludvik(24), as a re- sult of electron microscope studies of Trichotnonas foetus, reported that this organelle appeared as a “club-like shadow on the anterior part of the nucleus.”

Kofoid and Swezy(21) were of the opinion that the parabasal body of trichomonads varied in size according to the metabolic demands of the organism. \Ven- rich(43) suggested that the distal portion of the parabasal body of Trichomonas inuris may become de- tached and serve some function in metabolism. nu- boscq and GrassC(l0) were of the opinion that this organelle was secretory in function and homoloqized it to the Golgi apparatus.

With the aid of the electron microscope. a number of investigators observing protozoan and metazoan cells have demonstrated a group of filaments which they have called basophilic material( 2,9,33,49). Porter (33) has termed this filamentous, basophilic material the endoplasmic reticulum, and believes it to be a system of vesicles and tubules. The components of the parabasal body of Trichomonas miiris seem to pos- sess a similar structure and coincide in size and form to the endoplasmic reticulum as described by Porter (31,33) and other workers.

Mitochondria. Palade( 28.29,30), investigating mitochondria of various rat tissues. reported them to consist of a double limiting membrane and a system of internal ridges or cristae mitochondriales that protrude perpendicularly from the inside surface of the limiting membrane toward the interior of the organelle. This author also has reported the same mitochondria1 structure for two flowering plants, Lemizn sp. and Sicot iana and two protozoa, Euglena grocilis and Potcriockro- monas stipitata. Other workers have reported this same internal structure for different types of cells( 11. 36,42).

Powers, Ehret and Roth(35) reported the mitochondria of Paramecium aurelia and P. bursaria to be cylin-

ELECTRON JIICROSCOPY O F Trichonzonas 123

drical, approximately 9100 -3 long and 5400 .i in diameter. They reported the internal structure to consist of an osmiophilic continuum interrupted by small tubular spaces of about 140 ‘4 in diameter. Sedar and Porter(40) reported that the mitochondria of P. iizulti- i~rjfronzi~lcatmnz are limited by a membrane from which microvilli project into the mitochondrial matrix. Beams and Tahmisian( 3) found that the mitochondria of the male germ cells of Helix aspersn consisted of longitudinally oriented filaments or lamellae inside a limiting membrane. The structure herein described for the mitochondria of Trichonzonas muris is not in agreement with any of the previous reports on the submicroscopic structure of mitochondria.

Chromatic g r a d e s . Buttrey( 7 ) believes the chromatic granules of Tritrichoiizonns augusta to be waste products of the cell. This he assumes on the basis of the fact that the granules tend to accumulate in the posterior portion of the organism, and a t the time of autotoniy, the portion that is lost includes a number of these granules. Alexeieff (1) called the chromatic granules mitochondria. This is doubtful, for they are seen following fixation with Schaudinn’s solution. which is known to destroy mitochondria. Obviously the true nature of the chromatic granules is not known.

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3. Bearms, H. W. & Tah,misian, T. N. (1954). Structure of the mitochondria in the male germ cells of Helix as revealed by the electron microscope. Expll. Call Rrsrarch, 6, 87-93.

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13. Gabel, J. R. (1954). The morphology and tasonomy of the intestinal protozoa of the .4merican woodchuck, Marntota nzonar Linnaeus. J . Morphol., 94, 413-549.

Contribution a I’ttude des flagell&s parasites.

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16. Hogue, M. J. (1926). Studies on Trichoinonus Duccnlis.

1 7 . Honigberg, B. M. (1951). Structure and morphogenesis of Trickontoncis prowawki (Alexeieff) and Trichotnonns brunzpti (Alexeieff). l Jn iv . Calif. (Berkeley) Pitbls. Zool., 55, 33i-394.

18. Houwink, 4. L. (1951). An electron microscope study of the flagellum of Euglpna gracilis. Proc. Koninkl. 5 ~ d . .+lknd. Welrnschap., 54, 132-137.

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21. Kofoid, C. A. 8: Swezy, 0. (1915). Mitosis and multiple fission in trichomonad flagellates. Proc. A m . Acad. A r t s Sci., 51, 289-378.

2 2 . Kraneveld, F. C., Houwink, A. L. 8: Keidel. H. J. W. (1951 ) . Electron microscopical investigations regarding the structure of Trypanosonia evansi. Proc. Koniiikl. -Ved. d k a d . Wetrnschap., 51, 393-399.

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124 MORPHOLOGY AND DIVISION OF E . neglcctuni

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J. PROTOZOOL, 2, 114-134 19551.

Observations on the Morphology and Division of Eudiplodinium neglectum Dogiel (Ciliata Entodiniomorpha) from the Stomach of a Moose

(Alces arnericana) .* SERHIJ KKASCHENINNIKOW

I ’ i t iwrs i ty of Punnhylvania, Departmtwt of Zoology, Pkiladclphia, Pennzyivanm

SUMM.4RY. The material containing the ciliate Ezidiplodiniuirt neglecttun Dog. from the rumen o i a moose was fixed with 4% formalin solution and studied both unstained and after staining with methylene blue or methyl green.

This ciliate is closely related to Eudiplodinizmz neglectzim Dog. (1925) formae spectubile Dog. (1925) and impalae Dog. (1925), bu t differs from them in the presen’ce of a transverse cuticular line. The form of the ma,cronucleus of the investigated ciliates is regularly elongated clavate, the anterior end being wider than the posterior one. The following measurements were obtained from 14 specimens: length 81.3 p (67.2-112 p ) , width 66.2 p (48.8-84 p ) .

During division the anterior daughter gets the old skeletal plate and a new one arises de novo in the posterior daughter. The anlage of a new skeletal plate for the posterior daughter was observed in earlier and h t e r telophases, first as an indistinct structure, later as a group of a variabl:. number of granules, more often about 30. ‘Phis anlage gradually grew into a typical skeletal plate. The anlape of the pharynx in the posterior daughter was seen in later telophases. A part of the distal end of the old skeletal plate (one, two or five rectangular prisms belonging to this orgamlle) and a part of the distal end of the old oesophagus are possibly cut off during division and dissolved in the protoplasm of the posterior daughter. A crossing of the distal end of the old skeletal plate by the divi,sion plane was observed only in those specimens in which this part consisted of one row of the rectangular skeletal prisms a n d was not found in the ‘specimens with a shaft of the skeletal plate consis,ting of two rows of polygons. In laster division stages a bending of the old skeletal plate takes place.

The morphological plasticity of the skeletal plate (loss of the distal part and subsequent bending of this organelle during the division of the ciliate) observed by the present writer and its dy- namic reversibility (disappearing during starvation and thickening under satisfactory feeding condi- tions, reported by Westphal) confirm the assumption that the skeletal lamina functions primarily as a storage organelle. Some cases of destruction and defective development of the old skeletal plate were observed.

The presence of fa t in some specimens of E . neglectuut Dog. is noted.

HIS PAPER is the report of a study of the ciliate Eirdiplodininin neglrctima Dogiel from the moose

(AIces arn f r i rana) . The moose 167’ years old, was killed on October 12,

19.52, a t Lac Ganiain, Abitibi, Quebec, Canada, by Mr. Jean Croteau to whom I express my sincere gratitude for the opportunity to study this material. I am in- debted also to Dr. D. H. Wenrich of the University of Pennsyl\.ania and to Dr. C. \Y. Rletz for their many courtesies and assistance.

The data published in this paper had been reported at Fif th Annual Ale:ting of the Society of Protozoologists held in Madi- son, \Yisc(insin. September 7-9, 1953. See Proceedings of the Society of Protozoologists. Vol. I , pp. 5-6, 1953.

The material was taken from the rumen in the field and fixed in 470 formalin solution. Afterwards it was washed and observed in temporary mounts ( i n water).l At times these mounts were stained with methylene blue or methyl green acetic acid to learn the shape of the nuclei.

SYSTERIATIC STATUS OF E . ncglrctirin DOGIEI, nogiel(7) gives the following forms of E . ncglcc-

turn: n@ectuin, bovis, spcctabile, itnpalae, insigne, gigantcum, inonolobi{iiz, rugosuiiz, dilobziin.

1 Chlor-zinc-iodine caused great deformation of the ciliates and therefore could not be used for this investigation.

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