Cytoplasmic budding of a nuclear polyhedrosis virus and comparative ultrastructural studies of envelopes.

The processes of cytoplasmic budding in Euproctis subflava nuclear polyhedrosis virus (NPV) were investigated, and comparisons were made among three types of envelopes which were acquired by, 1) de novo morphogenesis in the nuclei, 2) nuclear budding, and 3) cytoplasmic budding. The direction of nucleocapsids in the envelope was the same in these three modes of envelopment; the envelopment seemed to occur from a nipple end which was at one extremity of the nucleocapsid. After the envelopment, electron-dense materials were seen between the envelope and nucleocapsid, though their contents and morphological features were different among the three types of envelopes. However, these materials seemed to function similarly as a mediator between the envelope and nucleocapsid as have been observed in many vertebrate viruses which acquire envelopes. A marked difference among the three types of envelope was the characteristic cap-shaped structures with spikes which were seen only on the surface of envelope derived from the plasma membrane. After cytoplasmic budding, nucleocapsids enveloped by this way were located on the basement membrane or liberated in the hemocoel, and then they appeared to enter neighboring healthy cells via viropexis with the spike end at the head. At the sites where these spikes came into contact with healthy cells, coated vesicle-like structures were observed inside the plasma membrane. Occasionaly, incomplete particles which lacked nucleocapsids were also budded through the plasma membrane and released into extracellular space.     

Electron-dense endoplasmic reticulum-like profiles closely associated with mitochondria in glomus cells of the carotid body after fixation with oxalate

Fixation in the presence of oxalate was used to demonstrate the electron-dense Ca2+ precipitates in the endoplasmic reticulum in glomus cells of the carotid body. Glomus cells in intact carotid bodies or cells dissociated from the organ by treatment with collagenase were studied electron microscopically. In the intact organ as well as in dissociated glomus cells, electron-dense endoplasmic reticulum-like profiles were seen closely associated with mitochondria, while these lacked reaction product. The interspace between mitochondria was occupied by electron-dense, slightly distended ER, which appeared to contact the outer membrane of the mitochondria. Occasionally, a mitochondrion was in contact with several ER profiles or the ER formed an electron-dense 'cap' on the mitochondrion. The electron-dense precipitates could be removed from ultrathin sections with the calcium chelator ethyleneglycol-2(2-aminoethyl tetra-acetic acid) (EGTA). It is tentatively suggested that the endoplasmic reticulum could be involved in intracellular buffering of Ca2+ in the glomus cell, as has been previously suggested for neurons.     

鸭病毒性肠炎病毒强毒株的形态发生学与超微病理学研究

应用透射电镜和超薄切片技术,研究鸭病毒性肠炎病毒(duck enteritis virus,DEV)CH强毒株人工感染成年鸭后,病毒在宿主细胞内的形态发生及各组织器官的超微结构变化.结果表明,感染后不同时间剖杀及发病后死亡鸭的肝、肠、脾、胸腺、法氏囊等组织器官中,均观察到典型的疱疹病毒粒子.病毒主要的靶细胞为淋巴细胞、网状内皮细胞、成纤维细胞、巨噬细胞、血管内皮细胞、肠道上皮细胞、肠道平滑肌细胞和肝细胞等.DEV的核衣壳有空心型、致密核心型、双环型和内壁附有颗粒型四种形态,存在胞核和胞浆两种装配方式.病毒核衣壳可在核内获得皮层,通过核内膜获得囊膜成为成熟病毒;也可通过内外核膜进入胞浆,在其中获得皮层,然后在各种质膜上获得囊膜,最后成熟病毒释放到细胞外.伴随着病毒的复制、装配和成熟,细胞中出现多种核内和胞浆包涵体、核内致密病毒核酸颗粒、微管和中空短管以及胞浆内膜包裹的电子致密小体、双层管等病毒相关结构.超微研究表明,组织细胞有坏死和凋亡两种变化.坏死细胞肿胀甚至破裂,线粒体肿胀空泡化,粗面内质网扩张,核糖体脱落,有的细胞器甚至完全崩解,染色质或固缩或溶解.凋亡细胞则染色质聚集,胞浆凝聚深染,细胞膜上有大量空泡,并有凋亡小体形成.细胞坏死与凋亡往往同时存在,疾病发生过程中,脾、胸腺、法氏囊以及小肠固有层中的淋巴细胞凋亡数量明显增多.     

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.     

Cytologic and ultrastructural findings of a peculiar alteration in cervical cells from patients with human papillomavirus infections

Inclusion bodies were previously found in Papanicolaou smears of patients infected by the human papillomavirus (HPV). To ascertain their origin, 12 biopsies from patients with colposcopic evidence of papillomatous lesions were studied by cytology and electron microscopy. In several instances, koilocytotic atypias and electron-dense masses included within epithelial cells were observed. Some of these epithelial cells appeared to be surrounded by other cells in a sort of concentric arrangement. The electron-dense masses were composed of intermediate filaments, vacuoles and electron-dense material. They seemed to be dyskeratotic cells. They can be compared with apoptotic bodies and may be related to a disturbance of the involucrin expression caused by the HPV infection.     

Ultracytochemical localization of the vacuolar marker enzymes alkaline phosphatase, adenosine triphosphatase, carboxypeptidase Y and aminopeptidase reveal new concept of vacuole biogenesis in Saccharomyces cerevisiae

Logarithmic cultures of Saccharomyces cerevisiae strains LBG H 1022, FL-100, X 2180 1A and 1B were studied together with the mutants pep4-3, sec18-1 and sec7-1. The necessary ultrastructural observations showed that, as a rule, juvenile vacuoles were formed de novo from perinuclear endoplasmic reticulum cisternae (ER) packed and inflated with electron-dense (polyanionic) matrix material. This process was disturbed solely in the sec18-1 mutant under non-permissive conditions. The vacuolar marker enzymes adenosine triphosphatase (ATPase) and alkaline phosphohydrolase (ALPase) were assayed by the ultracytochemical cerium precipitation technique. The neutral ATPase was active in vacuolar membranes and in the previously shown (coated) microglobules nearby. ALPase activity was detected in microglobules inside juvenile vacuoles, inside nucleus and in the cytoplasm as well as in the membrane vesicles and in the periplasm. The sites of vacuolar protease carboxypeptidase Y (CPY) activity were assayed using N-CBZ-L-tyrosine-4-methoxy-2-naphthyl-amide (CBZ-Tyr-MNA) as substrate and sites of the amino-peptidase M activity using Leu-MNA as substrate. Hexazotized p-rosaniline served as a coupler for the primary reaction product of both the above proteases (MNA) and the resulting azo-dye was osmicated during postfixation. The CPY reaction product was found in both polar layers of vacuolar membranes (homologous to ER) and in ER membranes enclosing condensed lipoprotein bodies which were taken up by the vacuoles of late logarithmic yeast. Both before and after the uptake into the vacuoles the bodies contained the CPY reaction product in concentric layers or in cavities. Microglobules with CPY activity were also observed. Aminopeptidase was localized in microglobules inside the juvenile vacuoles. These findings combined with the previous cytochemical localizations of polyphosphates and X-prolyl-dipeptidyl (amino)peptidase in S. cerevisiae suggest the following cytologic mechanism for the biosynthetic protein transport: coated microglobules convey metabolites and enzymes either to the cell surface for secretion or enter the vacuoles in all phases of the cell cycle. The membrane vesicles represent an alternative secretory mechanism present in yeast cells only during budding. The homology of the ER with the vacuolar membranes and with the surface membranes of the lipoprotein condensates (bodies) indicates a cotranslational entry of the CPY into these membranes. The secondary transfer of a portion of CPY into vacuoles is probably mediated by the lipoprotein uptake process.     

pH-dependent influence of a quaternary ammonium salt and an aminoester on the yeast Saccharomyces cerevisiae ultrastructure

Quaternary ammonium salts inhibited the growth of yeast especially at pH higher (pH 8) than optimal. It was postulated that compounds integrate with the cell membrane and interfere with its functions. The yeast cell ultrastructure investigated under an electron microscope confirms this hypothesis. A relatively high percentage of cells treated at pH 6 with the quaternary ammonium salt of alanine derivative (DMALM-12) at the minimal inhibitory concentration showed an irregularity in the cell shape. No such irregularity was observed in the control. Besides, in the cells treated with the drug, practically no lipid droplets were seen at all. Inside the control cells, electron-dense round bodies were clearly seen and interpreted as vacuoles. These bodies were absent in the cells treated with DMALM-12. Although the yeast cells growing at pH 8 showed a more or less normal shape, they seemed to have difficulty in budding - no fully developed buds were found in the preparations. Only some convexities of the cell wall were seen that could be the beginning of budding which stopped early after the start. Some changes in the round bodies interpreted as vacuoles were visible: they were less dense and full of granules.     

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.     

Electron microscopic observations of FL cells infected with herpes simplex virus. III. Dense bodies.

FL cells infected with the -GCr Miyama strain of herpes simplex virus at an adsorbed multiplicity of approximately 10 were fixed at late stages of infection and examined by electron microscopy. Dense bodies containing electron-dense material and surrounded by a limiting membrane were occasionally observed in the perinuclear disternae and in intranuclear vacuoles. Budding of electron-dense material to the cisternae with acquisition of a limiting membrane at the inner nuclear membrane was also occasionally observed. These findings are in constrast with observations that in cells infected with cytomegalovirus numerous dense bodies and their budding process were observed only in the cytoplasmic area.     

大菜粉蝶颗粒体病毒的形态发生及细胞病理的超微结构观察

本文叙述感染大菜粉蝶颗粒体病毒后,病虫脂肪体细胞超微结构的改变,大菜粉蝶感染后24小时,病虫脂肪体细胞开始出现明显的病变,整个病程是,在开始时细胞核内出现清晰区并出现病毒发生基质,核膜多点成套增生,其后核膜断裂,大量膜样结构聚集在病毒发生基质的周围,核衣壳大量产生,有一部分核衣壳从这些病毒发生基质四周的膜样结构碎片上获得套膜,荚膜蛋白沉积形成成熟的病毒荚膜,或称包含体;另一部分则排列在胞浆内的空泡边缘上;其余的核衣壳则从细胞边缘“芽突”而获得套膜,另外还描述环孔片层及线粒体改变。     

Electron microscopy of human skin fibroblasts in situ during growth in culture

We have studied the electron microscopic (EM) appearance of cultured diploid human skin fibroblasts during logarithmic and confluent stages of growth using a simple technique which permits in situ visualization of individual cells in monolayer. By comparison, cells disrupted from a monolayer and pelleted showed drastic distortion of the cell surface and appearance of organelles: mechanical scraping produced massive dilatation of the rough endoplasmic reticulum (RER); and trypsin-produced multiple blebs of the plasma membrane and cytoplasmic vacuoles. In situ, these changes in trypsinized cells disappeared within 1 h after plating. Six hours later, microtubules and microfilaments had surrounded the nucleus and were oriented in longitudinal bundles beneath the plasma membrane during rapid growth. At confluence these cytoskeletal elements seemed to extend beyond the plasma membrane at intercellular junctions. Pinocytotic vesicles were abundant at those surfaces devoid of filaments. Mitochondria, aligned with the long axis of the cell, were extremely long and narrow. During logarithmic growth there were many free ribosomes, polysomes, and some flat cisternae of RER. At confluence most ribosomes were found in spirals on dilated saccules of RER which contained electron-dense material. Lysosomes of several types were present during all phases of growth and varied in number from cell to cell. Late in culture the lysosomes tended to be larger, often occupying whole areas of cytoplasm or even extruding from the cell, and resembled those seen in lysosomal storage diseases. Understanding the in situ ultrastructure of normal human fibroblasts during growth in culture will permit systematic examination of such cells in a variety of pathological states.     

Development of spherical organelles from the endoplasmic reticulum in the nucellus of some Euphorbia species

Summary The development of characteristic cytoplasmic inclusions from endoplasmic-reticulum (ER) cisternae in nucellar cells of some species of Euphorbia has been studied by electron microscopy. The formation of these organelles is preceded by the appearance of rough ER cisternae filled with an electron-dense material and forming complicated networks. Vesicular structures are formed which grow rapidly to give electron-dense, spherical dilations. On the outer surface of their limiting membrane numerous ribosomes and often polysomes are present. This membrane can be seen to remain continuous with the membranes of one or more cisternae of the rough ER up to when the dilations have a maximum diameter of 2.5–3 . At this time, continuity between the ER cisternae and the spherical dilations ceases. After this the new cell organelles remain unchanged in size, shape, and electron-density until the cell is disintegrated by the growing embryo-sac. The fate of the contents of these organelles is discussed.This work was supported by a grant of the Italian National Research Council (C.N.R.). We acknowledge with appreciation the excellent technical assistance of Mr. Sergio Casini.     

Endocytotic pathways in the melanotroph of the rat pituitary

Summary The internalization of the extracellular markers horseradish peroxidase (HRP) and cationized ferritin (CF) by the melanotrophs of the intermediate lobe of the rat pituitary was studied during short-time incubation of mechanically dissociated cells or in cell culture after 5 days. After a 30 min exposure, the tracers were found in electron-lucent granules or vacuoles of approximately the same size as the secretory granules, situated 200–500 nm from the cell membrane. In the cultured cells, which showed a higher rate of tracer uptake, internalization was followed for 1, 2 and 5 min after labelling and during 2 h of exposure. Initially, the label was seen only in coated pits and coated vesicles at the cell membrane. Larger vacuoles were first seen after 2–5 min of incubation. After 2 h of exposure the labelling pattern was distinctly different for the two tracers. CF was found in larger vacuoles of varying morphology, in dilatations at the base of cilia, within Golgi saccules and at the edge of the electron-dense core of forming secretory granules. HRP was found in an extensive array of tubulovesicular structures extending throughout the cytoplasm. The Golgi complex and forming granules were, however, not labelled with HRP. The study identifies part of the electron-lucent granules or vacuoles in the melanotroph as endosomes, and shows that the melanotrophs sort CF and HRP via diverting pathways after internalization, suggesting that granule membrane, and possibly its functional components, can be recycled in these cells.     

Ultrastructural observations in hepatitis C virus-infected lymphoid cells

It is currently unclear whether the hepatocellular damage in chronic hepatitis C virus (HCV) infection is produced through the intrahepatic action of the anti-HCV immune response or through a direct cytopathic effect. In order to investigate the features of HCV replication (morphogenesis and cytopathic effect), we studied the infection of a permissive lymphocytic B cell line, Daudi cells, which were infected with sera of HCV-positive patients, and were examined after various time points under electron microscope. Viral genomic RNA was detected by in situ hybridization, and apoptosis with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The amount of viral genomic RNA was observed to increase during infection. HCV replicated rapidly, since characteristics of viral morphogenesis resembling those of yellow fever virus in a hepatoma cell line could be found 2 days after infection. These included the following: a) several viral particles identical in size (about 42 nm) and structure (a spherical 30-nm-sized electron-dense nucleocapsid surrounded by a membrane) to yellow fever virus were present in the cytoplasm of cells displaying already typical signs of the early stage of apoptosis; b) numerous membrane-bound organelles and in particular the endoplasmic reticulum and vacuoles were observed; c) proliferation of membranes was apparent; and d) intracytoplasmic electron-dense inclusion bodies which have been demonstrated to correspond to nucleocapsids for other flaviviruses were detected. Several cells presented electron-dense areas in the endoplasmic reticulum displaying 30-nm circular structures lying among an amorphous material. Striking cytopathic features with ballooning, extremely enlarged vacuoles and signs of apoptosis were found in cells often containing sequestered aggregates of virus-like particles. By in situ hybridization we found that such enlarged cells contained HCV RNA. Our results thus indicate that the ultrastructural features of HCV viral particles and their morphogenesis resemble that of yellow fever virus and dengue virus. In Daudi cells, HCV infection seems to rapidly trigger apoptotic cell death, and efficient release of viral particles does not seem to take place.     

The fine structure of the chinchilla placenta.

The structure of the placental labyrinth, interlobular or "coarse" syncytium, visceral (splanchnopleuric) yolk sac, giant cells and subplacenta of the chinchilla was studied with the electron microscope. The fine structure of the interhemal membrane of the placental labyrinth was found to be hemomonochorial, consisting of a single layer of syncytial trophoblast. In this respect, the placental labyrinth was similar to that of another caviomorph rodent, the guinea pig. The labyrinthine trophoblast had pinocytotic vesicles as well as larger vaculoes and multivesicular bodies. The interlobular syncytium contained granular endoplasmic reticulum, and in one case from early in gestation there were intracisternal granules in the ER. The visceral endodermal cells of the inverted yolk sac placenta had a well-developed system of apical vesicles and tubules as well as larger cytoplasmic vacuoles. Their appearance was similar to that of endodermal cells found in other rodents which are known to absorb proteins and other substances from the uterine lumen. Towards term the giant cells were often vacuolated and contained large deposits of glycogen as well as lipid droplets. The syncytial trophoblast of the subplacenta contained numerous moderately electron-dense granules which may be secretory in function; cytotrophoblastic cells lacked these granules. The subplacental syncytium often surrounded spaces or lacunae which contained an electron-dense granular material.     

Ultrastructural evidence of endoplasmic reticulum changes during the maturation of the olive pollen grain (Olea europaea L., Oleaceae)

Characteristic features of rough endoplasmic reticulum (rER) distribution and proliferation were noted during olive pollen (Olea europaea L.) development, suggesting the physiological significance of this organelle. Initially scarce in the young microspore, ER increases as cytoplasmic vacuoles form. At the vacuolated microspore stage the cytoplasm contains numberous polysomes and elongated rER cisternae arranged preferentially in stacks, with an average intracisternal width of 0.07 µm. Stacks persist in the bicellular pollen grain but consist of fewer, shorter, dilated cisternae (mean intracisternal width 0.1 µm) containing a considerable electron-dense matrix. Cisternae in the mature grain are fragmented, leaving behind an ER of swollen pockets. Pockets of ER containing a material of greater electron density are evenly deposited along the plasmalemma, in close relation with it. A dense material is seen in the tubules of the apertural region, which was lacking in earlier stages. Our results show that ER may be involved in protein transport to the intine.     

Human cytomegalovirus: development and progression of nuclear inclusions by primary clinical isolates and laboratory-adapted strains

The morphogenesis of cytomegalovirus (CMV) nuclear inclusions (NIs) was investigated using unadapted clinical isolates and adapted laboratory strains. Both adapted and unadapted strains of CMVs induced NIs whose morphologic appearance was similar in human fibroblastic cells. Early NIs appeared as ring-like structures composed of dense granular and fibrillar material, while late NIs appeared to consist of multiple electron-lucent areas containing coarse granules and bounded by electron-dense fibrillar material (cellulae). Capsids and nucleocapsids were associated primarily with the electron-dense fibrillar material; however, developing nucleocapsids were most often observed at the interface of the electron-dense and -lucent areas. Although there was some variation in the rate of development and maturation of the NIs with the intensity of infection, all CMVs examined produced late NIs with similar organizational patterns consisting of cellulae. Substitution of human fibroblastic cells derived from various tissues as cellular substrate did not appreciably affect the results. Thus, the unique organization of the CMV late NI, consisting of multiple cellulae, appears to be an intrinsic feature of CMV replication since it seems to be independent of the extent of laboratory adaptation, the virus strain, the intensity of infection, or the cell type.     

Ultrastructure of spermatogenesis in the sea star,Asterina minor

The ultrastructural features of spermatogenesis were investigated in the hermaphroditic sea star Asterina minor. The primordial germ cells in the genital rachis contain small clusters of electron-dense material (nuage material) and a stack of annulate lamellae. They also have a flagellum and basal body complex situated close to the Golgi complex. After the development of the genital rachis into the ovotestis, spermatogenic cells increase in number and differentiation begins. Nuage material is observed in spermatogonia, but it gradually disappears in spermatocytes. The annulate lamellae do not exist beyond the early spermatogonial stage. By contrast, a flagellum and basal body complex are found throughout spermatogenesis. The Golgi-derived proacrosomal vesicles appear in the spermatocyte and coalesce to form an acrosomal vesicle in the early spermatid. The process of acrosome formation is as follows: (1) a lamella of endoplasmic reticulum (ER) continuous with the outer nuclear membrane encloses the posterior portion of the acrosomal vesicle; (2) the vesicle attaches to the cell membrane with its anterior portion; (3) periacrosomal material accumulates in the space between the acrosomal vesicle and the ER; (4) the nucleus proper changes its features to surround the acrosome; (5) amorphous, electron-dense material is deposited under the electron-dense disk; and (6) the nucleus forms a hollow opposite the electron-dense material.     

Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp

Various Naegleria strains were examined to determine the possible origin and significance of membrane-bound black bodies that were found in all exponentially growing cell populations. The bodies, 40–80 nm in diameter, were distributed randomly in the cytoplasm of Naegleria with ultrastructural features typical of trophozoites. No evidence was obtained that the contents of the black bodies were synthesized in the rough endoplasmic reticulum (ER) and packaged by membranous components, which could be a primitive “Golgi complex” in these amoebae. Examination of cells in various stages of encystment indicated that at least some of the cyst wall material was synthesized and packaged by the rough ER. After condensation into amorphous granules in the cisternae, the cyst wall material appeared in vesicles of the rough ER; these were frequently seen in close proximity to the cell membrane in the vicinity of developing cyst wall. Amorphous granules (～100 nm in diameter), which had variable densities and did not appear to be membrane bound, were seen in the cytoplasm of encysting cells. The substance of these granules also seemed to be incorporated into the cyst wall. The membrane-bound black bodies appeared to be destroyed in lysosomal elements during encystment. The membrane-bound black bodies were concluded to be characteristic of trophozoites and unrelated to encytment of Naegleria.     

Ultrastructural characterization of capsulated Haemophilus influenzae type b and two spontaneous nontypable mutants.

Capsulated Haemophilus influenzae type b and two spontaneous mutants (classes I and II variants) were characterized by transmission and scanning electron microscopy. When cells were treated with type b-specific antiserum prior to manipulations for electron microscopy, sectioned capsulated cells had electron-dense, fibrous capsular antigen-antibody complexes around them. In negatively stained preparations, the complexes appeared as electron-transparent zones surrounding cells. In contrast, only residual electron-dense, extracellular material was seen in sectioned, untreated, capsulated cells, and electron-dense "bridges" connected adjacent cells in negatively stained preparations. No extracellular capsular material was seen around the class I and II variants. Characteristic electron-translucent regions were always observed within the cytosol of the class I cells, both in thin sections and by negative staining. These areas were located adjacent to the cell envelope separating the plasma membrane from the dense cytoplasmic matrix. At times, electron-dense, thread-like material extended from the dense cytoplasmic matrix to the plasma membrane. No such regions were seen in the capsulated and class II cells. Class I cells fixed with methanol or suspended in NaCl or phosphate-buffered saline prior to treatment with fluorescein-tagged type b-specific antiserum (FTA reagent) exhibited, by immunofluorescence, patches of capsular antigen along their sides. However, when fixed with glutaraldehyde or OsO4 or suspended in tris-(hydroxymethyl)aminomethane plus Ca2+ buffer prior to treatment with FTA reagent, no patches of capsular antigen were seen. Subsequent exposure of the latter cells to methanol followed by treatment with FTA reagent resulted in the reappearance of the patches of capsular antigen. Thus, in the class I variant the capsular antigen is unlikely to be surface located. Scanning electron microscopy revealed that class I and II variant cells within undisturbed colonies were regularly aligned side-by-side, whereas cells within colonies of the capsulated strain were randomly distributed.