Ultrastructure of the vegetative cell ofBrassica napus pollen with particular reference to microbodies

Summary The ultrastructure of the vegetative cell ofBrassica napus tricellular pollen grains, just before anthesis with standard chemical fixation, is reported. The vegetative cell may be regarded as a highly differentiated and metabolically active fat-storage cell. It contains many mitochondria with a well developed internal membrane system, starchless plastids, microbodies, lipid bodies, dictyosomes and numerous vesicles thought to originate from the dictysomes. Rough endoplasmic reticulum organized in stacks of cisternae is also spatially associated with certain organelles, mainly lipid bodies, microbodies and plastids. There are also randomly distributed polyribosome areas. The microbodies are mainly polymorphic in shape and are often observed in contact with lipid bodies. The above spatial relationship implies that the microbodies may have a glyoxysomal function. In the late period of vegetative cell maturation, the microbodies are probably involved in the process of glyconeogenesis in which the conversion of lipid reserves to sugar takes place.Abbreviations VC vegetative cell - VN vegetative nucleus - SC sperm cell - M mitochondria - MB microbodies - L lipid body - P plastid - D dictyosomes     

Changes in Parenchyma Cells of Poplar Xylem during Transition from Growing to Wintering Stages

Ultrastructural changes in xylem parenchyma cells during transitionfrom growing to wintering stages were investigated. Changesin the fine structures of parenchyma cells in differentiatedxylem started in mid-August. At this time, dictyosomes werefrequently found and vesicles were abundant in the cells inwhich active formation of cellular structures took place. Thecytosol was filled with polysomes. In mid-September, the cytoplasmof the parenchyma cells gradually began to fill up with microbodies,and the endoplasmic reticulum cisternae had already startedto decrease. Dictyosomes and vesicles were still abundant atthis stage and microbody-associated vesicles could be seen,indicating participation of the vesicles in the formation ofmicrobodies at this stage. A direct structural connection withrespect to the biogenesis of the microbodies remained unclear.In the early stage, the microbodies showed a dense center surroundedby a less dense outer surface, and in the latter stage in November,the outer surface exhibited an increased accumulation of osmiophilicmaterials. There were few further changes in the overall patternof the fine structures until mid-winter. (Received July 2, 1987; Accepted November 30, 1987)     

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds

Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.     

向日葵幼叶及其培养的愈伤组织细胞的超微结构研究

通过超微结构的观察,向日葵幼叶及其经培养后10天的愈伤组织细胞之间有明显区别。叶肉细胞的细胞质、细胞器及核的结构和发育都比较完整。当外植体组织发生变化和愈伤组织形成时,观察到线粒体相互连接成链状围绕在叶绿体周围,而叶绿体有的围绕在核的周围;线粒体的嵴和基质,叶绿体的基粒和片层结构常发生退化或解体,细胞质稀薄,核糖体和胞质凝成线状或网状,微体和高尔基体消失,液泡化程度高并含有较多的次生物质;而细胞核在后期才发生明显变化,轮廓不够清晰。     

WHAT ARE THE CHROMIDIA OF POLYPHAGUS EUGLENAE?

Zoospores, prosporangia, and asexual sporangia were studied with electron microscopy to determine the ultrastructural identification of “chromidia,” granular masses surrounding nuclei that classical mycologists believed to be extruded chromatin used for lipid synthesis. In the zoospore the nucleus was enclosed by an aggregation of ribosomes. In other developmental stages the behavior of microbodies was identical to that described for “chromidia.” A microbody network with interspersed ER surrounded nuclei in young prosporangia. As the prosporangium matured, lipid globules became associated with the microbodies. When the single, large nucleus migrated into the elongate asexual sporangium, microbodies still surrounded the nucleus; but after the nucleus divided and a multinucleate sporangium formed, microbodies were scattered throughout the cytoplasm. When incubated in the diaminobenzidine medium for the cytochemical detection of catalase, reaction product was found in these microbodylike structures, confirming that “chromidia” described in prosporangia and asexual sporangia by classical mycologists are really microbodies. Rather than giving rise to lipid, these microbodies are probably involved in the metabolism of the lipid globules with which they are associated. The “chromidia” in zoospores are not extruded chromatin as suggested earlier, but correspond in their location around the nucleus to an aggregation of ribosomes.     

Occurrence of microbodies in the green algaBracteacoccus cinnabarinus grown heterotrophically

Summary A comparative study was made of the ultrastructure and abundance of microbodies in the green algaBracteacoccus cinnabarinus grown photoautotrophically and heterotrophically on a conventional culture medium containing sodium acetate, potassium acetate and glucose. Several changes were observed in the cells maintained under these conditions. Most noticeably, cells grown on acetate in both light and dark were packed with lipid bodies. Microbodies were found to be closely appressed to the lipid bodies in cells grown heterotrophically in the dark on sodium acetate and potassium acetate. The average number of microbody profiles per cell was in general threefold greater in cells grown on sodium acetate than those grown on potassium acetate. No microbodies were observed in cells maintained photoautotrophically on the three carbon sources or in cells maintained photoautotrophically on Bristol's inorganic medium alone. Cytochemical staining with 3,3-diaminobenzidine indicated the presence of catalase in the microbodies. The presence of microbodies suggests that the organelle may be performing functions similar to glyoxysomes in higher plants, namely the net conversion to succinate of acetyl CoA derived from lipid degredation. It is also apparent thatBracteacoccus can grow well as a heterotroph in the dark when acetate is included in the culture medium as a source of carbon.     

CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.     

Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules

Summary Nitrogen-fixing peanut root nodules are characterized by their unique structural organization, distinct from other legume nodules. The focus of this study has been in and around the hostsymbiont interface, where the bacterioid and the host cell surface (peribacteroid membrane envelope) interact during symbiosis. The infected nodule cells have revealed the presence of lipid bodies (oleosomes) in intimate association with the peribacteroid membrane, which encloses the large spherical bacteroids with a relatively narrow peribacteroid space. Electron dense structures, referred to as dense bodies have been found attached to the bacteroid outer membranes at the host-symbiont interface. The dense bodies are osmiophilic, amorphous and 3,3-diaminobenzidine positive. The isolated intact bacteroids with dense bodies attached to their cell wall showed significant catalase activity. Many microbodies showing DAB-positive reaction have been found in the host cytoplasm, associated closely with the peribacteroid membrane. These ultrastructural and cytochemical characteristics of peanut root nodules suggest that lipids are utilized during symbiosis and the dense bodies and microbodies may be involved in the catabolic process.Abbreviation DAB 3,3-diaminobenzidine     

The Ultrastructural Feature and Distribution of Microbodies in Mature Embryo Cells of Pinus bungeana

The material of pine seeds used in this investigation was collected in 1982 from Peking. The microbodies of mature embryo ceils are very well developed and their diameter averages about 2–3 μm, even up to 4.3 μm. The appearance is usually ovoid or elliptic. The microbodies are essentially glyoxysomes. The microbody matrix is composed of two types of substances, one type is of a finely granular material in a densely arrangement (Plate Ⅲ Fig. 6); the other is of coarsely granular or flocculant in appearance and the elements of the matrix are loosely distributed. These matrices usually contain an amorphous inclusion or crystalline arrays in regular arrangement. The inclusion sometimes occupies a small portion of the microbody matrix (Plate Ⅲ, Figs, 5, 6) and sometimes the inclusion occupies nearly the entire glyoxysome (Plate Ⅱ, Fig. 3). It is interesting that the “pockets” frequently appear in the microbodies of mature embryo cells, and those are actually as a result of invagination in microbodies (Plate Ⅱ, Fig. 4). In addition, an electron-transparent “oil body-like space” occurs occasionally in microbody (Plate Ⅰ, Fig. 1). The periphery of “space” is a constitutive part of matrix or continuing with the matrix. This “space” may be due to the degradation in a part of the matrix. While the periphery of the pocket is membranaceous and an electron-opaque cytoplasmic groundplasm was found within the pocket. The microbodies of mature embryo cells in Pinus are mainly distributed in pericolumn cells of the root cap and cortical cells of the hypocotyl. Besides the dominant organelles of lipid bodies in the cells of above mentioned tissues, there are also microbodies, amyloplasts, mitochondria, plastids, endoplasm reticulum and Golgi apparatus, of which the microbodies are the most aboundant organelles. In contrast, the microbodies and other organelle are rare in the parenchyma of the cotyledons in Pinus. Their common and outstanding characteristics in various tissues of mature embryo is that the entire cytoplasm of the cells is almost full of the lipid bodies, and each organelle is directly surrounded by a number of lipid bodies (Plate Ⅰ—Ⅲ, Figs. 1–6). Because of the other organelles are rare in parenchyma of the cotyledons, the lipid bodies are so appressed with each other that the inlaid periphery of lipid bodies frequently occurs in some degree. To sum up, based upon 'the state of distribution of microbodies in mature embryo tissues, cotyledons of Pinus could be considered as the main storage organ of nutrient substances, while the root cap and hypocotyl are the important sites of glyoxysome metabolism. The function of glyoxysomes is to convert lipid into the carbohydrates and to transfer the latter to embryos for growth.     

Relationship between mitochondria and the development of specific lipid droplets in capillary endothelial cells of the respiratory tract in patients with sarcoidosis

The aim of this study was to morphologically evaluate damage in the capillary endothelial cells of the respiratory tract in patients with sarcoidosis. We examined tissues of the bronchus and lung obtained from 16 patients with sarcoidosis consisting of 2 stage 0, 10 stage I and 4 stage II patients, and 11 controls. The morphology of capillary endothelial cells was studied using electron microscopy. In the samples from patients with sarcoidosis, lipid droplets exhibiting dark monophasic density (unsaturated fatty acids) were surrounded by abundant lysosomes in the capillary endothelial cells, and the double-membrane structure of the mitochondria attached to these lipid droplets was lost in three cases. Biphasic lipid droplets with dark and lucent (saturated fatty acids) densities were also observed, accompanied by a few lysosomes containing the residual bodies of undigested lipid-containing substances. Lucent monophasic droplets were also detected in the tissues from patients with sarcoidosis. The plasma membrane was more often damaged in capillary endothelial cells containing biphasic lipid droplets, lucent monophasic droplets as well as in those with dark monophasic droplets. However, no lipid droplets were detected in capillary endothelial cells obtained from the control subjects, except in a single case. This study demonstrated that a large number of mitochondria were mobilized and showed notable morphological changes including swelling in the capillary endothelial cells in patients with sarcoidosis. A close relationship between mitochondria and lipid droplets was observed in capillary endothelial cells of the respiratory tract, and this relation may be involved in the pathogenesis of sarcoidosis.     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae)

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.     

The ultrastructure of polymorphic pollen grains of Canna indica L.

Summary Our investigations on Canna indica L. indicate that the pollen of this species is polymorphic: there are two types of pollen — a larger type and a comparatively smaller type. Transmission electron microscopy (TEM) revealed the presence of small vacuoles containing tannic substances in the generative cell (GC) of the larger grains: the GC of the mature grain contained a higher quantity of tannins than the GC of the immature grain. Mitochondria, lipid bodies, rough endoplasmic reticulum (RER) and microtubular bundles were present in the cytoplasm of the GC. Numerous mitochondria, lipid bodies and plastids were also present in the vegetative cell (VC), with the mitochondria clustered around the vegetative nucleus. The plastids were observed to be associated with the RER cisterns. During the maturation process, the number of starch grains contained in the plastids decreased.     

Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function. We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A- gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.     

Changes in the lipid inclusion/Sertoli cell cytoplasm area ratio during the cycle of the human seminiferous epithelium

The area occupied by Sertoli cell lipid inclusions--electron-lucent lipid vacuoles (LLV) and electron-dense lipid droplets (DLD)--at each stage of the cycle of the seminiferous epithelium was measured on electron micrographs in young adults and elderly men, and expressed as the ratio "area occupied by lipid inclusions/area occupied by the Sertoli cell cytoplasm". For LLV this ratio increased from stage I to stage III, and decreased from stage IV to stage VI in young adults. These results suggest that the development of LLV is synchronized with the spermatogenic process: the residual bodies released in stages I and II are phagocytized by Sertoli cells and transformed into LLV; the amounts of LLV decrease in the subsequent stages of the cycle and increase again when new residual bodies appear. In elderly men the ratio LLV/Sertoli cell cytoplasm was 1.9-2.9 times higher than in young adults at each stage of the cycle. This increase may be related to the increased germ-cell degeneration observed in ageing testes, DLD were less abundant than LLV and the DLD/Sertoli cell cytoplasm ratio did not undergo cyclic changes in young adults or elderly men.     

PEARL BODIES OF A NEOTROPICAL TREE,OCHROMA PYRAMIDALE: ECOLOGICAL IMPLICATIONS

Multicellular, spherical or club-shaped pearl bodies characterized by large intracellular lipid vesicles are produced on leaves and stems of juvenile Ochroma pyramidale, a tree of second growth vegetation in lowland wet forest of the neotropics. Several lines of evidence suggest that pearl bodies are linked to maintenance of foraging ants on the plant: (1) their production is closely associated with foliar nectar secretion; (2) they are abundant on saplings grown in the glasshouse (averaging 402 bodies leaf^–1) but were not observed in the field where ants are predictably associated with Ochroma samplings; (3) pearl bodies are energy- and lipid-rich averaging 27.80 kJ/g dry wt^–1 and 74.4% lipid; (4) they are constricted at the base and easily detach from the leaf; (5) four ant species collect pearl bodies from artificial depots and return them to their nests. Chelaner sp. detaches pearl bodies from the leaf and returns them to the nest. Production of pearl bodies represents about 25% of the energy allocated to foliar nectar by saplings. Characteristics of the pearl bodies of Ochroma are consistent with those of a widespread group of trichomes and leaf emergences suggesting a common ecological role as ant food for these structures.     

Ultrastructure and isolation of glyoxysomes (microbodies) in zoospores of the fungusentophlyctis sp.

Summary A correlative approach, involving light and electron microscopic, cytochemical, and biochemical techniques, was used to study the structure and function of microbodies in zoospores ofEntophlyctis sp. The same population of microbodies already existing in the zoosporangium appeared to be segregated into zoospore initials during cytoplasmic cleavage. Microbodies laid at the anterior end of zoospores and were part of an organized assemblage of organelles, the microbody-lipid globule complex. In the microbody-lipid globule complex, endoplasmic reticulum occurred on the surface of the lipid globules toward the zoospore's exterior, and the microbody, subtended by mitochondria, was appressed to the opposite surface of the lipid globule. The organization of the microbody-lipid globule complex changed as the zoospore swam and encysted. As lipid globules coalesced, the microbody-lipid globule complex became disorganized. After lipid globule coalescence was completed, the microbody-lipid globule complex regained its order, and several microbodies were clustered adjacent to a single lipid globule. The microbodies persisted even in the encysted zoospore, but they were found on all sides of the lipid globule.Microbodies isolated from zoospores contained catalase as well as malate synthase and isocitrate lyase, two enzymes of the glyoxylate cycle. When zoospores encysted greater activities of these glyoxylate cycle enzymes could be detected. The presence of glyoxylate cycle enzymes and the close association between the microbody and lipid globule suggest that microbodies function as glyoxysomes in zoospores and encysted zoospores. The functional significance of the morphological organization of the microbody-lipid complex is discussed in terms of energy production and the conversion of storage lipid into structural components of the cell.     

Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity.

Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.