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Ultrastructural studies on blood leukocytes of the channel catfish, Ictalurus punctatus, show the presence of heterophils (neutrophils), small lymphocytes, monocytes, and thrombocytes. Monocytes cannot always be distinguished from large lymphocytes. Cells resembling macrophages or transitional forms between monocytes and macrophages are occasionally seen. Blood eosinophils and basophils are not found. Thrombocytes and small lymphocytes are the most abundant leukocytes, while monocytes are the least frequently encountered leukocyte. Glycogen, present in all leukocytes, is most abundant in heterophils and least abundant in monocytes. Although monocytes are similar to heterophils in size and shape, a greater amount of rough endoplasmic reticulum, free ribosomes, and fewer granules are observed in monocytes. Heterophils possess oval or elongate granules, which often contain a crystalline or striated structure; small tubules which resemble smooth endoplasmic reticulum, and cristae which traverse the long axes of the mitochondria are frequently seen. Small lymphocytes are characterized by the presence of pseudopodia, many free ribosomes, numerous large mitochondria, dictyosomes (Golgi), and long profiles of rough endoplasmic reticulum. The dictyosomes are often associated with a large zone of exclusion. Bundles of microtubules are observed near the elongated ends of thrombocytes. Deep indentations of the plasmalemma, which give the appearance of vacuoles, are also seen in thrombocytes.

Epoxy resins are mixtures of several components formulated in exact proportions to assure proper polymerization. It is necessary that this composition be maintained throughout the tissue being infiltrated to avoid embedding artifacts. Unfortunately, the separate resin components will not penetrate the tissue at the same rate unless they all have the same viscosity and molecular size. Failure to meet these criteria will result in component separation during tissue infiltration and may result in severe embedding defects. This and several ancillary problems such as low tissue contrast, damage to diamond knives, and poor postsection staining are explored in this brief discussion.

Epoxy resins are the principal embedding media for the preservation of tissues to be sectioned and examined by transmission electron microscopy. Their primary advantages are good ultrastructural preservation, little or no shrinkage, ease of sectioning, and reasonable stability in the electron beam. However, epoxy resins also have disadvantages; namely, some are toxic, they may mask antigenic sites to a greater extent than do some other embedding resins, and they do not penetrate tissues as well as less viscous embedding formulations. Some unusual characteristics may also be revealed, for example, as shrinkage of organelles, as problems in poststaining sections, and as movement of tissue elements within the block and section. Some of the properties of epoxy resins are discussed in this report. © 1993 Wiley-Liss, Inc.

In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.

In 1898, Camillio Golgi reported a new cellular constituent with the form of an extensive intracellular network (the apparato reticolare interno), which now bears his name. However, the history of Golgi's apparatus is replete with controversy regarding its reality, what components of the cell should be included under its aegis, and what terminology should be used when referring to it. Electron microscopy has resolved many of these controversies and it is appropriate that this volume emphasize that aspect of Golgi apparatus discovery. The principal structural component of the Golgi apparatus is the stack of cisternae, or dictyosome. As determined both biochemically and at the level of electron microscopy, the dictyosome is a highly ordered and polarized structure. The maintenance of order within the stack is thought to result from either intercisternal bonding constituents, or filamentous structures (or both) that bridge the space between adjacent cisternae. Mechanisms proposed for movement of membrane and product into and out of the dictyosome (i.e., the Golgi apparatus stack) include a serial mode which functions exclusively by the formation, displacement, and loss of cisternae from the stack, and a parallel mode which functions exclusively by the movement of membrane, product, or precursor molecules directly into the peripheral edges of the cisternae. In the parallel mode, all cisternae can be accessed either singly or simultaneously, at least in theory, at any position within the stack. It is probable that both the serial and the parallel modes function concomitantly and need not be mutually exclusive. Finally, the peripheral tubules of the cisternae represent a major membranous constituent of the cell with potentially unique functions. These tubules interconnect cisternae of adjacent stacks and may represent the major site of receptors for the shuttle (i.e., parallel) type of transfer among cisternae. Peripheral tubules as extensions of the cisternal lumina into the cytoplasm presumably have other functions, but these, like the tubules themselves, have only rarely been accommodated into functional models of Golgi apparatus dynamics in secretion or membrane flow.

The membrane maturation (flow differentiation) model of Golgi apparatus function embodies concepts of saccule formation at one face of the Golgi apparatus from membranes derived from endoplasmic reticulum and utilization of saccules in vesicle formation at the opposite face for delivery to the plasma membrane as existing saccules are displaced from one position within the stack to another. Derivation of the model came almost entirely from light and electron microscopy. Especially important were observations that passage through the Golgi apparatus was accompanied by differentiation of membranes from endoplasmic reticulum-like to plasma membrane-like across the polarity axis of the stacked saccules. The concept of coparticipation of endoplasmic reticulum and/or nuclear envelope, transition, and secretory vesicles and other pre- and post-Golgi apparatus structures through the operation of an integrated endomembrane system was essential to the model. Dynamic aspects confirmed initially by autoradiographing and cell fractionation studies have been corroborated in newer approaches of fluorescent labeling and with living cells.

The membrane maturation (flow differentiation) model of Golgi apparatus function embodies concepts of saccule formation at one face of the Golgi apparatus from membranes derived from endoplasmic reticulum and utilization of saccules in vesicle formation at the opposite face for delivery to the plasma membrane as existing saccules are displaced from one position within the stack to another. Derivation of the model came almost entirely from light and electron microscopy. Especially important were observations that passage through the Golgi apparatus was accompanied by differentiation of membranes from endoplasmic reticulum-like to plasma membrane-like across the polarity axis of the stacked saccules. The concept of coparticipation of endoplasmic reticulum and/or nuclear envelope, transition, and secretory vesicles and other pre- and post-Golgi apparatus structures through the operation of an integrated endomembrane system was essential to the model. Dynamic aspects confirmed initially by autoradiographing and cell fractionation studies have been corroborated in newer approaches of fluorescent labeling and with living cells.

Biochemical and electron microscopic studies have indicated that the Golgi apparatus responds to retinol. The purpose of this investigation was to visualize and record with living cells the rapidity of the response to retinol. A rapid response of the Golgi apparatus to retinol (1.75-17.5 mumol/L) added to the culture medium was observed using video-enhanced light microscopy with bovine mammary epithelial cells. The response was manifested within 1 min as a marked movement of membranes within the Golgi apparatus zone. In subsequent electron microscope preparations of the cells, only minor changes were observed and were restricted to increased numbers of normal-appearing membranes and vesicles associated with the trans Golgi apparatus face of the retinol-treated cells.

Introduction: This is a personal account of the author's attempts to solve some of the problems associated with the fixation and embedding of difficult biological materials such as plant tissues, almost all insect tissues, and large pieces of tissues including whole organs.Historically, the search began in the 1950's with the study of the maize root tip using the relatively new technique of electron microscopy. This system of cells, separated by thick and partially impermeable walls, did not respond well to the procedures most suitable for animal tissues. New approaches were needed and, in time, some were found that were at least useful if not ideal. A brief account of several of these is given in the following paragraphs.Fixation of plant cells: Potassium permanganate was chosen very early in the study primarily because it worked better for plant cells than did any of the fixation procedures currently available.

The recent note by J.D. Watson in Science (1993) is another of the periodical advice columns in which someone of renown gives his or her perspective of how to succeed in this most difficult of fields. And a very good article it is. I find no fault with any of the four rules presented which I quote as follows: 1) “To succeed in science you have to avoid dumb people.” 2) “To make a huge success, a scientist has to be prepared to get into deep trouble.” 3) “Be sure you always have someone up your sleeve who will save you when you find yourself in deep s—.” 4) “Never do anything that bores you.” Unfortunately, these four rules are directed to a level of scientist that clearly bypasses most of us irrespective of how many good intentions we may have. Therefore I feel obliged to submit a revised list of thoughts for us average beings who plow through the days in an average research environment.

There are several factors that were requisites for the biological applications of electron microscopy. One of these was, clearly, the development of a matrix substance that could infiltrate biological material and encapsulate tissue components so that they could be thin sectioned and examined under an electron beam. Without such matrix substances, examination of tissue as we know it today, would not be possible.Perhaps the start of practical electron microscopy in the context stated above was the application of methacrylate resins for tissue embedment. Methacrylates have a very low viscosity and maintain this viscosity until polymerization is initiated (for days or weeks if one should wish). The two most commonly used methacrylates were n-butyl (soft) and n-methyl (hard) which could be mixed in various proportions to yield almost any block hardness. Methacrylates can be cross-linked with divinyl benzene to improve stability but this was not commonly done. Methacrylates are very easy to section which was an important aspect at the time since sectioning had to be done with glass knives.

Various means have been devised to preserve biological specimens for electron microscopy, the most common being chemical fixation followed by dehydration and resin impregnation. It is intuitive, and has been amply demonstrated, that these manipulations lead to aberrations of many tissue elements. This report deals with three parts of this problem: specimen dehydration, epoxy embedding resins, and electron beam-specimen interactions. However, because of limited space, only a few points can be summarized.Dehydration: Tissue damage, or at least some molecular transitions within the tissue, must occur during passage of a cell or tissue to a nonaqueous state. Most obvious, perhaps, is a loss of lipid, both that which is in the form of storage vesicles and that associated with tissue elements, particularly membranes. Loss of water during dehydration may also lead to tissue shrinkage of 5-70% (volume change) depending on the tissue and dehydrating agent.

Epidermal and outer rootcap cells of maize root tips were treated with the sodium selective ionophore, monensin, and the ultrastructural changes were studied. In the presence of 10−5 to 10−3 M monensin, dictyosomes became distorted, cisternae separated from the stack, and secretory vesicles were released. Released secretory vesicles disappeard from the cytoplasm suggesting that their transport to, and fusion with, the plasma membrane was unaffected. Monensin did not inhibit cytoplasmic streaming of the outer rootcap cells. No new secretory vesicles were formed on the remaining dictyosomes or dictyosome fragments. In contrast to results with animal cells, swelling of plant dictyosome cisternae was observed only after fixation in glutaraldehyde-osmium tetroxide and not after fixation in potassium permanganate. Other cell components were not altered structurally by monensin. The effects of monensin on the Golgi apparatus were reversible, and dictyosomes were either repaired or new dictyosomes were formed after the root tips were removed from the monensin.

Guinea pig testes were processed for electron microscopy by prefixing in phosphate-buffered aldehyde and postfixing in 1% OsO 4 plus 0.05 M potassium ferricyanide [(K 3 Fe(CN) 6 )]. The K 3 Fe(CN) 6 enhanced the staining of glycogen, which was located predominantly in the Sertoli cells, and decreased the staining of ribosomes. The K 3 Fe(CN) 6 also selectively stained some of the small vesicles associated with the forming faces of spermatid Golgi apparatus, the nuclear envelope under the acrosome, and the ground substance of spermatid and spermatozoa mitochondria. This last staining reaction was subdued or absent from spermatocytes, spermatogonia, Sertoli cells, and Leydig cells.

The effects of hexachlorobenzene (HCB) on the ability of male rats to adapt to a crowded environment were investigated. Eighty male rats were individually housed (1000 cm2 floor space per rat) and given either feed containing 250 ppm HCB or control feed. After 4 w, half of the rats from each diet group were transferred to smaller cages with four rats per cage (100 cm2 floor space per rat). After 1, 2, 4, 7, and 10 d, four rats each from the four treatment groups were sacrificed and organs were removed for analysis. The HCB diet alone had no effect on the body weight of the rats. Crowding resulted in severe loss of body weight, an effect that was potentiated by the HCB diet. The HCB diet resulted In increased liver and kidney weight but had no effect on brain weight. Livers and kidneys of crowded rats weighed less than those of singly housed rats. Rats fed HCB then crowded had higher tissue residues of HCB and higher mortality than rats given HCB and not crowded or rats crowded but not given HCB.

The effects of ochratoxin A (OA), a nephrotoxic mycotoxin, were investigated in partially nephrectomized (PN) rats (approximately 70% reduction in renal mass) following compensatory hypertrophy of the renal remnant. Renal function stabilized 27 d after surgery. PN rats compensated for the initial loss of renal function except for glomerular filtration rate (GFR, inulin clearance); this remained significantly impaired. Sham-operated (SO) rats cleared inulin and p-aminohippurate (PAH) at rates of 3.84 and 7.49 ml/min, respectively, while compensated PN rats cleared inulin at 2.51 and PAH at 8.84 ml/min. Daily administration of low levels of OA produced decreased urine osmolality and body weight with a modest increase in urinary protein of PN versus SO rats. OA-treated rats cleared inulin, creatinine, and PAH at rates significantly lower than nontreated controls: 0.89 and 1.96 ml/min for inulin, 0.35 and 0.56 ml/min for creatinine, and 2.29 and 6.23 ml/min for PAH. Histopathological findings indicated a considerable increase in renal tubular necrosis and subcellular damage (i.e., loss of cytoplasmic ground substance, vacuolization, degeneration of mitochondria, and reorganization of endoplasmic reticulum) in PN animals versus controls, concurrent with alteration in renal function. These results verify that the nephrotoxic action of OA is elicited mainly in renal proximal tubules and is enhanced in the PN rat.

A method is described for eliminating a form of contamination called embedding pepper that appears in, or on, thin sections following poststaining in lead citrate. The pepper is present most often in the matrixes of mitochondria, peroxisomes, and red blood cells. Formation of pepper can be prevented by treating the section with 0.5 N HCl or 1.0% ethylenediaminete-traacetic acid (EDTA) for 1–5 min before staining in uranyl acetate or lead citrate.

Point source enlargers may cause unusual types of printing defects. One type is a large spot in the center of the enlarged picture field that sometimes appears when the edges of negatives are not adequately masked during printing. Another type is a blurry image caused by a defect in the polycontrast filter. The defect appears in the filter as a small spot of about 1/8-inch diameter, formed, presumably, by heat from the focused beam of the point source light. A spot defect of this type is difficult to see by a cursory visual examination of the filter and may develop unnoticed and persist for months before it is finally recognized.

1. 1. Sucrose density gradients were used to characterize lysosome-containing fraction from 0 hr white prepupae of Stomoxys calcitrans (L.). 2. 2. The acidic glycosidases (α-glucosidase, α-galactosidase, α-mannosidase, β-glucosidase, β-galactosidase, β-glucuronidase and N-acetylglucosaminidase) equilibrate at the same density as does acid phosphatase. 3. 3. The mitochondrial marker enzyme cytochrome c oxidase also equilibrates at the same density. Gomori staining showed the presence of acid phosphatase in the lysosomes. 4. 4. The 10,000 g fraction contains mostly mitochondria with lysosomes and RER.

Many factors (e.g., resolution of microscope, type of tissue, and preparation of sample) affect electron microscopical images and alter the amount of information that can be retrieved from a specimen. Of interest in this report are those factors associated with the evaluation of epoxy embedded tissues. In this context, informational retrieval is dependant, in part, on the ability to “see” sample detail (e.g., contrast) and, in part, on tue quality of sample preservation. Two aspects of this problem will be discussed: 1) epoxy resins and their effect on image contrast, information retrieval, and sample preservation; and 2) the interaction between some stains commonly used for enhancing contrast and information retrieval.

Three independent methods, one cytological and two biochemical, were used to estimate contributions of plastids and plastid fragments to various membrane fractions. In thin sections viewed by electron microscopy, KMnO(4) selectively enhanced the images of plastid membranes in situ as well as in isolated fractions. The amounts of plastid fragments in isolated membrane fractions were determined by electron microscopic morphometry of fractions fixed with KMnO(4) in conjunction with analysis of galactolipids and carotenoids. Monogalactosyl and digalactosyl diglyceride contents were directly correlated with the amount of plastid membranes in the fractions identified by electron microscope morphometry. Amounts of carotenoids also correlated with plastid membranes except at very low levels where estimates based on carotenoids exceeded those based on morphometry.

The effects of monensin on plant growth were evaluated using ryegrass seedlings. Monensin inhibited germination by ca. 8% at 10-5 M (ca. 7 mg/L) and ca. 91% at 10-4 M. At 10-4 M monensin, only the shoot emerged from the few seeds that germinated. Similarly, growth was partially inhibited by monensin at 10-5 M and severely inhibited at 10-4 M. Roots were affected more severely than shoots. Ultrastructural analyses of root cells showed alterations in dictyosomes and in the transport of secretory product into the cell wall. These alterations were more severe at 10-4 M than at 10-5 M monensin. At 10-5 M monensin, only the outer cells of the root were affected, whereas at 10-4 M monensin, even the innermost cells of the root showed effects. In addition, at 10-4 M monensin, but not at 10-5 M, ca. 20% of the root tip cells died with prolonged treatment.

Broiler chickens can be contaminated by Salmonella typhimurium, which is a food safety concern. It has been previously shown that D-mannose blocks S. typhimurium adherence to chicken intestine in vitro. One-day-old broiler chickens were fed normal drinking water or drinking water supplemented with 2.5% mannose for 10 days. On Day 3, both groups were challenged orally with 1 x 10(8) S. typhimurium [ST-10 (Animal Diagnostics Laboratory, Ames, IA)] resistant to Nal and Nov (Sigma, St. Louis, MO). On Day 10 the birds' caecal contents were examined for the antibiotic-marked S. typhimurium. Two additional groups of birds were provided normal drinking water or mannose but were not challenged with the bacteria. Salmonella-challenged control chickens were 78, 82, and 93% colonized whereas Salmonella-challenged mannose-treated chickens were only 28, 21, and 43% colonized. Moreover, the mean log10 counts of control and mannose groups were significantly (P less than .001) reduced by at least 99%. Mannose-supplemented drinking water had no effect on weight gains. Certain carbohydrates may provide a means to reduce S. typhimurium contamination in broilers.

Pierce's disease bacteria formed aggregates in the xylem of susceptible grapevines that appeared to hinder the flow of water and mineral nutrients through the plant. Similar aggregates were present in tolerant grapevines, but in substantially lower numbers. Pierce's disease iunduced the formation of tyloses and gums in the tracheary elements of infected grapevines. Thwe frequencies of tyloses and gums in tolerant muscadine and wild grapevines were 7 or 8 times those in susceptible bynch grapevines. These tyloses and gums often encapsulated the bacteria and occasionally filled the infected elements of the xylem. The results suggest that gums and tyloses restrict the spread of infecting bacteria, thus imparting tolerance to Pierce's disease in certain types of grapevines.

Ease of cutting thin sections with glass knives is markedly improved if the embedding resin contains a surfactant such as lecithin. With lecithin, it is possible to cut 50–100 thin sections from the same place on the knife edge even after facing off the block with 1–2-μm-thick sections. Image quality is similar to that of the unmodified resin if the resin formula is optimized. If not, some chatter or a “mottled” appearance of the tissue image may be present. Lecithin does not significantly affect sectioning with a diamond knife or the appearance of the section in the microscope. The increased ease of sectioning with glass presumably will be translated to diamond knives in the form of an increased useful life of the cutting edge.

The modulating effect of preinoculation and postinoculation treatment with a single oral 4.0 mg/kg dosage of T-2 toxin on cell-mediated resistance was studied in mice inoculated with Listeria monocytogenes. Toxin treatment caused significant decreases in thymus and spleen weights, bone marrow cellularity, and in the total number of circulating leukocytes, lymphocytes, and neutrophils. Enhancement or suppression of resistance to listeriosis was dependent on the time of toxin administration relative to the time of Listeria challenge. Preinoculation treatment on Day 2 or 4 prior to Listeria challenge significantly enhanced resistance and decreased mortality due to listeriosis by as much as 50%. In contrast, resistance was suppressed and mortality was increased by 50% in mice that were treated with toxin after Listeria challenge. Enhanced resistance to listeriosis was accompanied by a significant increase in the influx of macrophages into Listeria-elicited peritoneal exudates. In addition, in vivo phagocytosis of sheep red blood cells by peritoneal macrophages was significantly increased in toxin-treated mice that were sensitized with sheep erythrocytes. The data indicate that T-2 toxin has a modulating effect on cell-mediated resistance and that enhancement of resistance to listeriosis in mice pretreated with T-2 toxin is associated with increased migration/activation of macrophage effector cells.

During seed germination, lamellar bodies appear within the cytoplasm of maize embryo and bean and pea cotyledon. They are most prominent when endoplasmic reticulum (ER) is increasing most rapidly. Two unit membranes from the lamellar bodies appear to act as transition cisternae to provide continuity between the lamellar bodies and the ER. The transition cisternae are smooth surfaced (without ribosomes), and the lumina are narrow. The transition between the connecting cisternae and the ER occurs abruptly over a distance of less than 0.1 μm. At the region of transition, the lumen of the ER increases to a width characteristic of rough ER, and ribosomes are found attached to the ER. The lamellar bodies appear to be derived from the globoids of the protein bodies, from protein bodies conjoined with lipid bodies, or perhaps both. Formation of lamellar bodies coincides with the disappearance of stored protein and lipid reserves and the formation of new ER membranes during germination. Disappearance of the lamell...

Euglena gracilis was treated in 10-5 M monensin for various times from 2 minutes to 24 hours, and then processed for electron microscopy by fixation in glutaraldehyde and osmium tetroxide or potassium permanganate. Monensin affected the mature (trans) half of the cisternae but not the forming (cis) half of the cisternae. After glutaraldehyde and osmium tetroxide fixation, the affected cisternae appeared swollen, whereas after potassium permanganate fixation, the affected cisternae were distorted but not swollen. The monensin effect was first noticeable after 5 minutes of treatment and the maximum effect was observed after only 10 minutes of treatment. No additional monensin effects were observed up to 24 hours of treatment; however, by 24 hours there was variability in dictyosome form and some dictyosomes appeared relatively normal. The first noticeable effects at the 5 minute treatment time involved either the most mature (trans) cisterna or cisternae in the middle of the stack. Thus, inEuglena, the region of the Golgi apparatus that responds to monensin by cisternal dilation is restricted to the mature (trans) half of the dictyosomes, with the initial response given by specific cisternae in the stack.

Structures superficially resembling dictyosomes (DLS) are present in guinea pig spermatocytes. They are first visible in late stages of spermatogonial development as single, irregularly shaped saccules. DLS saccules continue to form, at what appears to be a much accelerated rate, during the first stages of spermatocyte development. After formation, the saccules mature and aggregate into a stacked, or dictyosomal, configuration. DLS reach their maximum numbers in spermatocytes just prior to the formation of proacrosomal granules and, at this time, constitute more membrane than Golgi apparatus and as much as 25% of the total endomembrane of the spermatocyte. The DLS then decline in numbers and only a few remain in spermatids. DLS reappear just prior to spermiation and become conspicuous features of the residual body and cytoplasmic droplet. DLS membranes have structural and cytochemical similarities to the membranes of the mature (trans) faces of the Golgi apparatus, and especially the thick cisternae of the spermatid Golgi apparatus. They are similar, also, to the membranes of the acrosome, the cell surface, multivesicular bodies (MVB), and a class of vesicles whose membranes appear “thin” following fixation in tannic acid. Both the thin membrane vesicles (i.e., the tannic acid asymmetrical vesicles-TAAV) and the MVB may act as membrane pools for the generation and/or dissipation of DLS.

The effects of epoxy resin formulation on tissue contrast and organelle size were evaluated by examining thin sections of rat liver in the electron microscope and by doing a simple morphometric analysis of peroxisome and nuclear size. Contrast was greatest for tissues embedded in the Quetol formulation of Kushida and least for tissues embedded in the Ladd formulation. Epon 812, Epon-Araldite, and Spurr formulations gave results intermediate to those of the Quetol and Ladd formulations. Organelle size was greatest for tissues embedded in the Ladd formulation and smallest for tissues embedded in the Spurr formulation. The maximum size differences were approximately 15%. Epon 812, Epon-Araldite, and Quetol formulations gave results (for organelle size) intermediate to those of the Ladd and Spurr formulations. The gross shrinkage properties of the epoxy resin blocks were all the same and, therefore, could not account for the differences in organelle size.

During the imbibition of aleurone layers of wheat grains (Triticum aestivum L `Arthur'), lamellar bodies appeared within the cytoplasm of aleurone cells. Previous morphological studies have implicated lamellar bodies as intermediates in the de novo formation of endoplasmic reticulum (ER). Lamellar bodies of wheat were pleomorphic Strongly osmiophilic, they consisted most often of closely spaced whorls of paired membranes in configurations reminiscent of animal myelin In situ, the lamellar bodies were associated with spherosomes, aleurone grains, and putative nascent ER. In electron micrographs of pellets prepared from cellular homogenates, lamellar bodies were readily recognizable even when not associated with other organelles Following gibberellic acid (GA3)-treatment of aleurone layers, the numbers and size of lamellar bodies increased, and their distribution among fractions prepared by differential centrifugation changed. Upon sucrose gradient fractionation of the differential pellets, lamellar bodies ...

The cells of the follicular epithelium of the ovarioles of the mosquito, Aedes aegypti (L.), and of the stable fly, Stomoxys calcitrans (L.), have been found to be interconnected by cytoplasmic bridges. Cytoplasmic bridges have been reported between sibling gamete cells of both male and female insects, but these bridges have not been previously reported to exist between somatic cells of insects.

Responses of cellular organelles to purposely-induced mechanical injury, at or near the surfaces produced by excision of sections from maize root tips, have been studied. These responses are apparent in sections fixed within a few seconds of excision, hence they are essentially immediate. The pores in the nuclear envelope are enlarged and, frequently, proliferation of the nuclear envelope is apparent. The nuclear ground substance appears more granular; the nucleolus appears unchanged. Structural modifications are seen in the mitochondria, endoplasmic reticulum, and the plasma membrane, but there are no striking changes in the Golgi apparatus or the ground substance of the cytoplasm. Certain types of associations, not ordinarily seen in uninjured cells, between some cellular organelles, particularly mitochondria with mitochondria, mitochondria with proplastids, and both of these organelles with “lipid” bodies, are also injury responses.

The ultrastructure and distribution of chloroplasts, mitochondria, peroxisomes, and other cellular constituents have been examined in cross sections of leaves from plants with either high or low photosynthetic capacity. Photosynthetic capacity of a given plant cannot be correlated with the presence or absence of grana in bundle sheath cell chloroplasts, the presence or absence of starch grains in bundle sheath or mesophyll cell chloroplasts, the chloroplast size in bundle sheath or mesophyll cells, or the location of chloroplasts within bundle sheath cells. We conclude that the number and concentration of chloroplasts, mitochondria, and peroxisomes in bundle sheath cells is the most reliable anatomical criterion presently available for determining the photosynthetic capacity of a given plant.

At least two kinds of lipid vesicles are present in pea and bean cotyledons which can be recognized at seed maturity on the basis of whether they do or do not interassociate into lipid vesicle sheets. Those that do interassociate into sheets are also characterized by (a) their association with plastids or plasma membranes during dormancy, and (b) the unique transformation into flattened saccules that they undergo during the first few days of seed germination. These interassociated (or composite) lipid vesicles have been found in only a few seeds and may be restricted to certain classes of plants and/or certain states of cellular development. Lipid vesicle-to-saccule transformation is predominantly confined to the germinating seed. However, some lipid vesicle-derived saccules are already present in some cells even before the seed reaches maturity. These partially transformed vesicles and saccules remain unchanged over dormancy, and then resume their transformation when the seed is germinated. This suggests that some stages of seed germination are already underway before the seed reaches maturity and are only resumed at seed germination. The lipid vesicles that do not interassociate into sheets (i.e., the simple lipid vesicles) are present in all tissues at all states of cellular development. These vesicles do not undergo any conspicuous structural changes during development.

Modification of the Golgi apparatus has been studied in rootcap cells through progressive states of normal cellular development. Large vesicles, with contents of considerable electron density, are produced by the hypertrophied portions of the Golgi cisternae of outer rootcap cells. These undergo change as they pass from the apparent site of their origin to and through the plasma membrane. This passage does not seem to involve discontinuities in the plasma membrane and it is assumed that the membranes of the Golgi-produced vesicles are incorporated into the plasma membrane. There is close packing of these bodies between the surface of the protoplast and the surface of the definable cell wall, with accompanying displacement of the protoplast around the masses. The discrete nature of the bodies is finally lost but their position is still discernible. Ultimately there is a disappearance of these masses, an increase in cell wall volume, and a return to smooth contour by the protoplast surface, paralleled by a return of the Golgi apparatus to an unhypertrophied state.

Isolated Golgi apparatus fractions and total homogenates from rat testis were examined for acid phosphatase activity (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2). A comparison of pH optima and substrate specificity of the acid phosphatase activity both in homogenates and in the purified Golgi apparatus fraction suggested an enrichment of certain enzymes during the fractionation procedure. Polyacrylamide disc gel electrophoretic patterns of Triton X-100 solubilized enzyme stained for acid phosphatase activity indicated that the Golgi apparatus possessed an unique acid phosphatase. Chromatography of the Triton X-100 solubilized Golgi apparatus proteins on DEAE-cellulose columns also suggested the presence of an unique Golgi apparatus acid phosphatase. Fractions off thd DEAE-cellulose column containing this acid phosphatase were further examined and found to be inhibited by low levels of both NaF and sodium tartrate. This enzyme was also partially inhibited by Cu2+ but not Cd2+ or formaldehyde; exhibited a broad pH optima between pH 3.5 and 6.0 and was stable at incubation temperatures up to 40 °C.

Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4.

Maize root tips were fixed in potassium permanganate, embedded in epoxy resin, sectioned to show silver interference color, and studied with the electron microscope. All the cells were seen to contain an endoplasmic reticulum and apparently independent Golgi structures. The endoplasmic reticulum is demonstrated as a membrane-bounded, vesicular structure comparable in many aspects to that of several types of animal cells. With the treatment used here the membranes appear smooth surfaced. The endoplasmic reticulum is continuous with the nuclear envelope and, by contact at least, with structures passing through the cell wall. The nuclear envelope is characterized by discontinuities, as previously reported for animal cells. The reticula of adjacent cells seem to be in contact at or through the plasmodesmata. Because of these contacts the endoplasmic reticulum of a given cell appears to be part of an intercellular system. The Golgi structures appear as stacks of platelet-vesicles which apparently may, under certain conditions, produce small vesicles around their edges. Their form changes markedly with development of the cell.

A method for the isolation of the Golgi apparatus from stem tissues of onion is described. Preparations that consisted mainly of morphologically identifiable Golgi apparatus have been obtained. The best preparations were obtained from tissue homogenized under conditions of minimum shear, and in the presence of sucrose and certain additives which aid in preservation of the integrity of the Golgi membranes. Those additives, which had a pronounced stabilizing effect on the isolated apparatus, included both monovalent and divalent ions (sodium and calcium) and dextran. A large portion of the Golgi apparatus did not appear to change microscopic appearance upon isolation, but were observed to fuse into large aggregate structures not unlike those occurring naturally in certain animal or insect cells (12). Fusion occurred both at the edges of the cisternae and in register, but the integrity of the individual cisternae was not destroyed. The major contaminants of the Golgi apparatus fraction were numerous small and large spherical vesicles. At least some of these vesicles appeared to have been derived from the Golgi apparatus; others may have been fragments of the cell membrane, the endoplasmic reticulum, or other cell debris. By utilizing this procedure, it has been possible to obtain fractions of Golgi apparatus from plant tissues other than onion stem. However, at the present time it is only with onion that the Golgi apparatus has been isolated in a form that would warrant further purification for biochemical analysis.

Raphide-forming cells in Vanilla planifolia and Monstera deliciosa differ from those of adjacent cells, and from other known cell types. The cytoplasm of cells destined to produce raphide crystals can be differentiated very early in development. Clear coincident patterns of change in plastids, endoplasmic reticulum, vacuoles, and crystal complexes indicate an interrelationship among these subcellular components in the production of raphide crystals. A previously unknown plastid form, apparently a modification of major portions of existing plastids, is initiated with the early stages of raphide formation. Ultimately the modified portion of these plastids appear to atrophy and are lost. An internal product accumulates in the latter stages of raphide cell development. This product appears to be evolved from elements of the endoplasmic reticulum but its composition is unknown.