Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution.

Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium.     

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.     

PRESERVATION OF MYELIN LAMELLAR STRUCTURE IN THE ABSENCE OF LIPID : A Correlated Chemical and Morphological Study

The fine structure of myelin was studied in glutaraldehyde-fixed rat sciatic nerves depleted of lipid by acetone, chloroform:methanol (2:1 v/v), and chloroform:methanol:concentrated HCl (200:100:1, v/v/v). One portion of each of these nerves, plus the extracts, was saponified and analyzed by gas-liquid chromatography for fatty acids. The remainder of each nerve was stained in osmium tetroxide in CCl₄ (5g/100cc) and was embedded in Epon 812. Thin sections, examined in the electron microscope, revealed the preservation of myelin lamellar structure with a 170 A periodicity in nerves depleted of 98% of their lipids. Preservation of myelin lamellar structure depended on glutaraldehyde fixation and the introduction of osmium tetroxide in a nonpolar vehicle (CCl₄) after the lipids had been extracted. It is concluded that the periodic lamellar structure in electron micrographs of myelin depleted of lipid results from the complexing of osmium tetroxide, plus uranyl and lead stains, with protein.     

A detection method for lactic dehydrogenase activity in the inner ear

A method for the detection of lactic dehydrogenase enzymatic activity in outer hair cells of the rabbit is described. The membranous labyrinth with temporal bone was prefixed in glutaraldehyde. After being placed into the incubation medium, it was postfixed in osmium tetroxide. Specimens of the organ of Corti were removed. Then the specimens were embedded in water-soluble glycol and cut with a cryostat for light microscopy, and also they were embedded in Epon and cut for light and electron microscopy. Sectioning of the membranous labyrinth was very easily made when the specimens were embedded in both the water-soluble glycol and the Epon. The structures of the frozen sections as well as the Epon-embedded ones were well preserved. In the frozen sections the preservation and localization of reaction products were thoroughly kept, but monoformazan of the Epon-embedded sections was soluble.     

Ultrastructure of infected cells in the actinorhizal root nodules ofGymnostoma papuanum (Casuarinaceae) prepared by high pressure freezing and chemical fixation

Summary Actinorhizal root nodules ofGymnostoma papuanuum (Casuarinaceae) were examined with transmission electron microscopy after being either fixed with glutalaldehyde and osmium tetroxide or frozen with liquid nitrogen at high pressure and freeze-substituted. Much better preservation was obtained by the cryopreservation method. Mitochondria, plastids, membranes und ribosomes were much better preserved in the frozen specimens than in the chemically fixed tissues. No nucleoids were observed in the microsymbiont in frozen specimens. In contrast nucleoid regions were present in chemically fixed specimens. The actinomycete microsymbiont differentiates small spherical-shaped symbiotic vesicles after the hyphae have grown and penetrated into most regions of the cytoplasm.Dedicated to the memory of Professor Oswald Kiermayer     

Agar-Screen Specimen Carrier for Bulk Processing of Biopsy Materials for Electron Microscopy

A specimen carrier for processing large numbers of biopsy materials for epoxy embedding and electron microscopy is described. Commercially available 18-mesh stainless steel or 16-mesh aluminum wire screening is used. The screening is cut into 1 × 3-inch strips. One corner is snipped off for orientation purposes. Four drops of warm 4% agar is placed on a prewarmed standard microscopic glass slide. A thin agar support film is formed on the bottom side of the horizontally held wire screen by lightly running it against the agar. Tissue blocks trimmed to 1 mm³ are blotted on filter paper and placed in a prearranged order on the top surface of the support film. A thin top coating of agar is applied on the specimen by touching it with the tip of a pasteur pipette containing warm 4% agar. The agar-screen unit with the mounted specimens is stabilized in 4% buffered formalin and rinsed with Sorenson's phosphate buffer, pH 7.4, with 6.8% sucrose. It is then processed as a unit through routine osmium tetroxide postfixation, alcohol dehydration, and Epon 812 infiltration. The tissue blocks are plucked off the agar support film with fine-tipped tweezers and embedded in individual capsules. No difficulty in thin sectioning was encountered and examination of the sections under the electron microscope showed good infiltration by the epoxy resin.     

Histochemistry of glycogen in the inner ear

Synopsis The effect of fixation and processing upon the morphological appearance of glycogen within the outer hair cells of the guinea-pig was investigated using two methods. In each method, tissue was fixed for 12 h in cold phosphate-buffered 4% paraformaldehyde and eventually dehydrated in ethanol, embedded in Epon 812, and cut into 4 m sections. In procedure A, after complete processing, the sections were tained using the periodic acid-Schiff reaction (PAS) or the periodic acid-thiocarbo-hydrazide-osmium tetroxide (PATCO) reaction which resulted in the appearance of listinct, coarse granules in the cytoplasm of the outer hair cells. Diastase digestion on one of the two matched sections after Epon removal and prior to staining, confirmed the granules to be glycogen. In procedure B, after primary fixation, the tissue was post-fixed in 1% osmium tetroxide and then processed exactly as in procedure A. Here, unless the Epon and osmium was remoyed, there was no staining of the outer hair cell cytoplasm. However, after Epon removal there was diffuse, grainy appearance of the outer hair cell cytoplasm which we considered to be due to glycogen although diastase confirmation was not possible. We have concluded that osmium tetroxide (1) inhibits PAS or PATCO staining, (2) prevents diastase digestion, and (3) prevents the appearance by light microscopy of distinct granules of glycogen.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

New view of the surface projections of Chlamydia trachomatis.

Two kinds of surface specializations of chlamydiae have been described: hemispheric projections and spikelike rods. We undertook the present studies to demonstrate chlamydial ultrastructure in greater detail in conventional thin-sectioned specimens. Chlamydia trachomatis (LGV strain L2/434/Bu), cultured for 40 h in L929 mouse fibroblasts, was fixed in glutaraldehyde-acrolein, p-formaldehyde-glutaraldehyde, or glutaraldehyde-osmium tetroxide mixtures, postfixed in osmium tetroxide, stained in uranyl acetate, dehydrated in ethanols, and embedded in Epon. By the use of fixatives that penetrate and fix rapidly, chlamydial outer and plasma membranes were clearly revealed. Our results indicate that the hemispheric projections are specializations of the plasma membrane of elementary bodies. The spikelike projections are found in intermediate forms, originate beneath depressions of the plasma membrane, and extend through the periplasmic space and outer membrane to end with pointed tips. Improved preservation of chlamydiae provides a new, informative view of their complex structure. Significant interactions between chlamydiae and host cells might be influenced by the surface structures shown in this study.     

Preparation of tissues for localization of sex hormones by autoradiography

Summary Tissues of rats given ³H-oestradiol were prepared for autoradiography according to methods commonly used in light and electron microscopy.By formalin fixation large amounts of radioactive material were lost, both in the fixative and during dehydration. Altogether 78.6±7.5 per cent was extracted from uterine tissue, while 49.0±4.6 per cent was lost from liver tissue removed 15 minutes after the injection. Significantly more radioactivity was lost in the fixative from liver tissue than from uterine. In the former fixation accounted for about 60 per cent of the loss, whereas in the latter it was responsible for about 25 per cent.Osmium tetroxide fixation was found to retain the radioactivity of liver and uterine tissue almost completely. However, large amounts were invariably extracted during dehydration. Although only 3.9±1.2 per cent of the radioactivity of uterine tissue diffused into the fixative, 72.8±12.4 per cent was extracted during ethanol dehydration. A heavy loss was also registered when dehydration and infiltration were carried out in glycol methacrylate.Glutaraldehyde perfusion and postfixation with osmium tetroxide retained almost completely the radioactivity of uterine and pituitary tissue. Nevertheless, nearly all of it was extracted during ethanol/propylene oxide dehydration and Epon embedding.The methods studied are not adequate for accurate autoradiographic localization of oestradiol.This work was supported by grants from The Norwegian Cancer Society and by Nordisk Insulinfond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

A rapid technique for preparing microorganisms for transmission electron microscopy.

A rapid and efficient method of preparing microorganisms for transmission electron microscopy is reported. In developing the method Salmonella, streptococcal, and protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

A Rapid Technique for Preparing Microorganisms for Transmission Electron Microscopy

A rapid and efficient method of preparing microorganisms for transmission electors microscopy is reported. In developing the method Salmonella, streptococcal, ad protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

Differentiation of the membrane system in the cells of imaginai discs

Summary Imaginal discs from developing larvae of the fly Calliphora erythrocephala fixed in permanganate or osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. When the larval growth ceases, the differentiation manifests itself through an enlargement of the endoplasmic reticulum, by which continuous membrane contact is established between all cell organelles. During the same time mitochondria swell up and transform into lipid granules and the intercellular contacts weaken.I am indebted to Mrs Mariann Carleson and Miss Brita Nilsson for technical aid.     

Ultrastructure of sea urchin calcified tissues after high-pressure freezing and freeze substitution

The improvements brought by high-pressure freezing/freeze substitution fixation methods to the ultrastructural preservation of echinoderm mineralized tissues are investigated in developing pedicellariae and teeth of the echinoid Paracentrotus lividus. Three freeze substitution (FS) protocols were tested: one in the presence of osmium tetroxide, one in the presence of uranyl acetate, and the last in the presence of gallic acid. FS in the presence of osmium tetroxide significantly improved cell ultrastructure preservation and should especially be used for ultrastructural studies involving vesicles and the Golgi apparatus. With all protocols, multivesicular bodies, suggested to contain Ca(2+), were evident for the first time in skeleton-forming cells. FS in the presence of gallic acid allowed us to confirm the structured and insoluble character of a part of the organic matrix of mineralization in the calcification sites of the tooth, an observation which modifies the current understanding of biomineralization control in echinoderms.     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

Immunoelectron microscopic demonstration of thyroglobulin and thyroid hormones in rat thyroid gland

We have developed a post-embedding immunogold technique for electron microscopic localization and quantitation of thyroglobulin (TG), thyroxine (T4), and triiodothyronine (T3) in rat thyroid. Labeling for TG was located on rough endoplasmic reticulum, Golgi apparatus, exocytotic vesicles, luminal colloid, colloid droplets, and lysosomes, whereas labeling for thyroid hormones was located on luminal colloid, colloid droplets, and lysosomes. We tested different procedures of fixation, dehydration, embedding, polymerization, and immunoincubation to optimize ultrastructural preservation and immunolabeling. Fixation with glutaraldehyde and osmium was possible with retained antigenicity. Dehydration temperature and the choice of embedding resin were the two crucial factors for good immunolabeling. Low-temperature dehydration greatly improved immunolabeling and could be combined with embedding in the methacrylate LR White or the epoxide Agar 100 (equivalent of Epon 812) polymerized at 40-60 degrees C, as the temperature during subsequent embedding and polymerization was of little importance for the immunoreactivity. Labeling on LR White sections was always higher than on Agar 100 sections. Various etching procedures were tested without improved specific labeling. Etching with hydrochloric acid gave nonspecific labeling of certain cell compartments.     

An analysis of the contribution of the preparatory technique to the appearance of condensed and orthodox conformations of liver mitochondria

The structure and volume of isolated mitochondria embedded for electron microscopy during different respiratory states were analyzed in thin sections. Three different embedding methods were compared; osmium tetroxide fixation/acetone dehydration, glutaraldehyde fixation/acetone dehydration, and glutaraldehyde fixation-osmium tetroxide postfixation/acetone dehydration. Analysis of fresh mitochondria, isolated in a sucrose medium, revealed the presence of a homogeneous population with respect to structure when any of the three methods were applied. After fixation with osmium alone, or in combination with glutaraldehyde, nearly 100% of the mitochondria were in a "condensed" conformation. Mitochondria fixed with glutaraldehyde alone resulted in a population of mitochondria that had large spaces separating the two membranes of the cristae which corresponds to the condensed conformation as observed after osmium fixation. Transfer of the mitochondria to the incubation medium led to the appearance of two classes of mitochondria with respect to size. One class had a volume close to that observed when suspended in sucrose, and another class was present that was 30-45% larger. In osmium fixed or in double-fixed preparations, these small and large classes corresponded to "condensed" and "orthodox" forms of mitochondria respectively. When glutaraldehyde was used alone as the fixative, the two size classes were also present. However, the mitochondria were homogeneous with respect to structure. In these mitochondria, the width of the space that separated the cristae membranes had become reduced when compared to mitochondria suspended in sucrose. The two size classes were also present in samples of mitochondria prepared during both states 3 and 4. State 4 conditions did not lead to any significant increase of the number of condensed mitochondria. In state 3 preparations, 65-70% of the population were condensed. The condensed and orthodox forms could be related to normal and swollen forms of mitochondria. Conditions that led to a swelling also led to an increase in the number of orthodox mitochondria in osmium-fixed material. The different appearance of the mitochondria is explained by the different conditions for fixation of the mitochondria that exist when nonswollen and swollen mitochondria are fixed. This difference is particularly crucial in the case of osmium tetroxide due to the unique way this fixative, among generally used fixatives, denatures proteins.