INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : II. Enzymatic and Other Hydrolytic Treatments

The effects of proteolytic enzymes, ribonuclease, and deoxyribonuclease upon a fibrous component of chick embryo mitochondria, which was previously shown to have many fixation and staining properties characteristic of the bacterial nucleoplasm, are reported. Pepsin digestion of formaldehyde-fixed tissues removed the membranes and matrices of mitochondria, but a pepsin-resistant fibrous material remained which was heavily stained by uranyl and lead ions. Experiments on a DNA "model system" showed that DNA treated with osmium tetroxide can be depolymerized by deoxyribonuclease. Zinc ions strongly inhibited the depolymerization of DNA. Digestion of osmium tetroxide-fixed tissues (fixed only briefly) with deoxyribonuclease for 1 hour greatly reduced the Feulgen staining of the nuclei, and after 4 hours the Feulgen reaction was completely abolished. The reduction and the disappearance of the Feulgen reaction in nuclei was paralleled by partial to complete digestion of the mitochondrial fibers in the regions studied (after 1 and 4 hours, respectively), without any other obvious changes in cellular structures. When deoxyribonuclease was inhibited by the addition of zinc ions, the nuclear Feulgen reaction was not diminished, nor were the mitochondrial fibers removed. Buffer control incubations for deoxyribonuclease and ribonuclease did not alter the structure or staining properties of the mitochondrial fibers, nor did incubation with ribonuclease. The latter reaction digested the cytoplasmic and nucleolar ribosomes after a 4-hour incubation period, in parallel with the abolishment of toluidine blue staining. The results contribute further evidence that these mitochondria contain deoxyribonucleic acid.     

Marchi's Staining Method: II. Fixation

Following experimental lesions, spinal cords of cats and rabbits were fixed with acid, neutral, and alkaline solutions. Staining was limited to a chromate-osmic (Marchi's) solution and a chlorate-osmic solution. The following conclusions were drawn:

The presence of an acid in the fixative caused normal myelin sheaths to stain. This effect was reduced by washing tissues before staining, by adding acetic acid to the stain, or by employing a non-formalin fixative. It was, however, at no time entirely obviated.

A study was made of the granular deposits which occur in nearly all Marchi preparations and which are especially confusing if very light backgrounds are obtained.

The staining reactions of the granular deposits were very similar to those of degenerating myelin but some suppression of the granules was obtained by adding KCIO₃ to the formalin fixative.     

Fixation and Staining of the Bacterial Nucleus

An examination of some early methods in bacterial cytology shows that technics for demonstrating the nucleus of the Eubacteriales were available for at least twenty years before the current era of investigation, which began in 1942. Although the bacterial nucleus reacts in many respects like the chromatin elements of higher plants, it shows certain peculiarities in its staining reactions. For example, hematoxylin, methyl green, and several preparations used to stain chromosomes, apparently do not exhibit the same affinity for the bacterial nucleus that they do for chromosomes in higher plants. Not only is the selection of a fixative important in nuclear studies, but also the manner in which the fixation is obtained. For example, when bacteria are fixed and processed in a completely wet state it is generally impossible to stain their nuclei. None of the special fixatives studied revealed any unusual organization in the bacterial cell or exhibited any advantage over the fixatives now in common use by bacteriologists. In view of the properties of osmium tetroxide vapor, particularly its relative lack of interference with positive nuclear staining, there can be little doubt of its superiority as a nuclear fixative. It appears that the basophilic material removed from the bacterial cell by hydrochloric acid is not only in the cytoplasm, but that a very significant amount of it is in close contact with the cell wall.     

Alcoholic Bouin Fixation of Insect Nervous Systems for Bodian Silver Staining. I. Composition of 'Aged' Fixative

Alcoholic Bouin (Duboscq-Brasil) fixative being 'aged' at 60 C to improve tissue preservation and subsequent staining was sampled at various stages to determine its histological effectiveness and chemical composition. Histological performance was tested using ventral nerve cord ganglia of the cockroach Periplaneta americana and the locust Schistocerca gregaria. Chemical analysis was by ultraviolet spectroscopy, thin layer and gas-liquid chromatography, and mass spectrometry. Histological performance improved rapidly during the first 7-10 days and composition changed correspondingly. The rate of change then slowed as a more stable condition was approached. Fully aged solutions, after about 40 days, giving optimum fixation and staining, contained little more than half the amounts of the volatile components (formaldehyde, ethanol, and acetic acid) in the original mixture, together with ethyl acetate and a formal, diethoxymethane, as the principal reaction products, but picric acid content showed little change. Older ('overaged') solutions, fully aged and then kept at room temperature for 1-2 yr, gave poorer fixation and staining and contained still less of the original volatile constituents and correspondingly more of the reaction products. A 'simplified synthetic aged alcoholic Bouin' (15 ml 40% formaldehyde, 35 ml ethanol, 3.5 ml acetic acid, 5 ml ethyl acetate, 15 ml diethoxymethane, 0.46 g picric acid, and water to 100 ml) closely simulated the performance of the fully aged orthodox fixative without the need for aging.     

Fixation and Staining of Planaria for Histological Study

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acld-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.     

INTRAMITOCHONDRIAL YOLK-CRYSTALS OF FROG OOCYTES : I. Formation of Yolk-Crystal Inclusions by Mitochondria during Bullfrog Oogenesis

Electron microscope examination of thin sections of bullfrog (Rana catesbeiana) ovarian oocytes has shown the presence of mitochondria containing yolk-crystal inclusions in oocytes of all sizes, from 160 to 1500 µ mean diameter. The hexagonally shaped yolk-crystals have major periodicities of 73.8 ± 10.7 A (n = 100). Several forms of modified mitochondria, observed in the smaller oocytes, may be arranged into a series of structurally intermediate forms between standard oocyte mitochondria and the typical mitochondria with yolk-crystal inclusions. The observation of such intermediate forms is consistent with proposals that the yolk-crystal inclusions arise within a limited portion of the oocyte chondriome by a complex process of mitochondrial differentiation.     

Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

STUDIES ON SEEDS : I. Fixation of Seeds

Several fixation procedures were studied to determine those most suitable for preservation of seeds during late stages of development and early stages of germination. These are the periods when the tissues are partially dehydrated and are most difficult to fix for electron microscopy. It was found that a prefixation with a mixture of glutaraldehyde, reconstituted formaldehyde (i.e. paraformaldehyde), and acrolein, followed by a postfixation in OsO₄ or KMnO₄, gives very acceptable images. The results also indicate that glutaraldehyde is necessary for preservation of cell shape, paraformaldehyde for stabilization of reserve proteins, and acrolein for rapid penetration of tissues. Phosphate, cacodylate, and collidine are all acceptable buffers, although collidine gives the most consistent results.     

Photosynthetic Reactions in the Marine Alga Codium vermilara: I. CO(2) Fixation and Hill Reaction in Isolated Chloroplasts

Chloroplasts were isolated from the marine alga Codium vermilara (Siphonales). The isolated chloroplasts were active in CO₂ fixation in the light at a rate comparable to the rates obtained by fragments of thalli. Maximal rates of CO₂ fixation by isolated chloroplasts from Codium were obtained in the presence of salt or sorbitol isoosmotic with sea water. The conditions of isolation of Codium chloroplasts are much less stringent than those required for active chloroplasts from higher plants. The isolated chloroplasts comprise a homogeneous population of the intact “class I” type, as based on microscopic observations and on their inability to reduce ferricyanide unless osmotically shocked. The intact chloroplasts are able to reduce p-benzoquinone at a high rate.     

Iron Hematoxylin Chelates. I. The Weil Staining Bath

The procedure for the preparation of the staining solution for the Weil myelin sheath stain was systematically varied in respect to pH, concentration, time, temperature and relative proportions of the ingredients. The results were explainable on the basis of the presence of a number of iron hematoxylin chelates in the staining bath. Compounds of the form of [Fe_nHem]^m+ are nuclear stains, those of the form of [FeHem_n]^m± are myelin sheath stains while the precipitate is probably [Fe_nHem_y]_x^m±. The following procedure for the stain is recommended. Mix equal portions of a 0.25% solution of ripened hematoxylin prepared from a 10% alcoholic solution and 1% ferric ammonium sulphate and use immediately. Preferably, the solutions should be at a temperature of about 5 C and the staining done in the refrigerator, but room temperature may be used. Higher temperatures are contraindicated. Hematein should not be substituted for ripened hematoxylin; the resulting stains are too weak to be usable. The absorbance of hematein is no measure of the concentration of the component that stains myelin sheaths. Hematein apparently consists largely of a sparingly soluble highly colored inactive compound.     

An Apparatus Designed for Holding Cover Slips During Fixation and Staining

The need of an apparatus for fixing and staining cover slips upon which cell cultures are grown, led to the construction of the glass cover-slip holder here described. The holder is made with grooves on the sides and on the bottom into which the cover slips fit. The holder illustrated is for eight cover-slips, but may of course be made for any number.     

Localization of single-chain interruptions in bacteriophage T5 DNA I. Electron microscopic studies.

Bacteriophage T5 DNA was examined in an electron microscope after limited digestion with exonuclease III from Escherichia coli. The effect of the exonuclease treatment was to convert each naturally occurring single-chain interruption in T5 DNA into a short segment of single-stranded DNA. The locations of these segments were determined for T5st(+) DNA, T5st(0) DNA, and fragments of T5st(0) DNA generated by EcoRI restriction endonuclease. The results indicate that single-chain interruptions occurr in a variable, but nonrandom, manner in T5 DNA. T5st(+) DNA has four principal interruptions located at sites approximately 7.9, 18.5, 32.6, and 64.8% from one end of the molecule. Interruptions occur at these sites in 80 to 90% of the population. A large number of additional sites, located primarily at the ends of the DNA, contain interruptions at lower frequencies. The average number of interruptions per genome, as determined by this method, is 8. A similar distribution of breaks occurs in T5st(0) DNA, except that the 32.6% site is missing. At least one of the principal interruptions is reproducibly located within an interval of 0.2% of the entire DNA.     

Studies on Effect of Certain Quinones: I. Electron Transport, Photophosphorylation, and CO(2) Fixation in Isolated Chloroplasts

The effect of quinone herbicides and fungicides on photosynthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2,3-Dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these inhibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction.     

Nitrogen Turnover in Marine and Brackish Habitats: I. Nitrogen Fixation

Potential nitrogen-fixing genera were found to be abundant intwo natural populations of blue-green algae, one from a rockycoast and the other from a sand-dune slack. ¹⁵N studies confirmedthat these populations fixed nitrogen in the laboratory andin the field. Preliminary quantitative data on Fixation in thefield suggest that the algae contribute appreciable quantitiesof fixed nitrogen to the environments in which they occur.     

PAP染色法在微生态学中应用的研究——Ⅰ.细菌染色方法的建立

在微生态学中应用过氧化物酶—抗过氧化物酶(PAP)染色法鉴定细菌的研究还未见报道。本文报告了用埃希氏大肠杆菌(O_(111)B_4)腹腔感染小鼠,取其多种脏器制石蜡切片,建立PAP染色程序。确定了第一抗体(兔抗埃希氏大肠杆菌O_(111)B_4型血清)最佳染色滴度为1:800～3200。观察到埃希氏大肠杆菌(O_(111)B_4)定位于组织器官上的状态。在细菌鉴定上PAP染色法较其它方法更具有优点。     

Supravital Staining and Fixation of Brain and Spinal Cord by Intravascular Perfusion

Forty single and 13 combinations of dyes were tested for concomitant supravital staining and fixation of brain and spinal cord of rats, cats and squirrel monkeys by intravascular perfusion in 3 steps: (1) 60 or 80 ml of physiological saline containing 40 mg/100 ml of NaNO₂ as a vasodilator; (2) 250 or 550 ml of stain-fixative solution consisting of either: A—2 parts of dye solution in a concentration from 0.001% to 0.05% dissolved in saline containing 40 mg/100 ml of NaNO₂ and 1 part undiluted formalin; or B—2 parts of dye solution in a concentration from 0.001% to 0.05% dissolved in distilled water acidified with 1.5 ml of glacial acetic acid per 100 ml of water, and 1 part undiluted formalin; and (3) 100 or 150 ml of 6% dextrose in distilled water. Complete staining and fixation was accomplished in 53 min for rats and 41 min for cats and monkeys. Brains and spinal cord were frozen sectioned, and the cut surfaces of the frozen tissue were photographed similar to the procedure described by Gasteiger et al     

Alcoholic Bouin Fixation of Insect Nervous Systems for Bodian Silver Staining. II. Modified Solutions

A previously devised synthetic equivalent of 'aged' alcoholic Bouin (Duboscq-Brasil) fixative was modified in various ways to discover which of the chemical changes brought about by aging were important in improving fixation and staining. Effects were tested with ventral nerve cord ganglia of the cockroach Periplaneta americana, locust Schistocerca gregaria, and honey bee Apis mellifera. Formation of reaction products, chiefly ethyl acetate and diethoxymethane, seemed to play only a subsidiary role: neither individually appeared essential as long as a sufficient quantity of one or the other was present. In place of diethoxymethane, ethyl acetate concentration could be increased to 25% with little effect on results. Reduction in concentration of two of the original constituents, formaldehyde and ethanol, appeared to be the principal factor in improving fixation. Varying the concentration of each original constituent individually revealed that formaldehyde mainly increased glial staining, ethanol increased tissue shrinkage and reduced overall staining intensity, acetic acid improved preservation, and picric acid decreased glial staining but produced few other effects within a wide range of concentrations, though its omission seriously impaired overall preservation and staining. Varying the ethanol and acetic acid concentrations simultaneously confirmed that they acted in opposite ways. A decrease in ethanol and an increase in acetic acid both improved results. The optimum mixture, 'improved synthetic alcoholic Bouin' (40% formaldehyde 0-15: ethanol 25: acetic acid 5: ethyl acetate 5: diethoxymethane 15: picric acid 0.5: water to 100), gives better preservation and more intense staining, and formaldehyde content can be varied to give the degree of glial staining required. Without formaldehyde glial staining is virtually eliminated, while preservation and staining of the neurons appears unaffected. This modification seems to offer a valuable advance in technique.     

Staining Plant Cells with Silver. I. The Salt-Nylon Technique

A technique is described for selectively silver staining nucleoli, active nucleolus organizers, nucleolar material attached to chromosomes, kinetochores, synaptonemal complexes, and chromosome cores in plant cells. The technique, called salt-nylon silver staining, involves spreading cells on glass slides, treating the cells with a solution of saline sodium citrate, and incubating the cells in a silver nitrate solution covered with nylon screen. Selected variables important for achieving reliable silver staining are considered.     

Fine structure of Bacillus subtilis. I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO(4), OsO(4), or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO(4) in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO(4) fixation appeared to provide much better definition of the boundaries of various structures than did OsO(4). With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO(4) fixation) as two thin lines. In cells fixed first with OsO(4) solution, and then refixed with a mixture of KMnO(4) and OsO(4) solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO(4). One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.