Rapid Preparation of Thick Sections of Fleshy Plant Tissues

Methods are described for permanent micro-slide preparations of soft, large-celled plant tissues such as ripe fruit. Thick sections (200-800 [t) cut on a sliding microtome are aspirated in an aqueous killing agent; after fixing and washing, the sections are dehydrated and cleared in an alcohol-xylene series. Infiltration with 20, 30, and 40% solutions of mountant prior to mounting the sections is necessary to avoid too abrupt changes in the cleared tissues. Several staining methods have been successfully used for different purposes. The final preparations showed nearly perfect preservation of intact cells and intercellular spaces in their 3-dimension-al structure.     

Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture

Leydig cells prepared routinely (glutaraldehyde--osmium) for ultrastructural studies are generally found to be lacking in subcellular detail as a result of poor membrane preservation and a dense cytoplasmic matrix. A method modified after that of Karnovsky (1971), utilizing a ferrocyanide--osmium mixture for post-treating glutaraldehyde fixed tissued, was found to yield routinely excellent preservation of Leydig cells. The primary advantages of this method were the enhancement of contrast within the Leydig cell and greatly improved membrane preservation. In addition, the smooth endoplasmic reticulum always appeared as an extensive network of interconnected tubules of uniform diameter; mitochondria, lysosomes, peroxisomes, multivesicular bodies, and Golgi were especially prominent. Glycogen and microfilaments, not readily seen in routine preparations, were found to be abundant in these cells. New observations on the numbers and distributions of subcellular organelles are described and are discussed in relation to their possible role in the steroidogenic process. In view of the greatly improved tissue preservation observed in this study, it is suggested that this treatment be used routinely for preservation of rat Leydig cells.     

An improved method of toluidine blue counterstaining of neurons in immunocytochemical preparations

Immunocytochemical methods can identify individual neurons and processes made immunoreactive by virtue of the antigens they contain. However, frequently it is also useful to visualize surrounding nonimmunoreactive cells, but immunocytochemical procedures often interfere with the quality of subsequent counterstaining. This report describes an improved method of counterstaining immunocytochemical specimens with either aged (at least 1 year) or concentrated solutions of toluidine blue. This technique combines well with immunocytochemical preparations of at least two antigens, i.e., choline acetyltransferase and glutamic acid decarboxylase, to delineate nonimmunoreactive somata. Additionally, a method of photographing these color preparations is described that, by the use of an appropriate filter, allows one to illustrate sections essentially with and without blue counterstain in black and white photomicrographs.     

An Improved Method of Toluidine Blue Counterstaining of Neurons in Immunocytochemical Preparations

Immunocytochemical methods can identify individual neurons and processes made immunoreactive by virtue of the antigens they contain. However, frequently it is also useful to visualize surrounding nonimmunoreactive cells, but immunocytochemical procedures often interfere with the quality of subsequent counterstaining. This report describes an improved method of counterstaining immunocytochemical specimens with either aged (at least 1 year) or concentrated solutions of toluidine blue. This technique combines well with immunocytochemical preparations of at least two antigens, i.e., choline acetyltransferase and glutamic acid decarboxylase, to delineate nonimmunoreactive somata. Additionally, a method of photographing these color preparations is described that, by the use of an appropriate filter, allows one to illustrate sections essentially with and without blue counterstain in black and white photomicrographs.     

Non-aqueous permanent mounting for immunofluorescence microscopy

It is generally assumed that an aqueous mounting medium is necessary for the preservation of immunofluorescent-labelled microscopical preparations and polyvinyl alcohol-based solutions (e.g. Mowiol) being the most frequently used mounting media; however, both the quality and intensity of the fluorescence signal in most immunolabelled preparations after aqueous mounting slowly diminish with time, and finally, samples become unsuitable for examination. In the present work, we describe a very simple and rapid non-aqueous mounting procedure for cultured cells and tissue sections, which preserves the fluorescent signal in an excellent way after immunodetection or use of other specific labelling methods. It is based on the current histological protocol in which, after fluorescence labelling, preparations are dehydrated in ethanol, cleared in xylene and mounted in DePeX. Using this non-aqueous mounting medium, the fluorescent signal remains high and stable, allowing a suitable and permanent preservation of labelled and counterstained microscopical preparations.     

Conservation and management of brown trout, Salmo trutta, stocks in Wales by the Welsh Water Authority

SUMMARY. 1. The diversity, value and status of the trout resource within the Welsh Water Authority area is described.
2. Reported angling catches of sea trout have increased in recent years but there is an apparent decline in brown trout stocks. Factors affecting the distribution, status and diversity of the trout resource are identified and discussed.
3. Investigations are being carried out to evaluate the apparent problems and to provide information required to formulate management solutions. A management strategy is proposed which allows for the maintenance and development of the resource, whilst ensuring the preservation of strains of trout which have conservation value in their own right.     

Ultrastructural visualization of unfixed and unstained whole mounts by high-voltage electron microscopy at low temperatures

A physical procedure for the visualization of cellular fine structures is described as an alternative to chemical preparative techniques. It consists of fixation by fast freezing followed by controlled etching in the cryostage of a million-volt transmission electron microscope. Whole mounts were thus observed under stable conditions with no use of chemical fixatives, solvents, or stains, with no exposure to the atmosphere, and with the improved penetration and resolution in thick specimens that characterize high-voltage electron microscopy. The preservation, contrast, and resolution exhibited by images of preparations obtained by this procedure are discussed.     

STUDIES ON FORMALIN FIXATION FOR ELECTRON MICROSCOPY AND CYTOCHEMICAL STAINING PURPOSES

A study has been made of the preservation of fine structure, phospholipids, and the activity of acid phosphatase and esterase in rat liver fixed in various solutions containing 4 per cent formaldehyde. Examination of methacrylate-embedded preparations shows that calcium-containing fixatives result in poor preservation of fine structure, whereas veronal-treated or phosphate-buffered formalin gives excellent results if the tonicity of the solutions is suitably adjusted by addition of sucrose. Formol-phosphate, to which Versene has been added, causes deterioration of cellular morphology. Phospholipids are retained almost quantitatively in tissue fixed in formol-calcium, and in phosphate-, collidine-, or triethanolamine-buffered formalin. About 50 per cent of the activity of acid phosphatase and esterase are preserved after 24 hours exposure to these fixatives at 0–2°C, and the distributions of the enzymes and of phospholipids, as judged by cytochemical staining results, are not altered by any of these formalin solutions. Consideration of the morphological and biochemical integrity of the fixed tissue suggests that 4 per cent formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, and containing 7.5 per cent sucrose, is the most suitable of the fixatives for combined cytochemical staining and electron microscopical studies.     

Internally perfused Myxicola giant axons showing long-term survival.

An improved method for internally perfusing the Myxicola giant axon based on removing the axoplasm by dispersing it in KCl-KF salt solutions is described. Proteolytic enzymes are not introduced. With this improved method perfused preparations show long-term stability of their electrical properties and the ability to generate action potentials for many hours. Mean initial values for resting membrane potential, action potential amplitude, and peak inward current were -68 mV, 118 mV, and 3.62 mA/cm2, respectively. Mean resting membrane resistance was 75% of that in intact axons. In one series of voltage clamp experiments, perfused preparations remained excitable for a mean period of 5 1/2 h, but this period could exceed 10 h. 4 min are needed for exchange of internal solutions. At least 50 mM KF is required both in the axoplasm liquefying solution and in the standard perfusate to obtain stable preparations.     

Crush preparations of lesions of the central nervous system. A useful adjunct to the frozen section

A series of 32 cases in which crush preparations were used in addition to frozen sections for the rapid diagnosis of lesions of the central nervous system (CNS) is presented. The cytopathologic features in crush preparations of astrocytomas, glioblastomas multiforme and a pituitary adenoma are described. Excellent preservation of cellular detail was seen in the crush preparations. Frozen sections lacked cytologic detail but provided a better view of the tissue architecture. The crush preparations yielded the correct diagnosis in 29 of the 32 cases. In the other three, a secondary component of the neoplasms (oligodendroglioma and fibrosarcoma) was identified only in the paraffin sections. Use of both frozen sections and crush preparations is recommended for all cases in which an immediate diagnosis of a CNS lesion is required.     

Biodiversity,biological uncertainty,and setting conservation priorities

In a world of massive extinctions where not all taxa can be saved, how ought biologists to decide their preservation priorities? When biologists make recommendations regarding conservation, should their analyses be based on scientific criteria, on public or lay criteria, on economic or some other criteria? As a first step in answering this question, we examine the issue of whether biologists ought to try to save the endangered Florida panther, a well known “glamour” taxon. To evaluate the merits of panther preservation, we examine three important arguments of biologists who are skeptical about the desirability of panther preservation. These arguments are (1) that conservation dollars ought to be spent in more efficient ways than panther preservation; (2) that biologists and conservationists ought to work to preserve species before subspecies; and (3) that biologists and conservationists ought to work to save habitats before species or subspecies. We conclude that, although all three arguments are persuasive, none of them provides convincing grounds for foregoing panther preservation in favor of other, more scientifically significant conservation efforts. Our conclusion is based, in part, on the argument that biologists ought to employ ethical, as well as scientific, rationality in setting conservation priorities and that ethical rationality may provide persuasive grounds for preserving taxa that often are not viewed by biologists as of great importance.     

A New Technique Facilitating Studies of Scant Cell Specimens

A simple method is described by which multiple cytological and cytochemical studies can be done on a clinical sample that contains relatively few cells. The cells are concentrated by centrifugation. The cell pellet is fixed, frozen and embedded in plastic. Thin (2-μm) sections are cut from the plastic. Thus, each cell may appear in several sections and many slides can be made from a single specimen. The advantages of this method over cytospins and Millipore filter preparations of cell suspensions are optimal utilization of all cells, excellent morphological and immunological preservation and ease and reproducibility of this technique.     

A New Technique Facilitating Studies of Scant Cell Specimens

A simple method is described by which multiple cytological and cytochemical studies can be done on a clinical sample that contains relatively few cells. The cells are concentrated by centrifugation. The cell pellet is fixed, frozen and embedded in plastic. Thin (2-μm) sections are cut from the plastic. Thus, each cell may appear in several sections and many slides can be made from a single specimen. The advantages of this method over cytospins and Millipore filter preparations of cell suspensions are optimal utilization of all cells, excellent morphological and immunological preservation and ease and reproducibility of this technique.     

Using multivariate analysis to deliver conservation planning products that align with practitioner needs

This software note describes an extension to the conservation planning package Marxan in which multiple solutions can be evaluated instead of only relying on the measures of best solution and irreplaceability. For this extension we coupled Marxan with the statistical software R. The pool of possible conservation plans is transferred from Marxan into R – which returns an ordination plot, as well as a cluster dendrogram that can be used to evaluate similarity of solutions. Also, the most efficient solutions per group are flagged. We believe that identification of alternative planning options facilitates review and implementation of Marxan solutions as negotiating parties have multiple alternative starting points.     

A DIPPING APPARATUS FOR ESTIMATING THE TOXICITY OF INSECTICIDES IN LIQUID MEDIA

An apparatus and a technique are described for the study of the effect of liquid insecticidal preparations by dipping insects in the toxic material. The method is applicable to quick-settling suspensions and quick-creaming emulsions as well as to solutions. Dipping is performed at constant temperature with end-over-end shaking; and the technique has the advantage over the other dipping methods that have been described in that handling of individual insects whilst wet is eliminated.     

Subcellular fractionation of isolated rat hepatocytes a comparison with liver homogenate

An improved method for the homogenization and the subsequent subcellular fractionation of hepatocytes isolated from adult rat liver is described.The homogenization procedure developed in the present study allows the preservation of the integrity of subcellular structures, as demonstrated by measurement of the activities of representative enzymes as well as by determination of their latency.The activities of representative marker enzymes, as calculated on subcellular fractions obtained by differential centrifugation of the homogenate, are identical whether the homogenate arises from isolated hepatocytes or from the whole liver.Moreover, there is a close similitude between the kinetic parameters (K_{m and} V) of two microsomal cytochrome P₄₅₀-dependent mixed-function oxidases, namely aniline hydroxylase and aminopyrine demethylase determined on microsomal preparations obtained either from isolated cells or from the whole liver.     

From the Past to the Future: Natural Sound Recordings and the Preservation of the Bioacoustics Legacy in Portugal

The preservation of historical and contemporary data safeguards our scientific legacy. Bioacoustic recordings can have historical as well as scientific value and should be assessed for their conservation requirements. Unpreserved bioacoustics recordings are generally not referenced and are frequently at high risk of loss by material degradation and/or by misplacement. In this study we investigated the preservation status of sets of natural sound recordings made in Portugal from 1983 until 2010 inclusive. We evaluated the recordings on the basis of their rate of loss, the degree to which unpreserved recordings could be preserved, and their risk of loss. Recordists of animal sounds were surveyed (by questionnaire or interview) to identify sets of recordings and to collect information on their quality and state of preservation. Of the 78 recordists identified, we found that 32% of the recordings have an unclear status and that only 9% of the recordings are lost. Of the c. 6 terabytes of unpreserved sound recordings discovered, an estimated 49% were recoverable. Moreover, 95% of the recoverable sets of recordings were at high risk of loss by their being misplaced. These risks can be minimized if recordists are persuaded to deposit their material in an institution committed to long-term curation of such data (e.g. sound archives). Overall, the study identified a considerable body of unpreserved animal sound recordings that could contribute to our scientific heritage and knowledge of the biodiversity found in Portugal. It highlights the need to implement effective policies to promote the deposit of recordings for preservation and to reverse the present scenario so that scientific material can be preserved for future generations.     

Factors affecting DNA preservation from museum-collected lepidopteran specimens

Recent innovations in molecular genetics made DNA an intriguing molecule not only in molecular biology, but also in ecology and evolutionary and conservation biology. Despite this general interest, several discrepancies have been reported in the literature regarding the techniques for preserving insects for DNA analysis, prompting us to analyse the effects of different storage conditions on lepidopteran DNA preservation. In particular, in the present paper, adults of the cabbage moth, Mamestra brassicae (L.) (Lepidoptera: Noctuidae), were stored under various conditions in order to verify which method is the most suitable to preserve lepidopteran specimens for DNA studies. Mamestra brassicae adults were stored by rapid desiccation with silica gel, by preservation in acetone, 2-propanol, Carnoy's or ethanol (both at 75 and 100% concentrations) solutions, and finally by storage in an ultracold freezer and liquid nitrogen. Adults preserved by each method were used to extract DNA at the aim of verifying the size of the extracted DNAs, the extraction yield and the possibility of using these samples to amplify both short and long DNA sequences by polymerase chain reaction. The results were compared with those obtained using fresh samples acting as controls. Acetone preservation appeared to be the most recommendable method for moth specimens as it proved to be a good storage medium for DNA analysis, it is cost-effective, and it is applicable not only to field surveys, but also to obtain efficient and low-cost storage of lepidopteran specimens in museum collections.     

Comparison of ThinPrep and conventional preparations on fine needle aspiration cytology material

OBJECTIVE: To compare the various cytologic features on ThinPrep 2000 (TP) (Cytyc Corporation, Marlborough, Massachusetts, U.S.A.) and conventional preparation (CP) specimens from fine needle aspiration cytology (FNAC) material by a semiquantitative scoring system. STUDY DESIGN: In this prospective study a total of 71 consecutive cases were included. In each case, two passes were performed. The first pass was used for conventional preparations, with direct smears made and fixed immediately in 95% alcohol for Papanicolaou stain. For TP preparation a second pass produced material for processing in the ThinPrep 2000. The TP and CP slides were studied independently by two observers and representative slides of CP and TP compared for cellularity, background blood and necrotic cell debris, cell architecture, informative background, presence of monolayer cells, and nuclear and cytoplasmic details by a semiquantitative scoring system. Statistical analysis was performed by Wilcoxon's signed rank test on an SPSS program (Chicago, Illinois, U.S.A.). RESULTS: TP preparations contained adequate diagnostic cells in all cases and were tangibly superior to CP preparations concerning monolayer cells, absence of blood and necrosis, and preservation of nuclear and cytoplasmic detail (statistically significant, Wilcoxon's signed rank test, P < .000). CONCLUSION: TP preparations are superior to conventional preparations with regard to clear background, monolayer cell preparation and cell preservation. It is easier and less time consuming to screen and interpret TP preparations because the cells are limited to smaller areas on clear backgrounds, with excellent cellular preservation. However, TP preparations are more expensive than CP and require some experience for interpretation.