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Faecal measurements of oestradiol and testosterone allow the non-invasive estimation of plasma steroid concentrations in the domestic fowl.

1. Radioimmunoassays were validated for oestradiol and testosterone in faecal and plasma samples from domestic fowls. Faecal samples were prepared in phosphate buffer. A delay of up to 24 h between defaecation and the freezing of a dropping did not affect faecal steroid concentrations. 2. Differences in plasma concentrations of testosterone and oestradiol between cockerels and hens were clearly reflected in faecal hormone concentrations. Faecal concentrations of oestradiol were low in males and did not overlap with much higher concentrations in females, whereas there was some overlap between the sexes in faecal concentrations of testosterone. 3. The correlation coefficients (r2) between plasma and faecal steroid concentrations were 0.464 for testosterone and 0.852 for oestradiol (log-transformed data). The coefficients did not increase when the mean hormone concentrations for several droppings produced by each bird during a collection period were used. 4. Faecal steroid concentrations can be used as a measure of plasma steroid concentrations and hence of gonadal activity in chickens. The resolution of the faecal steroid method is less than if plasma measurements were used, but it offers a practical alternative to blood sampling that is non-invasive and does not require birds to be handled.

Animals↗

The baton dialyzer.

Macromolecular solutions (such as radioactive materials, sterile solutions, infectious materials) requiring special handling precautions for dialysis may be safely and easily handled using a baton-shaped dialysis device.

Dialysis↗

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Biomarkers↗

Artifacts in eighth cranial nerve biopsy.

The occurrence of artifactual morphological changes was investigated in human eighth nerve biopsies and corresponding changes were brought about in biopsies from rats by mechanical handling before fixation. At the very site of compression by a pair of forceps the biopsy stained lighter than in its immediate surroundings. Electron microscopy showed denuded and fragmented axons among vesicular debris in the area. Further away the myelin coats were greatly thickened, often to the degree that the axonal canal could no longer be identified. The myelin lamellae showed separation and were occasionally seen in convoluted forms. Also variations in the diameters of the nerve fibres and ruptures of the meylin coats could be produced after mechanical handling.

Animals↗

IF-40. A new polystyrene microscope tray for immunofluorescent studies.

A polystyrene plate is described which was designed to replace microscope slides for routine immunofluorescent examinations. The plate has 40 flat indentations of 8 mm diameter. The plates can be sterilized and stored in plastic bags. They are well suited for drop preparations or application of cryostat slices of biopsy and necropsy material, and tissue cultures can be grown directly in the indentations. When compared with glass microscope slides, which have long been used for these purposes, the polystyrene plates offer a variety of advantages. Cells can be grown directly in the indentations without washing or pretreating the trays in any way. The indentations prohibit mixing of samples or reagents and enable the use of smaller quantities of reagents. Up to 40 samples can be worked up simultaneously on one plate, or a plate rack can be used for the processing of up to 400 samples at once. The use of a special stage enables easy microscoping of all 40 spots without further handling of the tray.

Fluorescent Antibody Technique↗

The laboratory culture of Dichelobacter nodosus in a footrot eradication program.

As part of a program to eradicate virulent footrot from Western Australia, 2745 isolates of Dichelobacter nodosus were isolated from 5263 specimens from 1883 submissions. The virulence of each isolate was assessed using protease thermostability and isoenzyme zymogram. We describe changes to the materials and methods required to handle these specimens and to reduce the interval between submission and report to 8 days.

Aerobiosis↗

Handling and reporting of tumor-containing kidney specimens.

The pathologic features of RCC are the most valuable factors in predicting the prognosis and for planning surveillance and treatment protocols. Urologists and pathologists should optimize approaches in handling tumor-containing kidney specimens to allow for the best evaluation and reporting of such specimens. A pathologic report of a tumor-containing kidney specimen should include all established or potential prognostic factors, especially tumor types, size, grade, information for pathologic staging, and status of the surgical margin.

Biopsy, Needle↗

Mouse morphological phenotyping with magnetic resonance imaging.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, with both MRI physicists and biologists starting to use MRI to identify mouse models of human disease. The purpose of this chapter is to provide details of the animal handling necessary for routine and robust in vivo imaging with particular emphasis on multiple-mouse imaging. In addition, techniques for perfusion-fixation for postmortem imaging of specimens and whole mice are given.

Animals↗

Intrarenal venous glucose levels in the dog: an evaluation of the sampling technique.

Analysis of samples of intrarenal venous (IRV) blood from anesthetized dogs demonstrated that IRV glucose concentrations were greater than renal venous and arterial glucose in most samples. However, IRV glucose fluctuated with time such that this technique is unreliable for assessing changes in renal cortical glucose handling during experimental interventions.

Animals↗

In situ measurements of labile Cu, Cd and Mn in river waters using DGT.

The technique of diffusive gradients in thin-films (DGT) has been trialed in two river systems for in situ trace metal speciation measurements. This paper presents results for cadmium, copper and manganese concentrations in fresh and estuarine waters and demonstrates for the first time the effectiveness of using DGT to measure labile metal concentrations in such waters. This work has shown that even with very simple deployment systems the technique is sensitive and reproducible. The precision of in situ DGT measurements was typically 11% or better when the precision of subsequent analysis was good. The in-built metal pre-concentration procedure of DGT allowed Cd to be measured at concentrations below the detection limit of a direct determination by GF-AAS. The theoretically predicted linear increase in mass with time was obtained in river deployments of up to 72 h, confirming steady state river conditions and indicating no adverse effects due to biofouling over this period. Concentrations of DGT-labile Cu and Cd were equal to dissolved (0.45 micron) metal concentrations for the Ring and Stitt Rivers indicating the absence of tightly bound organic complexes or colloidal species. DGT-labile Cu and Cd were approximately 50% of the dissolved metal in the Que River and DGT-labile Cu was approximately 30% of dissolved Cu in the Savage River. A concentration-depth profile of labile manganese was obtained in a stratified estuary by deployment of a string of DGT devices at 0.3-m intervals across the redoxcline. Results revealed a large spike (maximum concentration = 1.4 mg/l) of DGT-labile Mn at the base of the redoxcline and demonstrate the utility of DGT to determine vertical changes in metal speciation across redox boundaries in stratified estuarine systems. It is in such dynamic systems as this that the in situ capabilities of DGT are likely to be most useful. DGT records multiple metal species as they exist in situ, overcoming the considerable problems of contamination often associated with sample collection and handling.

Australia↗

Serological testing in a microbiology laboratory of specimens from patients with suspected infectious disease.

AIMS: To determine how the microbiology laboratories of one region process serological requests from patients with suspected infectious illness, referred to as "clinical syndrome" type patients in this study; to consider areas where improvement in the associated serology service could be made. METHODS: A prospective two month collection of data on all serological requests from patients with suspected infectious illness was undertaken. A questionnaire on laboratory policies/procedures was also completed by the 10 departments taking part. RESULTS: Serology specimens from "clinical syndrome" patients accounted for 1-2% of total microbiology samples. There was significant variation in some of the policies/procedures carried out by the 10 laboratories when handling serological requests. Differences were seen in the use of laboratory protocols for test processing, range of tests performed, demand for second (convalescent) serum samples, storage of serum samples, and reporting of results. CONCLUSIONS: The laboratory management of "clinical syndrome" type requests is complex. Individual pathology departments vary in the way they handle serology specimens but this study highlighted areas which may contribute to improving the appropriateness of testing and the more efficient use of serology resources. These include improving (1) clinician education, (2) pathology request forms to encourage better clinical information, (3) appropriate laboratory protocols to aid decision making on test selection, (4) percentage of convalescent serum samples received together with longer serum sample storage, and (5) turnaround times of serology reports.

Clinical Protocols↗

Computer programs in cytology reporting and record keeping.

Large numbers of specimens are handled by cytology laboratories. There is great variation in their origin, a need for a name and disease index and for statistical data about the source and nature of the specimens. A computer program was devised to reduce this workload and to improve the accuracy of record keeping. Much of the tedium of this important aspect of laboratory function can thus be removed.

Computers↗

[Liquid handling with mechanical pipettes].

Dispensing very small amounts of liquid is essential in all science fields. A wide range of pipetting systems is now available, including air-cushion pipettes and positive-displacement pipettes. The precision and accuracy depends on not only the principle of pipetting systems but vapor pressure, density, viscosity or wetting behavior of the solution. Preventing contamination is also important issue. Basically, we should handle these pipettes according to maker's instruction manuals.

Chemistry↗