A rapid method for staining large chylomicrons

In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains.     

A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals

A simple, rapid procedure for dual staining of cartilage and bone in rodents, particularly in late gestation, has been developed for routine use. The procedure involves rapid, complete skinning of fresh eviscerated specimens following a 30 sec immersion in a 70 C water bath. The unfixed specimen is stained in a mixture of 0.14% Alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid. Specimens are then macerated in 2% KOH, cleared and hardened in 1:1 glycerin and distilled water, and stored in pure glycerin. Rapid staining of cartilage only is done in a mixture of 0.08% Alcian blue, glacial acetic acid, and ethanol, with subsequent maceration, clearing, and hardening as in the double staining procedure. Rapid staining of bone only, concurrent with maceration of soft tissue, can be done by placing fresh, unskinned specimens in a diluted mixture of alizarin red S in 2% KOH, with subsequent clearing and hardening in 1:1 distilled water and glycerin. Good quality fetal specimens can be prepared for examination by any of these procedures in a minimum of 11/2-2 days as compared to a minimum of 4-5 days for other procedures. Double stained specimens can be examined for abnormalities of the cartilage as well as bone.     

Detection of Pseudomonas aeruginosa in water samples using a novel synthetic medium and impedimetric technology

AIMS: To investigate whether the use of a novel synthetic medium in conjunction with impedimetric technology could provide a rapid and automated detection of Pseudomonas aeruginosa in water samples. METHODS AND RESULTS: A selective synthetic medium (Z-broth) in which the only carbon and nitrogen source is acetamide was applied in direct impedimetric examination for the selective isolation of P. aeruginosa. A total of 1036 tap-water, well-water, swimming-pool water and dialysis water samples were investigated, and any P. aeruginosa contamination was detected in 7-24 h. Neither false-negative nor false-positive results were observed. CONCLUSIONS: The results of the present evaluation demonstrate that impedance measurement with the use of Z-broth is suitable for the rapid and automatic detection of P. aeruginosa in water. SIGNIFICANCE AND IMPACT OF THE STUDY: The main advantages of the method: 240 samples can be examined in one step, the procedure is fully automated, the results are obtained quickly and the labour and media requirements are low.     

Analysis of the carbohydrate composition of glycoproteins by high-performance liquid chromatography

A procedure for rapid and sensitive analysis of carbohydrate in glycoproteins is described. After methanolysis and benzoylation of the monosaccharides and carbohydrates of a glycoprotein, the derivatized sugars were analyzed by reverse-phase high-performance chromatography using a Vydac C₁₈ stationary phase and a mobile phase composed of a water/acetonitrile gradient. The advantages of this procedure over previously described methods are (1) the simple binary solvent system which is used requires no buffering salts and (2) separate sets of peaks from individual sugars obviate the usual need to reacetylate sugar amino groups.     

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10^-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.     

New immunofluorescent method for the rapid determination of microbial antibiotic sensitivity

A new procedure for rapid determination of the levels of antibiotic sensitivity in pathogenic microorganisms with the use of fluorescent antibodies is described. The procedure was developed with the use of a model of the vaccinal strains of Bacillus anthracis. It is based on determination of the microbial antibiotic resistance with the method of serial dilutions on solid media. Still, the medium with an antibiotic is inoculated instead of the pathogen with the native material subject to the analysis. The antibiotic effect on the microorganism is estimated with the method of fluorescent antibodies. The replica preparations obtained as a result of the pathogen growth in a mixed culture on nutrient media containing definite concentrations of the antibiotic are examined with the method of luminescence microscopy. The modification of the immunofluorescent procedure for rapid determination of the microbial sensitivity to antibiotics does not require obligatory isolation of the pathogen as a pure culture. This makes the procedure more economic with respect to the time necessary for the analysis. The following conditions for performing rapid analysis with respect to Bacillus anthracis are required: the minimal concentration of the pathogen in the specimen (2.10(5) spores/ml), preliminary thermal treatment of the specimen for destroying the spore microflora, additional cultivation for 6-8 hours at 37 degrees C. The presence of the accompanying sporulating microflora, i.e. common microorganisms present in the atmosphere, soil and open water bodies does not prevent the performance of the analysis.     

Rapid Quantitative Method for Salmonella Detection in Polluted Waters

A procedure has been developed for the enumeration of salmonellae in polluted waters using several modifications of existing techniques. Confirmation of salmonellae is achieved within 48 hr. This procedure includes selective enrichment in m-Tetrathionate Broth (22 +/- 1 hr), plating on Brilliant Green Sulfa Agar (20 +/- 1 hr), and confirmation by flagellar (H) agglutination of the growth in a mannosecontaining medium (6 +/- 1 hr). An incubation temperature of 41.5 C was used throughout this procedure. Dilution to extinction techniques (most probable number) were employed to enumerate salmonellae. Large sample volumes were concentrated through the use of membrane filters. This technique proved to be rapid and reliable for the enumeration of salmonellae in water, waste water, and waste-water sludges.     

A simple bioluminescence procedure for early warning detection of coliform bacteria in drinking water

Traditional cultivation-dependent tests for coliform bacteria in food and drinking water take 18–24 h to complete. Bioluminescence-based enzyme assays can potentially reduce analysis time for indicator bacteria such as coliforms. In the present study, we developed a simple presence/absence (P/A) bioluminescence procedure for rapid detection of coliform bacteria in groundwater-based drinking water. The bioluminescence procedure targeting β-d-galactosidase activity in coliform bacteria was based on hydrolysis of 6-O-β-galactopyranosyl-luciferin. Bacteria immobilized on membrane filters were enriched for 6–8 h in selective media containing isopropyl-β-d-thiogalactopyranoside (IPTG) to induce β-d-galactosidase activity in coliform bacteria. The equivalent of approximately 300 E. coli cells was required for bioluminescence detection of β-d-galactosidase activity. In comparison, PCR based detection of E. coli in drinking water required approximately 30 target cells. Analysis of contaminated drinking water samples showed comparable results for coliform bacteria using traditional multiple-tube fermentation, Colilert-18, and the bioluminescence procedure. Aeromonas hydrophila or indigenous groundwater bacteria did not interfere with the procedure. The bioluminescence procedure can be combined with commercial substrates such as Fluorocult or Colilert-18, and will allow the detection of one coliform in 100 ml drinking water within one working day. The results suggest the bioluminescence assays targeting β-d-galactosidase activity may be used for or for early warning screening of drinking water and/or rapid identification of contaminated drinking water wells.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.     

Automated Differential Staining for Cartilage and Bone in Whole Mount Preparations of Vertebrates

An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days.     

Automated differential staining for cartilage and bone in whole mount preparations of vertebrates

An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days.     

Manual N-terminal microsequencing of proteins electroeluted from polyacrylamide gel slices

A simple and rapid procedure for preparation of proteins for manual microsequencing using sodium dodecyl sulfate gel electrophoresis is described. The procedure involves pre-electrophoretic labeling of the protein amino groups with a coloured Edman reagent, disk electrophoresis for purification or fractionation of the proteins, and reversed electrophoretic transfer of the separated protein from gel slices into a small volume of buffer (100 to 150 microliter) using a discontinuous conductivity gradient to recover the proteins. The pre-electrophoretic labeling facilitates location of the separated proteins in the gel and the monitoring of their complete electroelution. The isolated proteins are separated from excess of salts by acetone precipitation and solvent partitioning in pyridine/water (1:1) and subjected to manual DABITC/PITC degradation.     

RAPID CHEMICAL DEHYDRATION OF PLANT MATERIAL FOR LIGHT AND ELECTRON MICROSCOPY WITH 2,2-DIMETHOXYPROPANE AND 2,2-DIETHOXYPROPANE

A rapid and simple procedure was used for chemical dehydration of plant tissue during sample preparation for light and electron microscopy. Chemically fixed tissues were washed with distilled water and then rapidly dehydrated with either 2,2-dimethoxypropane or 2,2-diethoxypropane for 15 minutes. Light microscopic observation of paraffin-embedded tissue or tissue embedded in Spurr's plastic showed excellent preservation. Electron microscopic examination of plastic-embedded tissue showed well maintained ultrastructural morphology. The dehydration procedure was also successfully applied to plant tissue destined for examination in a scanning electron microscope.     

Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan™) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure.     

FLUORESCENCE CONCENTRATION IMMUNOASSAY FOR RAPID DETECTION OF CAMPYLOBACTER SPP. IN CHICKEN RINSE WATER

A total of 215 freshly processed post-chill whole chicken carcasses were assessed for Campylobacter spp. contamination by a fluorescence concentration immunoassay (FCIA) procedure. Whole chicken carcasses were sampled with low volume water rinses from which 5 ml portions were enriched with brucella enrichment broth with or without oxyrase supplement in a test tube enrichment system. After a 24h stationary incubation at 42C, each sample was assayed using a FCIA procedure for the presence or absence of campylobacters. The FCIA procedure indicated Campylobacter spp. contamination in 84% of carcasses using oxyrase supplemented enrichment, while only 47% of the chicken carcasses were positive from nonsupplemented enrichment. The corresponding incidence rates detected by culture method were 92% and 87% for oxyrase supplemented and unsupplemented samples, respectively. The FCIA procedure can be completed in less than 1 h with 48 samples including a positive and a negative control assayed on one plate. In summary, the test tube oxyrase-supplemented stationary enrichment system followed by the use of the FCIA procedure was found to be an effective, rapid method for the detection of Campylobacter spp. in chicken rinse water.     

Organism losses during ice melting: A serious bias in sea ice community studies

Summary When ice samples are melted, microorganisms living within the brine inclusions are subjected to rapid and extreme changes in salinities. This procedure results in substantial losses of flagellates and ciliates. Most of these losses can be prevented if ice samples are melted in larger volumes of sterile sea water to buffer salinity and osmotic changes. Since most studies on the ice biota have ignored, or have been unable to avoid this bias, current views of the composition and activity of sea ice communities are based on assemblages over-representing organisms with rigid cell material.     

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.     

Chemical Stabilization of Golgi Silver Chromate Impregnations

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100μm, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.     

Chemical stabilization of Golgi silver chromate impregnations.

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100 micrometer, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.     

A rapid and efficient method to remove labelled molecular probes from nucleic acids immobilized on solid supports

A rapid and efficient method is described for the removal of radio-active molecular probes from nucleic acids immobilized on nylon membranes. This method involves boiling in distilled water in a microwave oven. This procedure can be completed within ten minutes, does not require the use of any buffers or reagents, and produces results comparable with conventional buffer-wash procedures recommended by the suppliers of the transfer membranes.