A method for rapid abundance estimation of semiplanktonic meiofauna

Many meiofaunal copepods and plathelminths enter the tidal waters at night thus exhibiting a life-style intermediate between benthic and planktonic. At the same time, ostracods may leave their interstitial dwelling and move across the sediment surface. In laboratory experiments, the percentage of plathelminth populations emerging from the sediment varied with the species, temperature, light conditions, and the dimensions of the sediment cores studied, but not with tidal level, season, ambient density of conspecifics, or the sediment composition. Therefore, the swimming activity may be utilised for extraction of semiplanktonic meiofauna provided that the extraction procedure is standardised with respect to temperature, light and core size. For free-living plathelminths from the Wadden Sea intertidal a robust standard procedure is as follows: sediment cores 1.6 cm in diameter (2 cm² surface area) and 3 cm deep are fitted into cylindrical containers and submerged into aquaria containing filtered seawater (ambient salinity, room temperature, darkness) for 24 h. The sediment containers are then removed and the aquarian water filtered through appropriate meshes; the residue contains the emergent faunal component. For plathelminths, this procedure reduces sorting time by some 90% compared with the standard shaking–decantation method and thus makes it possible to process a high number of samples in a short time. Similar procedures may be developed for copepods and epibenthic ostracods. Received in revised form: 28 August 2000 Electronic Publication     

A rapid and sensitive method for measuring cell adhesion

We have adapted the CyQuant® assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant® assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (>1000) of adherent cells.     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

Immunoblot assay: a rapid and sensitive method for identification of salmonid fish viruses

An immunoblot assay was used to identify the viruses of infectious pancreatic necrosis, infectious hematopoietic necrosis, and viral hemorrhagic septicemia. Viral antigen in infected cell culture supernatant was adsorbed onto nitrocellulose membrane or Whatman 541 filter paper and detected by enzyme-linked immunosorbent assay techniques. The immunoblot assay took less than 4 hr to perform and required no special instrumentation. Assays using cell culture supernatant fluids showed immunoblot sensitivity was 10(5) - 10(6) PFU/ml. Assay sensitivity, determined using purified virus, is 0.85-4.0 ng of viral antigen. The immunoblot assay was used to detect and identify virus in cell culture fluids.     

A rapid and sensitive method for the screening of DNA intercalating antibiotics

An improved, rapid and inexpensive gel mobility shift assay was developed for the screening of anthracycline antibiotics. The assay based on the intercalation activity of these molecules into dsDNA was used to assess the activity of partially purified antibiotics. Detection limits were of 0.1 ng ml^–1 with an average run time of 2 h. The assay is potentially useful for high throughput screening in bioprospecting, for monitoring fermentation production phases and downstream purification process.     

A rapid and sensitive method for HPLC cholesterol determination in bile.

A relatively little time consuming simple method based on the treatment of bile with cholesterol oxidase and subsequent high performance liquid chromatography measurement of the 3-ketocholesterol produced in order to determine the level of the cholesterol concentration is described. The method avoids bilirubin interferences, has high reproducibility and recovery assays give 100% values. It is highly sensitive and suitable for use in the determination of cholesterol concentrations in bile and other bilirubin containing biological fluids.     

A method for the rapid identification of pathogenic yeast-like microorganisms

Summary A new rapid diagnostic method for the identification of pathogenic yeast-like microorganisms is described. By this method in the course of one week these genera can be identified:Candida, Torulopsis, Rhodotorula, Trichosporon, Geotrichum and seven species often found in infectious material, of the genusCandida: C. albicans, C. tropicalis, C. pseudotropicalis, C. krusei, C. guilliermondii, C. pelliculosa andC. parapsilosis.     

A PCR detection method for rapid identification of Paenibacillus larvae

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.     

A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G1 and G2 + M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

A rapid and sensitive radioimmunoassay for norethisterone

A direct radioimmunoassay for the measurement of norethisterone (NET) in unextracted serum samples was developed. The combined use of a highly specific unpurified antiserum and heat treatment of diluted serum samples obviated both extraction and chromatographic procedures. The direct NET assay fulfilled all the quality control parameters. When this assay was compared with other methods involving either solvent extraction and/or chromatographic purification procedures, no significant differences were observed. The overall results were interpreted as demonstrating that this simple, rapid and reliable NET assay can be used as a helpful tool in metabolic and pharmacokinetic studies of this contraceptive progestogen.     

A rapid and sensitive HPLC-MS method for the detection of plasma and cellular rifampicin

Rifampicin is active against both intracellular and extracellular Mycobacterium tuberculosis. The ability to measure rifampicin drug concentrations in both plasma and in cells may be useful in evaluating the suitability of dosage regimens for populations and individuals. Here a novel simple, precise and accurate method for the quantification of rifampicin in both cells and plasma is reported. Sample proteins were precipitated with acetonitrile containing the internal standard and then diluted with water. Aliquots of supernatant were then injected into the HPLC-MS system for chromatographic separation and detection. Rifampicin calibration curves encompassed concentrations from 100 to 12,800 ng/mL. Intra- and inter-assay precision and accuracy were determined using low, medium and high concentration quality control samples and was found to be within 10% in all cases. Rifampicin concentrations were found to be unaffected by freeze-thaw cycles, but were significantly affected by heat-inactivation (58 degrees C, 40 min). This assay was successfully utilised to determine the pharmacokinetic profile of rifampicin in plasma and peripheral blood mononuclear cells (PBMC) in 8 tuberculosis patients receiving rifampicin over an 8h period.     

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml^?1 of bacterial cells and spores, respectively. IPCR was found to be 2–3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.     

A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations.

By using the fluorescence enhancement of ethidium bromide when it binds to RNA, a very rapid, simple and sensitive assay for the concentration of ribosomal RNA in complex mixtures has been devised.