Rapid in-house method of CSF analysis utilising sedimentation direct from the spinal needle.

OBJECTIVES: To establish the utility of a novel in-house method of CSF analysis using sedimentation cytology direct from the spinal needle for the detection of laboratory-defined pleocytosis. MATERIALS AND METHODS: In dogs and cats undergoing routine CSF analysis for investigation of neurological signs, an additional preparation was made at the patient's side by inverting the spinal needle on a slide and sedimenting for at least 1 hour. Nucleated cellularity and differential counts were assessed and compared with "gold-standard" analysis. Variability of cell counts between observers and within slides using the new method was evaluated to optimise the procedure. RESULTS: Using a ×50 objective, at least 10 fields and an average of more than five cells per field were considered appropriate guidelines to achieve correct classification of samples (normal or pleocytosis). The new method had high sensitivity (89%) and specificity (100%) for the detection of laboratory-defined pleocytosis. Agreement on the type of pleocytosis was good. CLINICAL SIGNIFICANCE: Clinically useful information can be obtained from CSF samples in a patient-side setting without additional equipment. This technique may be of benefit if little fluid is available or if logistical constraints limit the availability of rapid specialist results.

Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters.

AIMS: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. METHODS AND RESULTS: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases). CONCLUSIONS: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. SIGNIFICANCE AND IMPACT OF THE STUDY: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.

Hyperbaric oxygen (HBO) as useful, adjunctive therapeutic modality in compartment syndrome.

The authors describe a compartment syndrome progressively developed after a long-term surgical procedure, with a patient positioned in supine position with calf rest, who was successfully treated with hyperbaric oxygenation. This approach saved the patient from a more invasive therapeutic intervention.

A 210-min solid phase cytometry test for the enumeration of Escherichia coli in drinking water.

A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies. The test involves membrane filtration through a 25-mm black polyester filter, induction of beta-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-beta-Dglucuronide as an enzyme substrate and laser scanning of the membrane filter. Scan results can be confirmed on-line by epifluorescence microscopy. Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated > or =90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar. In 5.4% of all samples examined, SPC detected between 1 and 11 E. coli per 100 ml, while the two plate methods yielded negative results. Cases of a negative SPC result but a positive E. coli count on both reference media were not observed. This test would primarily be useful for 'emergency' monitoring of drinking water when rapid results are crucial.

Rapid detection of fluorescent and chemiluminescent total coliforms and Escherichia coli on membrane filters.

The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-beta-D-glycosides as enzyme substrates and a UV-lamp for visualization. The most rapid procedures based on this approach for the demonstration of these indicator bacteria in water take 6-7.5 h to complete. As part of efforts to further reduce the detection time, an improved two-step procedure for the fluorescence or chemiluminescence labelling of microcolonies of E. coli/TC on a membrane filter has been developed. Essential features of this approach include a separation of the bacterial propagation and target enzyme induction from the actual enzymatic labelling, the use of improved fluorogenic, i.e., 4-trifluoromethylumbelliferyl-beta-D-glycosides and fluorescein-di-beta-D-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for beta-glucuronidase/beta-galactosidase, of enzyme inducers, of special membrane filters and of polymyxin B to promote the cellular uptake of the substrate. This labelling procedure has been applied in conjunction with different detection devices including a UV-lamp, CCD-cameras, X-ray film and the ChemScan((R)) RDI. Using the former three, microcolonies of pure cultures could be detected within 5.5-6.5 h, but waterborne E. coli/TC may fail to form microcolonies in this short time period, thus yielding poor sensitivity and a high false-negative rate. In contrast, a quantitative enumeration was feasible in less than 4 h with the ChemScan((R)) RDI, owing to its ability to detect both microcolonies and non-dividing single cells.

Effects of the composition of bacteriological growth media on a chemiluminometric assay of beta-galactosidase in Escherichia coli.

The effects of the composition of bacteriological growth media on the light output in a chemiluminometric assay of beta-galactosidase in Escherichia coli using 1,2-dioxetane substrates has been studied. In this assay a basic conflict exists between conditions that promote optimal bacterial growth and those conducive to maximal chemiluminescence. Common medium ingredients such as yeast or beef extract, protein hydrolysates and lactose suppress light emission and/or lead to high backgrounds. Quenching of light emission is probably partly due to light absorption by medium ingredients such as oxgall, and partly to interference with the reaction triggering the chemiluminescent process. Elevated backgrounds are caused by the presence of high concentrations of protein hydrolysates, which interact with the alkali in the accelerator solution. Only two purposely developed media, i.e. ILM and Colicult are shown to reconcile the requirements of growth support with that of optimal luminescent properties.

Limitations of highly sensitive enzymatic presence-absence tests for detection of waterborne coliforms and Escherichia coli.

This study presents evidence for the unfeasibility of enzymatic presence-absence tests to detect one total coliform or one Escherichia coli organism in 100 ml of drinking water within a working day. The results of field trials with prototype chemiluminometric procedures indicated that the sensitivity-boosting measures that are essential to achieve the required speed compromise the specificity of the tests.

Development of a Sensitive Chemiluminometric Assay for the Detection of (beta)-Galactosidase in Permeabilized Coliform Bacteria and Comparison with Fluorometry and Colorimetry.

Volume 61, no. 12, p. 4505, Introduction, column 1, line 13, and column 2, line 5: reference 19, which refers to a toxicity test based on the inhibition of (beta)-glucuronidase, not (beta)-galactosidase, in E. coli, should not be cited. [This corrects the article on p. 4505 in vol. 61.].

Development of a sensitive chemiluminometric assay for the detection of beta-galactosidase in permeabilized coliform bacteria and comparison with fluorometry and colorimetry.

We developed a chemiluminometric assay of beta-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate. Permeabilization of cells is required to ensure the efficient cellular uptake of this compound. By this method, one coliform seeded in 100 ml of sterile water can be detected after a 6- to 9-h propagation phase followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry afforded 4- and 1,000-fold increases in sensitivity and 1- and 6-h increases in the speed of detection, respectively.

Pharmacokinetics and anthelmintic efficacy of febantel in the racing pigeon (Columba livia).

This paper reports on the pharmacokinetics and the efficacy of febantel against Capillaria obsignata and Ascaridia columbae in the racing pigeon. Febantel was rapidly cleared from the circulation and highly metabolized. The efficacy of febantel against Capillaria and Ascaridia was studied on lightly and heavily infected pigeons. The efficacy against Ascaridia was 100% for the faecal egg count reduction (FECR) as well as for the worm reduction in all treated pigeons. After two treatments with febantel against Capillaria in heavily infected pigeons the FECR amounted to 96.7% and worm reduction was 95%. In order to effectively remove both parasites from the host, repeated treatments with febantel at a short interval could be the treatment of choice.