Rapid count of microbial cells in dialysate.

An apparatus for the non-culture method (NCM) of microbial cell count was formerly developed and named a bioplorer. The bioplorer NCM is based on the double staining of cells with 4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) and the automatic analysis of their fluorescent microscopic images. Viable cells can be stained with DAPI, while dead cells can be stained with DAPI and PI. In this study, the bioplorer NCM has been applied to the dialysate. The viable and dead cells in dialysate could be counted within 20 min. The detection limit expressed by log(10)[cells/100 mL] was 2.0. When cell-spiked dialysate samples containing prescribed number of Bacillus subtilis cells were assayed, the numbers of cells determined by the bioplorer NCM (N(VIA)(NCM)) and a conventional culture method (CM) on R2A medium (N(VIA)(R2A-CM)) were similar in the range of 2.6-4.6 within the 95% confidence interval (NCM-CM equivalent range). When test solutions sampled from a practical facility in a hospital were assayed, N(VIA)(NCM) was greater than, but comparable to, N(VIA)(R2A-CM). The endotoxin (ET) in the test samples were assayed as well using a test kit for limulus amoebocyte lysate assay. The results of microbial cells and ET concentration indicated that the dialysate supplying line was clean and well maintained. The bioplorer NCM can determine if the microbial contamination of dialysate supplying facilities is greater than 2.6 (398 cells/100 mL).

Rapid separation and counting of viable microbial cells in food by nonculture method with bioplorer, a focusing-free microscopic apparatus with a novel cell separation unit.

A nonculture method utilizing a novel apparatus, the bioplorer, was developed. The bioplorer is composed of an efficient cell separation unit, a focusing-free microscopic device, and an image analysis program. A meat or vegetable suspension is poured into the cell separation funnel, and insoluble matter in the sample suspension is trapped by prefilters. Microbial cells passing through the two prefilters are then trapped by the membrane filter (pore size, 0.4 microm). Trapped cells are double-stained with 4',6'-diamidino-2-phenylindole and propidium iodide, and the membrane filter is removed and set on the focusing-free microscope. A fluorescent image is then recorded. Total numbers of viable and dead cells on the membrane filter can thus be determined automatically. One assay can be performed within 10 min, which is much faster than the culture method. The results obtained with both the nonculture method and the culture method for meat and vegetable samples were highly correlated (r = 0.953 to 0.998). This method is feasible for the practical purpose of food safety control.

Rapid LED-based fluorescence microscopy distinguishes between live and dead bacteria in oral clinical samples.

Despite the existence of several methods for the diagnosis of oral infectious diseases, few rapid and quantitative methods exist for discriminating between live and dead bacterial cells in oral clinical samples. In this study, we characterized a light-emitting diode (LED) fluorescence microscopic technique for quantifying live and dead oral bacterial cells stained with 4',6'-deamidino-2-phenyllindole and propidium iodide. Four bacterial strains representative of the human oral microflora were used in this study. In addition, saliva and subgingival fluid specimens were collected from healthy volunteers. Saliva was obtained from the donors without stimulation, whereas subgingival fluid was obtained by inserting a sterile endodontic paper point into the subgingival sites of the first molar. The samples were cultured on agar plates and subjected to LED microscopy. The correlations between both methods were analyzed. The number of live bacterial cells as determined by LED-based fluorescence microscopy and standard colony counts on agar plates correlated well for the known oral bacterial strains and bacterial cells in the clinical specimens. The LED illumination method characterized in this study can be used for the rapid enumeration of living and dead cells. However, to show specificity, this method requires further innovations.