Application of laser scanning for the rapid and automated detection of bacteria in water samples

Abstract

It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using ‘viability markers’ may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z‐test, P < 0·05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 °C for 7 d was only 25·2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5‐cyano‐2,3,4‐tolyl‐tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z‐test, P < 0·05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro‐organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently‐labelled antibodies or nucleic acid probes.

Microbial Biofilms on Needleless Connectors for Central Venous Catheters: Comparison of Standard and Silver-Coated Devices Collected from Patients in an Acute Care Hospital

“…The viable microbial cell count assay, which is based on the activity of esterase enzymes within cells with intact cell membranes (23), indicated that more than 90% of untreated and silver-coated NCs contained viable cells. Our results are consistent with other published studies (24,25), indicating that solid-phase cytometry using ChemChrome will detect substantially higher numbers of viable bacteria in environmental samples than will culture-dependent methods. These results suggest that the culture-based methods used in this study select for organisms that can best compete under the growth conditions provided by the medium used for isolation, and the results may not represent the predominant organisms in the NC microbial communities.…”

Methods of Rapid Microbiological Assay and Their Application to Pharmaceutical and Medical Device Fabrication

“…A method may be required to provide quantitative or qualitative evidence of microbial presence survival of bioburden , some mechanism of contamination tracking, or to offer rapid confirmation of the absence of microorganisms. Few methods show complete promise in their range and relevance of reported applications Watling and Leech 1996, Anonymous 1996, 2 Bopp and Wachsmith 1981, Anderson et al 1986, 3 Wassall et al 1997, 4 Senior et al 1989, 5 Bussey and Tsuji 1986, 6 Blackburn et al 1989, Anonymous 2001, 7 Webster 1986, Woolridge 1989, Tanaka et al 1997, Newby 2000, 8 Kahn and FirstenbergEden 1984, Kaiserman et al 1989, 9 Connolly et al 1983, 10 Dal Maso 1998, 11 Watling and Leech 1996, 12 Wilkins et al 1980, 13 Palmgren et al 1986, 14 Denyer and Ward 1983, Denyer and Lynn 1987, 15 Ladd et al 1985, Bridgett et al 1993, 16 Connolly et al 1993, 17 Holah et al 1988, 18 Mittelman et al 1983, Newby 1991, Kawai et al 1999, 19 Newby 2000, 20 Anonymous 1995, 21 Wallner et al 1997, Gapp et al 1999, Reynolds and Fricker 1999, Newby 2000, 22 Alvarez et al 1994, 23 Jordan 2000, 24 Newby 2000, 25 Atlas 1991, Maiwald et al 1994 with biological indicators is necessary, which is required in the sterilization validation and GMP. To ensure that every reasonable opportunity is given for the recovery of stressed and i...…”

“…Sterilization protocols require regular microbiological validation; for some processes, continual efficacy monitoring fluorescently labeled antibodies may offer specificity to laser scanning cytometry Reynolds and Fricker, 1999 . It is particularly pleasing to see that in the reference of Wu et al 2001 , two technologies-phage interaction and ATP bioluminescence-come together in one application. The revolution in applied DNA technologies, particularly driven by medical diagnostics, offers major promise in the future for miniaturized nucleic acid-based detection systems; the recognition of gene families associated with particular microbial characteristics Stewart, 1997 offers a route to the detection of specific deteriogens and pathogens.…”

Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry

“…SPC has a theoretical detection limit of one cell per filtered volume 1, 2 , but SPC can also be used to determine the microbial load of highly contaminated samples 5 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 as it has a high dynamic range with an upper limit of approximately 10,000 cells per membrane filter 6 .…”