Limit of Resolution of Flow Cytometry for the Detection of Selected Bacterial Species

Abstract

The enumeration of bacteria in dental plaque samples is a vital but time-consuming procedure that uses standard cultural methods. Flow cytometry has proven to be a useful tool for the analysis of eukaryotic cells. In the present investigation, the utility of this technology for the enumeration of bacteria in mixtures was explored. Rabbit antisera were produced against the putative periodontal pathogens A. actinomycetemcomitans, B. intermedius, B. gingivalis, E. corrodens, W. recta, B. forsythus, as well as the frequently isolated supragingival species S. sanguis. Cross-reactive antibodies were removed by absorption, and the specificity of each antiserum was confirmed by being tested against a panel of 235 oral microbial strains (79 genera; 94 species) by means of ELISA. Conditions were established for the indirect immunofluorescent labeling of cells without agglutination with use of a goat anti-rabbit Ig-FITC second antibody. When an internal bead standard was used, it was found that unstained bacteria were enumerated by light-scattering parameters with poor efficiency (less than 3%). However, cells exposed to FITC either in the presence of specific or non-specific first antibody were enumerated with high efficiency (102.6 +/- 29.3%), indicating that a small amount of non-specific binding of fluorochrome facilitates bacterial detection. Clear discrimination between specifically- and non-specifically-stained bacteria was achieved with all six rabbit antisera. Mixtures of known composition were made (1) with pure cultures or (2) with a known species and supragingival plaque devoid of that species by culture. The results from both approaches with various species combinations revealed that the limit of resolution for accurate quantitation of a selected species was approximately 5%, although specific organisms could be detected qualitatively when present at approximately 1%.

Direct flow cytometry of anaerobic bacteria in human feces

“…While major attention has been paid to measurement of eukaryotic cells, only some attempts have been made to analyze bacterial populations. In this field, the focus has been on bacterial pure cultures (2,6,16,18,24), though in some studies noncultured mixed bacterial populations such as aquatic bacteria were characterized (21,23). Flow cytometry may be a potential technique to analyze fecal flora as well.…”

Recent advances of flow cytometry in fundamental and applied microbiology

“…The first applications involved detection of a particular species in a microbial mixture. However, the use of these crude reagents was disappointing when pilot studies with artificial blends of pure strains isolated and cultivated in the laboratory were extended to analyses of clinical or environmental samples (see for example [141,150]). Progress came with the increased use of monoclonal antibodies (mAbs), which are now the reagents of choice (ef table II).…”

“…Main applications of flow immunofluorescence analysis of microorganisms a.Monoclonal antibodiesMeasurement of the accessibility of epitopes of outer membrane proteins to antibodies as a function of the LPS length[15] Distribution of Gram-negative outer membrane proteins under different physiological conditions, in different strains and in different related species[101,130,164,181] Quantification of the distribution of different types of LPS structures[67,132,133,214] Variation of cell surface antigen density in relation to microbial pathogenicity[9, 88,113,147] Mutant selection[124] Surface antigen variation in parasitic protozoa[1, 3, 39] Quantification of binding of cytotoxic immunoconjugates to microbial pathogens[42] Identification of parasitic protozoa in infected tissues[69] Quantification of protein expression level[64] -Study of differentiation in slime moulds[12, 21,165,204,205] Polyclonal antibodiesDetection of microbial species in mixed populations[120,141,150,198] Cell surface distribution of antigens[43,125] Cell surface phenotypic variations of parasitic protozoa among clinical isolates [I, 2] -Specific anti-bacterial species antibody level in body fluids[58] Lectins b--Outer surface glycoproteins of parasitic protozoa [I, 11, 32, 131] -Mutant selection [25] -Pathogenic fungi characterization [82, 125] -Characterization of parasite populations in infected tissues [56]-Role and distribution of surface glycoproteins in plant-symbiotic bacteria association[210] …”

Characterization of Bacterial Cell Size and Ploidy using Flow Cytometry and Image Analysis

“…Some workers claim to have found these reliable for bacterial work (Amman et al 1990;Obernesser et al 1990), while others have found them unacceptable (Pinder et al 1990). Alternatives such as staphylococci (Robertson and Button 1989) have been investigated.…”

“…Characteristics of bacteria, including cell size, DNA and protein content, DNA base composition and immunofluorescence, have been studied by flow cytometry. Although most work has been carried out using Escherichia coli (Steen et al 1982(Steen et al , 1986Boye et al 1983;Scheper et al 1987;Steen 1989;Szabo and Damjanovich 1989;Amann et al 1990; E. Sahar Tel Aviv University, personal communication), some other bacterial species have been examined, including Desulphobacter (Amann et al 1990), Pseudomonas, Aeromonas, Enterococcus and Lactobacillus (Pinder et al 1990), and a variety of periodontal pathogens (Obernesser et al 1990). The aims of previous studies have included assessments of the feasibility of detecting bacteria (Amman et al 1990;Obernesser et al 1990; E. Sahar Tel Aviv University, personal communication), the identification of bacterial contaminants in food and water (Robertson and Button 1989;Howes 1992) and examinations of the effects of antibacterial compounds on bacterial cell size and DNA content (Steen et al 1982(Steen et al , 1986Martinez et al 1982;Boye et al 1983;Steen 1990a;von Freesleben and Rasmussen 1991).…”