Abstract
A new micromethod employing gel microdroplets (GMDs) and optical measurements can be used for rapid detection and enumeration of viable microorganisms (J. C. Weaver, G. B. Williams, A. M. Klibanov, and A. L. Demain, Bio/Technology 6:1084-1089, 1988) and has several potential applications in clinical microbiology. This method involves entrapping microorganisms in GMDs (10 to 100 microns in diameter) which are surrounded by a hydrophobic (low dielectric) fluid, subsequently distinguishing occupied and unoccupied GMDs with colorimetric or fluorescence indicators, counting both occupied and unoccupied GMDs, and applying Poisson statistical analysis. Acid-producing microorganisms were used to compare colorimetric and fluorescence pH indicator systems. Fluorescence systems were generally superior, particularly for detection before microbial growth occurred. Although colorimetric detection was reasonably fast for fast-growing microorganisms, significantly longer times were needed for slow-growing microorganisms. We investigated the dependence of the detection time on microbial division time, GMD size, and buffering capacity of the medium within GMDs. It was found possible to use a single preparation of GMDs, containing a range of GMD sizes, to simultaneously provide a viable enumeration of growing and nongrowing (e.g., stressed) cells. This was possible because small GMDs responded rapidly to both growing and nongrowing cells, while large GMDs, although slower, responded much more rapidly to growing cells than to nongrowing cells. Separate analysis of small and large GMDs in the same preparation yielded two enumerations, one of nongrowing cells and the other of growing cells. GMDs can also be used with conventional light microscopy to detect and enumerate fast-growing acid-producing bacteria much more quickly than conventional plating methods.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Cohen C. Y., Sahar E. Rapid flow cytometric bacterial detection and determination of susceptibility to amikacin in body fluids and exudates. J Clin Microbiol. 1989 Jun;27(6):1250–1256. doi: 10.1128/jcm.27.6.1250-1256.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Colombrita D., Ravizzola G., Pirali F., Manni M., Manca N., Savoldi E., Turano A. Evaluation of BACTEC system for urine culture screening. J Clin Microbiol. 1989 Jan;27(1):118–119. doi: 10.1128/jcm.27.1.118-119.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crokaert F., Lismont M. J., van der Linden M. P., Yourassowsky E. Determination of serum bactericidal activity against Escherichia coli by an automated photometric method. J Clin Microbiol. 1988 Oct;26(10):2069–2076. doi: 10.1128/jcm.26.10.2069-2076.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Libby J. M., Wada H. G. Detection of Neisseria meningitidis and Yersinia pestis with a novel silicon-based sensor. J Clin Microbiol. 1989 Jul;27(7):1456–1459. doi: 10.1128/jcm.27.7.1456-1459.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- Tenover F. C. Studies of antimicrobial resistance genes using DNA probes. Antimicrob Agents Chemother. 1986 May;29(5):721–725. doi: 10.1128/aac.29.5.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Williams G. B., Threefoot S. R., Lorenz J. W., Bliss J. G., Weaver J. C., Demain A. L., Klibanov A. M. Rapid detection of E. coli immobilized in gel microdroplets. Ann N Y Acad Sci. 1987;501:350–353. doi: 10.1111/j.1749-6632.1987.tb45735.x. [DOI] [PubMed] [Google Scholar]