Abstract
BackgroundThe power and simplicity of visual colony counting have made it the mainstay of microbiological analysis for more than 130 years. A disadvantage of the method is the long time required to generate visible colonies from cells in a sample. New rapid testing technologies generally have failed to maintain one or more of the major advantages of culture-based methods.Principal FindingsWe present a new technology and platform that uses digital imaging of cellular autofluorescence to detect and enumerate growing microcolonies many generations before they become visible to the eye. The data presented demonstrate that the method preserves the viability of the microcolonies it detects, thus enabling generation of pure cultures for microbial identification. While visual colony counting detects Escherichia coli colonies containing about 5×106 cells, the new imaging method detects E. coli microcolonies when they contain about 120 cells and microcolonies of the yeast Candida albicans when they contain only about 12 cells. We demonstrate that digital imaging of microcolony autofluorescence detects a broad spectrum of prokaryotic and eukaryotic microbes and present a model for predicting the time to detection for individual strains. Results from the analysis of environmental samples from pharmaceutical manufacturing plants containing a mixture of unidentified microbes demonstrate the method's improved test turnaround times.ConclusionThis work demonstrates a new technology and automated platform that substantially shortens test times while maintaining key advantages of the current methods.
Highlights
Since the 19th century, counting the visible microbial colonies grown on semi-solid agar-based growth medium has been the dominant method for quantitative microbiological analysis
Microbes are filtered onto a membrane which is placed on nutrient agar medium in a Growth CassetteTM (Figure S1), incubated for a substantially shorter time than the traditional test, and automatically counted
The membrane filters are composed of mixed esters of cellulose, the same material used for the traditional method, and are dyed black to minimize fluorescence background
Summary
Since the 19th century, counting the visible microbial colonies grown on semi-solid agar-based growth medium has been the dominant method for quantitative microbiological analysis. A key advantage of visual plate counting is its sensitivity for detecting growing cells; a visible colony will develop even when there is only a single culturable cell in a sample. Visual plate counting owes its 130 years of dominance in large part to cost effectiveness and ease of use. The power and simplicity of visual colony counting have made it the mainstay of microbiological analysis for more than 130 years. A disadvantage of the method is the long time required to generate visible colonies from cells in a sample. New rapid testing technologies generally have failed to maintain one or more of the major advantages of culture-based methods
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