The precision of bacterial quantification techniques on different kinds of environmental samples and the effect of ultrasonic treatment

• Microscopy, MPN and PCR product quantification have different precision.
• Precision depends on the sample complexity.
• Ultrasonic treatment increased precision but decreased MPN.
• PCR product quantification suitable for bacteria counting

The precision of cell number quantification in environmental samples depends on the complexity of the sample and on the applied technique. We compared fluorescence microscopy after filtration, quantification of gene copies and the cultivation based most probable number technique for their precision. We further analyzed the effect of increasing complexity of the sample material on the precision of the different methods by using pure cultures of Pseudomonas aeruginosa, fresh water samples and sediment slurries with and without ultrasonic treatment for analyses. Microscopy reached the highest precision, which was similar between pure cultures and water samples, but lower for sediment samples due to a higher percentage of cells in clusters and flocks. The PCR based quantification was most precise for pure cultures. Water and sediment samples were similar but less precise, which might be caused by the applied DNA extraction techniques. MPN measurements were equally precise for pure cultures and water samples. For sediment slurries the precision was slightly lower. The applied ultrasonic treatment of the slurries dispersed the cell clusters and flocks, increased the precision of microscopical and MPN measurements and also increased the number of potential colony forming units. However, the culturable cell number decreased by half. For MPN quantification of viable cells in samples with a high proportion of clustered cells we therefore recommend an optimization of ultrasonic treatment and a confirmation by microscopy and cultivation to reach highest possible dispersion of the cells with a minimum of inactivation. As a result of these observations we suggest a correction factor for MPN measurements to consider the effect of sonication on complex samples. The results are most likely applicable to other complex samples such as soil or biofilms.

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