Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry

Abstract

Background: In a wastewater treatment plant, the degradation process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment. The activated-sludge process is based on the formation of strong microbial flocs where many bacteria are attached to sludge flocs.Methods: Cytometric analysis requires an homogeneous cell suspension and so detachment of bacteria from flocs is required. In this study, sonication and homogenization were compared to find the most adequate pretreatment method for bacterial cytometric analysis in activated sludge samples. Bacterial viability was tested with a nucleic acid double-staining (NADS) protocol (Barbesti et al., Cytometry 2000;40:214-218) and on flow cytometry.Results: Each method showed a good efficiency in terms of bacterial detachment; thus finally, the choice of which could be the best treatment method was based on both viability results and analysis rapidity. On the basis of the degree of cell detachment and viability, the maximum value was obtained by sonication (2 Â 45@).Conclusions: The use of flow cytometry in conjunction with fluorescent dyes and an adequate pretreatment represents a useful method to rapidly detect and enumerate bacteria in activated sludge samples. q

Interaction between Physical Heterogeneity and Microbial Processes in Subsurface Sediments: A Laboratory-Scale Column Experiment

“…7 In 38 this sense, infiltration systems may be advantageous in many aspects; they may increase (by 39 recharge) groundwater supplies, provide further treatment to infiltrating water, and reduce 40 degradation of stream-water quality. 8 Infiltration systems may also enable water reuse thereby 41 primary mechanism for the microbial degradation of polymeric and macromolecular organic matter 48 into low-molecular-weight molecules which can then cross bacterial cell membranes, becoming 49 available for bacterial growth and nutrient cycles. 12 Extracellular enzyme activities are good proxies 50 to determine nutrient demands and decomposition capabilities of microorganisms, as well as to 51 characterize the quantity and quality of available dissolved organic carbon and nutrients in the 52 environment.…”

Biomass measurement by flow cytometry during solid‐state fermentation of basidiomycetes

“…When using small samples, both methods provided well separated but very weak signals. As observed by Falcioni et al (54) and Nebe-von-Caron et al (36), extended ultrasonic disruption reduced the proportion of intact cells which indicating a destruction of the cells. Particularly filamentous cells may react very sensitive to sonication treatment and not survive a 10 s treatment according to Nebe-von-Caron et al (36).…”

“…However, each such step reduces the yield of nuclei because they get trapped on the mesh or on tube walls. Another possibility would be to sonicate the samples after milling similar to the findings of Falcioni et al (54) to increase the release of nuclei from wood particles, since milling greatly increases the adsorptive surface of the sample. The sonication could be also performed in combination with other disruption methods, like wet blender treatment and wet shear based methods.…”

“…Wet disruption processes do not require sample drying and thus save time during pretreatment. Ultrasonic disruption and disruption using microwaves were regarded as promising wet disruption methods due to literature reports concerning the use of ultrasound to extract bacterial and fungal DNA (53,54) for PCR and the use of microwaves to extract fungal DNA (49,55). Both methods are very fast-for example, the suggested protocol for microwave disruption involves treatment times of 15 s to 1 min (56).…”

“…In addition, the reproducibility of the results obtained using these methods may be limited by inhomogeneities in their energy input and the formation of hotspots within the sample despite same geometry of the set-up and same material of the sample container. The microwave treatment was performed due to the rapidity and successful application according to the literature (47,53,54). However, this method includes many sources of error resulting in inhomogeneities of energy and temperature distribution and inconsistent and weak nuclei signals.…”