Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry.
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 x 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.
Center of Cytometry and Cytomorphology, University Carlo Bo, Urbino, Italy.
Tania
T
Falcioni
Anita
A
Manti
Paola
P
Boi
Barbara
B
Canonico
Maria
M
Balsamo
Stefano
S
Papa
D001419:Bacteria / Q000737:chemistry / Q000254:growth & development*; D001422:Bacterial Adhesion; D052160:Benzothiazoles; D015169:Colony Count, Microbial; D003959:Diamines; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008828:Microbiological Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D011419:Propidium / Q000737:chemistry; D011804:Quinolines; D012722:Sewage / Q000382:microbiology*; D013010:Sonication; D013194:Staining and Labeling / Q000379:methods
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012722:Sewage; C098022:SYBR Green I; D011419:Propidium