title: Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems.
abstract: Monitoring bacterial growth is an important technique required for many applications such as testing bacteria against compounds (e.g. drugs), evaluating bacterial composition in the environment (e.g. sewage and wastewater or food suspensions) and testing engineered bacteria for various functions (e.g. cellulose degradation). T?=1,^FigItem(1) ^ReloadFigure=Yesraditionally, rapid estimation of bacterial growth is performed using spectrophotometric measurement at 600nm (OD600) but this estimation does not differentiate live and dead cells or other debris. Colony counting enumerates live cells but the process is laborious and not suitable for large numbers of samples. Enumeration of live bacteria by flow cytometry is a more suitable rapid method with the use of dual staining with SYBR I Green nucleic acid gel stain and Propidium Iodide (SYBR-I/PI). Flow cytometry equipment and maintenance costs however are relatively high and this technique is unavailable in many laboratories that may require a rapid method for evaluating bacteria growth. We therefore sought to adapt and evaluate the SYBR-I/PI technique of enumerating live bacterial cells for a cheaper platform, a fluorimeter. The fluorimetry adapted SYBR-I/PI enumeration of bacteria in turbid growth media had direct correlations with OD600 (p>0.001). To enable comparison of fluorescence results across labs and instruments, a fluorescence intensity standard unit, the equivalent fluorescent DNA (EFD) was proposed, evaluated and found useful. The technique was further evaluated for its usefulness in enumerating bacteria in turbid media containing insoluble particles. Reproducible results were obtained which OD600 could not give. An alternative method based on the assessment of total protein using the Pierce Coomassie Plus (Bradford) Assay was also evaluated and compared. In all, the SYBR-I/PI method was found to be the quickest and most reliable. The protocol is potentially useful for high-throughput applications such as monitoring of growth of live bacterial cells in 96-well microplates and in assessing in vivo activity of cellulose degrading enzyme systems.
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK; Department of Biomedical Sciences, School of Basic & Biomedical Sciences, University of Health & Allied Sciences, Ho, Ghana. Electronic address: kduedu@uhas.edu.gh.
authors.forename: Kwabena O
authors.initials: KO
authors.lastname: Duedu
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK. Electronic address: c.french@ed.ac.uk.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: French
mesh_terms: D001419:Bacteria / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D052160:Benzothiazoles; D002482:Cellulose / Q000378:metabolism*; D015169:Colony Count, Microbial; D003116:Color; D004269:DNA, Bacterial / Q000032:analysis; D003959:Diamines; D004735:Energy Transfer; D004784:Environmental Monitoring / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D005516:Food Microbiology; D009696:Nucleic Acids; D009930:Organic Chemicals; D011419:Propidium; D011804:Quinolines; D013053:Spectrophotometry / Q000379:methods; D013194:Staining and Labeling; D062065:Wastewater / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D002482:Cellulose; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D009930:Organic Chemicals; D011419:Propidium; D011804:Quinolines; D062065:Wastewater; C098022:SYBR Green I
keywords: Cell density; Cellulose; Fluorimetry; Propidium iodide; Quantification of bacteria; SYBR Green