title: Rapid estimation of microbial numbers in water using bulk fluorescence.
abstract: Enumeration of microbial cells without culturing is an essential technique for microbial ecology and water quality evaluation. Here we show that bulk fluorescence using the SYBR Gold DNA stain can be used to rapidly quantify microbial cells per millilitre in fresh, marine and estuarine waters. The bulk fluorescence method is comparable to estimating cell concentrations in cultures using optical density; however, this enhanced method enables the user to estimate microbial numbers at lower concentration (> 10(5) cells ml(-1)) found in environmental samples. The technique worked in both single-cell and 96-well plate fluorescent spectrophotometers. Differences of approximately 10(5) cells per millilitre were discernible and the precision of the bulk fluorescence was higher than direct counting by epifluorescent microscopy. Treatment with DNase I increased sensitivity by lowering background noise attributed to free DNA. This technique is simple, rapid, inexpensive and adaptable for automatically estimating microbial numbers in water samples.
authors.affiliation: Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, USA. lwegley@gmail.com
authors.forename: Linda
authors.initials: L
authors.lastname: Wegley
authors.forename: Pamela
authors.initials: P
authors.lastname: Mosier-Boss
authors.forename: Stephen
authors.initials: S
authors.lastname: Lieberman
authors.forename: John
authors.initials: J
authors.lastname: Andrews
authors.forename: Amanda
authors.initials: A
authors.lastname: Graff-Baker
authors.forename: Forest
authors.initials: F
authors.lastname: Rohwer
mesh_terms: D002140:California; D003850:Deoxyribonuclease I / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods*; D005618:Fresh Water / Q000382:microbiology; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals / Q000737:chemistry*; D015203:Reproducibility of Results; D012623:Seawater / Q000382:microbiology; D014736:Vibrio parahaemolyticus / Q000302:isolation & purification*; D014871:Water Microbiology
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C474470:SYBR Gold nucleic acid gel stain; D003850:Deoxyribonuclease I