Rapid estimation of microbial numbers in water using bulk fluorescence.
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.
Wegley
Linda
L
Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, USA. lwegley@gmail.com
Mosier-Boss
Pamela
P
Lieberman
Stephen
S
Andrews
John
J
Graff-Baker
Amanda
A
Rohwer
Forest
F
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
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C474470:SYBR Gold nucleic acid gel stain; D003850:Deoxyribonuclease I