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.

Identification of a Novel Human Papillomavirus by Metagenomic Analysis of Samples from Patients with Febrile Respiratory Illness

“…Amplified DNA and cDNA were then purified using the NucleoSpin® Tissue Kit (Clontech, Mountain View, CA 94043, USA). Epifluorescence microscopy was used to ascertain the presence of microbes and viruses before and after chloroform treatment [23].…”

Enumerating Viruses by Using Fluorescence and the Nature of the Nonviral Background Fraction

“…Brook, creek, and river water samples were filtered (0.2-m pore size) and stored as described for the wastewater. Stored freshwater filtrates were subsampled (3 ml) and incubated for 15 min with 10 kU of DNase II (Sigma-Aldridge, Sydney, Australia) per ml as described previously (18). Viruses were then stained with SYBR gold (Molecular Probes Inc., Eugene, OR, USA) using the method of Patel et al (11).…”

“…No fixatives were used, as they cause rapid viral decay (20). Stored samples (3 ml of the 0.2-m-filtered filtrate) were incubated for 15 min with 10 kU of DNase II (Sigma-Aldridge, Sydney, Australia) per ml of sample as described previously (18). Viruses were then stained with SYBR gold (Molecular Probes Inc., Eugene, OR, USA) using the method of Patel et al (11).…”

“…A scanning fluorescence spectrophotometer has the ability to scan samples stained with SYBR at a range of excitations and emissions, thus allowing for greater accuracy in determining fluorescence peak intensities, positions, and shapes of the spectra. FEEMs have been used to detect bacteria in natural water samples stained with SYBR gold (18) to develop a rapid and inexpensive online and portable fluorimeter for measuring bacterial numbers in water. However, it falls short of measuring viral abundance because of what appears to be high background fluorescence.…”

A culture-free method for detection of Vibrio vulnificus from coastal seawater based on loop-mediated isothermal amplification targeting vcgC gene

“…So, the total DNA extracted from seawater samples might contain high background of DNA (Wegley et al, 2006). Sub-complementary sequences in the background DNA might interfere with the DNA amplification.…”