Format

Send to

Choose Destination
Int Microbiol. 2010 Jun;13(2):59-65.

Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454 pyrosequencing.

Author information

1
TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Microbial Genomics Group, Zeist, Netherlands.

Abstract

The preferential detection of cells with intact membranes by sample treatment with propidium monoazide (PMA) in combination with PCR amplification is gaining in popularity. This study evaluates the effect of PMA on 454 pyrosequencing profiles of environmental water samples from a canal in Amsterdam and seawater (with sediment) left untreated or exposed to elevated temperatures (50, 60, or 85 °C) for 10 min. Community analysis was based on the extraction of genomic DNA followed by PCR amplification of 16S rRNA genes using universal bacterial primers. Whereas the highest temperature in combination with PMA treatment completely suppressed PCR amplification, PCR products from the other samples were subjected to massively parallel tag sequencing. PMA treatment did not substantially affect the sequence profiles of non-heated samples, but heat exposure resulted in a clear difference in the relative proportions of certain groups. This difference was significantly more pronounced in heated seawater than in heated canal water. The effect of the chosen experimental conditions on the membrane integrity of cells was supported by BacLight LIVE/DEAD staining in combination with flow cytometry, which confirmed an increase in the uptake of propidium iodide in samples exposed to high temperatures.

PMID:
20890840
DOI:
10.2436/20.1501.01.111
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Springer Icon for Spanish Society of Microbiology
Loading ...
Support Center