Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication
- 1 January 2004
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 70 (1) , 25-33
- https://doi.org/10.1128/aem.70.1.25-33.2004
Abstract
Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.Keywords
This publication has 71 references indexed in Scilit:
- Higher Abundance of Bacteria than of Viruses in Deep Mediterranean SedimentsApplied and Environmental Microbiology, 2002
- A model for the distribution of particle flux in the mid-water column controlled by subsurface biotic interactionsDeep Sea Research Part II: Topical Studies in Oceanography, 2001
- Natural Communities of Novel Archaea and Bacteria Growing in Cold Sulfurous Springs with a String-of-Pearls-Like MorphologyApplied and Environmental Microbiology, 2001
- Particle settling rates increase with depth in the oceanDeep Sea Research Part II: Topical Studies in Oceanography, 2001
- Different methods for extracting bacteria from freshwater sediment and a simple method to measure bacterial production in sediment samplesJournal of Microbiological Methods, 2000
- Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteriaFEMS Microbiology Ecology, 2000
- Molecular methods for the study of methanotroph ecologyFEMS Microbiology Ecology, 1998
- Spatial and temporal dynamics in marine aggregate abundance, sinking rate and flux: Monterey Bay, central CaliforniaDeep Sea Research Part II: Topical Studies in Oceanography, 1998
- Comparison of methods for the concentration of bacterioplankton for in situ hybridizationJournal of Microbiological Methods, 1997
- Enumeration of microorganisms from deep-sea hydrothermal chimney samplesFEMS Microbiology Letters, 1997