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Applied and Environmental Microbiology, January 2004, p. 25-33, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.25-33.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication

Phyllis Lam^* and James P. Cowen

Department of Oceanography, SOEST, University of Hawaii at Manoa, Honolulu, Hawaii 96822

Received 9 June 2003/ Accepted 6 October 2003

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.

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* Corresponding author. Mailing address: Department of Oceanography, SOEST, University of Hawaii at Manoa, 1000 Pope Rd., Honolulu, HI 96822. Phone: (808) 956-2017. Fax: (808) 956-7112. E-mail: plam{at}soest.hawaii.edu.

SOEST contribution number 6301.

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Applied and Environmental Microbiology, January 2004, p. 25-33, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.25-33.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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