Filter-type and sample handling affect determination of organic substrate uptake by bacterioplankton

Natural assemblages of bacterioplankton (coasFiltration is used widely in many aspects of microbial tal seawater and seawater filtrates), treated with radiolabeled ecology research, therefore the performance of various organic substrates, were used to test the effectiveness of varfiltration techniques needs to be thoroughly underious types of 0.2 pm pore size filters in capturing labeled cell materials. The hiahest retention of cell matenal was observed stood. One is the use memd with filters made of either polyethersulfone (PES), mued celbrane filters to capture whole cells dunng uptake studlulose esters (MCE) or Nylon PES fllters retained up to 2 2 % ies emvlov~nu addltlon of radiolabeled substrates to . G less than MCE or Nylon filters in some comparisons, but in aquatic samples (Wnght & ~ ~ b b ~ ~ 1966, ~~.ff~~h~ et al. most cases these filters gave similar, apparently high retention of cell materials. In contrast, retention of labeled cell 1974, Azam & Hodson 1981, Hodson et al. 1981, Coffin material by track-etched polycarbonate (PC) or polyester (PE) 1989, Griffith et al. 1990, Middelboe et al. 1995, Rich et filters was 10 to 80% less than for PES, MCE or Nylon filters, al. 1996). Termination of incubations with radiolabeled even at low filtration vacuum (5 cm Hg). Radiolabel lost from substrates is often best achieved by filtering cells, PC filters was recovered as dissolved compounds in the filrather than addition of poisons, because poisons such trates, and tests showed that cell breakagelleakage occurred, not during filtration per se, but rather at the end of filtration HgC12 and strong acids can cause losses when filters ran dry. Higher filtration vacuum over the range label from the cells (Griffiths et al. 1974, Palumbo et al. 5 to 38 cm Hg caused greater losses with PC filters whereas retention of radiolabel by PES filters was nearly independent of vacuum. Losses on PC filters were greatest during early phases of incubations, when label in the cells was found mostly as untransformed parent substrate, and losses were greater for substrates known to function as lntracellular osmolytes (e.g. glycine betaine and dimethylsulfoniopropionate) than for substrates (e.g. leucine and glucose) that were rapidly incorporated into macromolecules. Treatment of PESfiltered cells by a cold osmotic shock procedure resulted in nearly identical losses to those which occurred with nonshocked cells on PC filters. Likewise, addition of formalin (2 % 1983). Filtration itself can be problematic, however, if conditions are harsh (high vacuum shear, or desication on dry filters), as this can cause release of dissolved organic compounds, especially from eucaryotes like microzooplankton and phytoplankton (Fuhrman & Bell 1985, Goldman & Dennet 1985, Nagata & Kirchman 1990). While most 0.2 pm membrane filters do effectively remove the majority of bacterial cells from water, as evidenced by insignificant numbers of acridine oranaeor DAPI-~ositive cells in the filtrates fHobbie d final concentration) to a seawater filtrate culture which had et 19771, few microbial ecology studies have already taken up I4C-glycine betaine caused rapid losses of 14C from particulate materials, resulting in the same amount addressed the efficiency of membrane filters for retenof loss as occurred ,,,,hefl live were filtcrection of all bacterial ce! r*ateria! s. Furthermcre, a wide onto PC filters. The resultssuggest that a major fraction of the variety of membrane filters have been used by investisoluble components of bacterioplankton can be selectively gators carrying out microbial uptake studies, yet little lost during filtration through polycarbonate-type membranes, information exists about the relative effectiveness of and by procedures which subject cells to osmotic shock and poisoning. Furthermore, our results indicate that PES, MCE the different and nylon filters function similarly well at retaining most cell We recently investigated uptake of the low molecumaterials. lar weight osmotic solutes, glycine betaine (GBT), and dimethylsulfoniopropionate (DMSP) by natural micro