Extraction of total RNA and DNA from bacterioplankton

Extraction of total RNA and DNA from bacterioplankton (Molecular Microbial Ecology Manual - Chapter 1.2.2)S TEFAN E ICHLER, M ARKUS G. W EINBAUER†, K ATJA D OMINIK ANDM ANFRED G. HÖFLE*GBF-German Research Center for Biotechnology, Dept. Environmental Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany*corresponding authorTel.: +49-531-6181-419, Fax: +49-531-6181-411, E-mail: mho@gbf.de†present address:Laboratoire d'Océanographie de Villefranche-sur-mer, Diversity, Biogeochemistry and Microbial Ecology Group, Villefranche-sur-mer, FranceIntroductionRecent advances in microbial ecology have been relying on the utilization of modern tools of molecular biology. Application of molecular techniques to study microorganisms in their natural environments not only led to the identification of new and uncultured species but also advanced our knowledge of interactions in complex microbial communities. The majority of the applications usually relies on analytical methods that circumvent cultivation, addressing the general paradigm that only 1% or less of the cells of a natural microbial population can be cultured on growth media in the laboratory [1]. The modern methods rely on molecular analysis of microbial nucleic acids that are used for the identification of microorganisms and their activities in natural environments. However, a crucial point for applicability, efficiency and precision of these modern technologies, is the reliability of extraction methods to obtain nucleic acids from a given environmental source. Microbial ecologists may have to cope with inefficient extraction methods, resulting in under-representation or underestimation of the role of certain members in a microbial community. Applying RNA-based technologies, researchers have the ability to analyze gene-expression or to assess the active fraction of a microbial community. On the other hand, DNA-based methods give insights into general biodiversity, genetic potential or abundant members of whole communities. As a conclusion, to answer many of the questions arising in microbial ecology, it would be beneficial to perform both RNA- and DNA-based analyses on the same environmental sample, thus making it necessary to utilize methods for the simultaneous extraction of both types of nucleic acids.A variety of nucleic acid based application is available to microbial ecologists for the analysis of community structure: 5S rRNA fingerprinting [13, 14], hybridization of probes to bulk extracted DNA [17] or to individual cells [4, 1], generation of 16S rRNA geneclone libraries [12], and sequence specific separation of 16S rRNA gene fragments by denaturing gradient gel electrophoresis (DGGE) [19], temperature gradient gel electrophoresis (TGGE) [20] or single strand conformation polymorphism (SSCP) [26]. Most of these techniques employ as targets the small sub-unit of ribosomes and their genes. Bacterial RNA in a community may be linked to activity or growth due to synthesis of ribosomal RNA usually constituting about 80% of total cellular RNA [27]. Concentration of DNA on the other hand can serve as an indicator for the total cell number [28] or biomass [22] of the whole community. Investigations on functional relationships and bacterial gene expression in communities are primarily carried out using mRNA probes, Northern blotting and specific (RT)-PCR assays [23, 30]. For many of these methods, nucleic acids have to be extracted from cells before analyses can be performed. An ideal extraction method should fulfill at least the following three requirements: 1) the amount of extracted DNA or RNA should be high, 2) the extraction efficiency should be similar for different species and 3) the extracted nucleic acids should be pure and intact to allow for molecular biology studies. Stable extraction efficiency is essential in order to avoid under- or overrepresentation of DNA and RNA from different populations.In the following, we focus on the simultaneous extraction of RNA and DNA from water samples for molecular analysis. It was necessary to an extraction method that allows good extraction of RNA and DNA from any bacterial biomass. Extraction efficiency is a problem with every technique applied to nucleic acids from the environment containing a mixture of bacteria. The extraction method should be feasible for a wide range of cell numbers, independent from the type of microorganism and guarantee the integrity of the obtained nucleic acids for eventual molecular biological studies.Bacterioplankton represents the bacterial communities of the water column of lakes and oceans. It is, considering the size of the world’s oceans, the largest microbial community and of general importance for the carbon flux in aquatic ecosystems [7]. Therefore, bacterioplankton is a good example of a natural microbial community of high relevance. Although a variety of nucleic acid extraction methods has been described for bacterioplankton [22], most of these methods put emphasis on the quality of obtained nucleic acids for eventual molecular biology applications, whereas their extraction efficiencies have not been tested meticulously. Therefore, we employed the mechanical extraction method of D OMINIK and HÖFLE [5], that has been developed and optimized to extract low-molecular-weight (LMA) RNA, i.e. 5S rRNA and tRNA, and adapted the method utilizing a pH shift for the simultaneous extraction of both RNA and DNA from the same filter. The method presented here, qualifies by its speed and reproducibility. Up to 12 samples can be handled simultaneously. P ICHARD and P AUL [24] showed for Escherichia coli that only 11% of the total RNA was recovered by using GF/F filters, in contrast to Durapore filters with low RNA binding capacity, which enabled the recovery of 70% of total RNA. They also showed in previous studies [23] that the GF/F filters could be used if larger volumes (liters) of water were filtered. We placed a glassfiber filter (GF/F) on top of a 0.2 µm pore size polycarbonate membrane filter. The use of such a sandwich of filters results in an increased filtration time but also a much better defined size fraction than without the membrane filter. By combusting GF/F filters (3 h at 450°C) before use, the retention of extracted nucleic acids to the filter was minimized. Furthermore, we could show that the use of glassfiber filters in combination with combusted glass beads has the advantage that the cells are disrupted not only in a chemical way but also mechanically. This is also advantageous for Gram-positive bacteria or other bacteria with rigid cell walls.Finally, we were able to significantly increase the extraction efficiency by using sodium lauryl sarcosine (SLS) as detergent instead of sodium dodecyl sulfate (SDS).Various studies concerning RNA extraction efficiency deal only with cells of Escherichia coli [23] or Pseudomonas sp. [15]. High extraction efficiencies (64-87%) can be obtained from bacterial isolates and cultures using RNA specific extraction methods [23, 31]. For DNA specific extractions, efficiencies for marine bacterioplankton ranged from 23 to 54% using the method described by F UHRMAN et al. [11]. With the method described here, we were able to repeatedly obtain extraction efficiencies for RNA and DNA between 43 to 61% for Gram-negative bacteria and bacterioplankton from pond water samples or 25-30% for Gram-positive microorganisms [34]. The evaluation of our improved extraction method was performed with 4 representative bacterial species. For the assessment of applicability on environmental water samples, we included pond water as natural bacterioplankton source. The methodology has been rigorously examined by comparing and testing several extraction protocols: replacement of SLS/phenol with DivoLab/phenol, or utilization of two commercially available extraction kits, InstaPur or RNA/DNA isolator. The extraction efficiency for the strains and natural bacterioplankton was assessed after sonication by determining the total RNA or DNA content of the bacteria by using the method described by J EFFREY et al. [16] and SYBRGreen II as dye [32] and by determining the amount of isolated RNA or DNA using fluorescence spectrometer with specific fluorochromes [34].Calibration with pure culturesFor the assessment of RNA extraction efficiency, the following representatives for aquatic bacteria were used: For Betaproteobacteria, Ralstonia eutropha, for Gammaproteobacteria, Escherichia coli, Aeromonas hydrophila, Pseudomonas fluorescens, for Cytophaga-Flavobacterium-group, Flavobacterium johnsoniae, for the high G+C Gram-positive Actinobacteria, Arthrobacter globiformis,and for low G+C Gram-positive strains,Enterococcus faecalis and Bacillus megaterium [5]. Figure 1 shows the extraction efficiency of three different extraction techniques, assuming that the orcinol method [21], which is based on an acid hydrolysis of the RNA, represents 100%. Compared with orcinol digestion, the utilization of the modified protocol resulted in an increase of the extraction efficiencies for RNA for Gram-negative strains from generally less than 10% to more than 40%.For calibration purposes for the simultaneous extraction methodology, the extracted amount of RNA and DNA was compared with extraction independent measurements of the nucleic acid content of the samples using sonication. The strains used were representatives of the Betaproteobacteria (Ralstonia eutropha DSM 531T), the Gammaproteobacteria (Escherichia coli DSM 613), of the Cytophaga-Flavobacterium-group (Flavobacterium johnsoniae DSM 2064T) and the high G+C Gram-positive Actinobacteria bacteria (Arthrobacter globiformis DSM 20124T). All strains were grown on nutrient broth agar (NB; 8 g/liter; Difco Corp.), transferred, re-grown at 30°C in liquid NB medium and collected during exponential growth. Table 1 shows bacterial abundance of reference material and nucleic acid concentrations based on SYBRGreen II staining and RNase digestion of sonicated samples. All four different extraction techniques are independent of cell number and reproducible, each having different efficiencies, assuming that sonication represents 100% (Table 2). The extraction efficiencies for RNA and DNA were considerably higher using SLS/phenol or DivoLab/phenol than for InstaPur or the RNA/DNA isolator. With the modified mechanical extraction technique utilizing SLS/phenol, the efficiency for RNA and DNA was 30% and 25% for A. globiformis and averaged at 57% and 49% for the tested Gram-negative bacteria, respectively. Replacement of SLS by DivoLab resulted in similar extraction efficiencies, for RNA reaching 20% for A. globiformis and about 57% in Gram-negative bacteria, for DNA we obtained 20% and about 52% for Gram-positive and Gram-negative test strains, respectively. The performance of the two tested commercially available kits was considerably less efficientcompared to the mechanical extraction methods utilizing SLS/phenol or DivoLab/phenol. Using the modified mechanical extraction method, the extraction efficiency for Gram-positive bacteria is greatly improved, although still about half of that of Gram-negative strains. Considering environmental studies, we deduce that extraction efficiencies varied only moderately within Gram-negative bacteria and between Gram-positive bacteria. This finding is significant since we demonstrated by transmission electron microscopy of water samples from a eutrophic lake that all cells showed a Gram-negative cell wall. Thus it is possible to conclude that Gram-positive cells in the pelagial seem to be negligible since the majority consists of Gram-negative pelagic bacteria. However, our data also showed that we were able to obtain 25-30% of RNA and DNA from the Gram-positive A. globiformis, suggesting, that this method could also be used in ecosystems with a significant amount of Gram-positive bacteria such as sediments or soil. Comparing both presented calibration experiments, we found disparities in RNA extraction efficiencies. However, it has to be considered that the spectrophotometrical RNA quantification using orcinol is not as specific as the utilization of fluorescent dyes. Orcinol is not specific for RNA, but for pentose sugars and reacts with compounds such as uronic acids, xylose and uronic acids [33]. Bacteria may contain significant amounts of arabinose, xylose and uronic acids [3, 9]. This may result in an overestimation of bacterial RNA content and the lower extraction efficiencies when comparing orcinol method with the utilization of fluorescent dyes.Extraction of total RNA and DNA from aquatic microbial communitiesHarvesting of bacterial biomass from aquatic microbial communities is done by using a sandwich of two filters that consists of a glassfiber filter on top of a membrane filter with a pore size of 0.2 µm. A 1-liter sample of a eutrophic lake is sufficient to get 1 to 10 µg of RNAand 0.01 to 3 µg of DNA. For seawater we recommend to harvest the biomass of 3 to 5 l water on the sandwich. The filter can be stored in a deep freezer until extraction (-70°C).The extraction procedure starts with cutting the filter sandwich into small pieces with a scalpel. After extraction with SLS, phenol and glass beads, cell and filter debris and phenol are separated from the aqueous supernatant by centrifugation. The debris is re-extracted by adding fresh SLS and phenol. After combination of both aqueous fractions they are purified by chloroform extraction. The final aqueous supernatant is precipitated overnight with 1 volume of isopropanol (RNA) or ethanol and sodium acetate (DNA), washed and is then ready for nucleic acid determination using fluorometry (see below) or for molecular studies such as PCR amplification of 16S rRNA genes, reverse transcription amplification of 16S rRNA or functional genes, or for SSCP, DGGE and 5S rRNA fingerprinting. However, it has to be considered, that humic and fulvic substances may inhibit Taq polymerases [36, 37] and are occasionally a reason for problems encountered with PCR amplifications, particularly regarding DNA extracts. Therefore, we suggest to use dilutions of the obtained DNA extract or to include a follow-up purification step utilizing commercially available DNA purification systems (e.g. Wizard DNA Clean-Up System; Promega, Madison, USA) prior to PCR.ProceduresHarvesting the bacterial biomass from water samplesIt is highly recommended to process the water samples after collection as soon as possible, minimizing detrimental storage effects on structure and activity of the natural microbial community. Ideally, filtration should be completed within 1-2 h. Depending on temperature and amount of water sampled, several hours are stilltolerable, in particular at low temperature and large volumes. Samples can be collected by filtration of water using combusted glassfiber filters (Whatman GF/F). Notice, that combustion of filters for 3 h at 450°C prior to filtration is minimizing the retention effects of nucleic acids to the glassfiber during the extraction procedure, resulting in a significant higher yield in both DNA and RNA. Water samples can be collected in clean plastic containers. A prefiltration is recommended to remove protozoa and algae. Therefore, we recommend an initial prefiltration using nylon net (mesh size 10 µm) followed by an additional filtration step using a membrane filter (Nucleopore; pore size 1 µm or 3µm). Filtering the water sample or liquid culture medium through a filter sandwich collects the microorganisms in the fraction smaller than 1 µm or 3 µm. The filter sandwich consists of a glassfiber filter (Whatman GF/F; diameter 90 mm) on top of a membrane filter (Nucleopore; diameter 90 mm, 0.2 µm pore size) that is placed in a glass filter holder with a fritted glass base as support. Filtration is performed by applying a mild vacuum (not more than 200 mbar). After filtration the filter sandwich is removed, folded and wrapped in aluminum foil, and immediately stored in a freezer at –70°C for later extraction of nucleic acids.Supplies• Glassfiber filter (Whatman Corp., Maidstone, England; # 1825090 type GF/F;diameter 90 mm)• Membrane filter (Nucleopore Corp., Pleasanton, USA; # 603, # 601, and # 6002, for the pore sizes 3 µm, 1 µm and 0.2 µm, respectively, diameter 90 mm)• Filter holder (Schott, Hofheim, Germany; filter head # 247209003, filter disc # 2520901)Isolation of total DNA and RNA from microbial communitiesThe protocol (Figure 2) is based on a combined mechanical and chemical extraction method [14, 5] utilizing a pH shift for the simultaneous extraction of both RNA and DNA from an individual filter [34]. Wear gloves while working and take proper laboratory safety measures, i.e. work under a hood. Autoclave all pipette tips, microtubes, extraction cells and solutions (except SLS, phenol and chloroform) before use to avoid contamination with DNases, RNases or contaminant nucleic acids. The used plastic ware is provided sterilized. The glass beads should be combusted for 4 h at 450°C and then siliconized [8]. Make sure to wear safety glasses and protective gloves, since phenol is highly toxic and corrosive.Extraction and phase separation1. Transfer 2 g of 2-mm and 2 g of 3-mm siliconized glass beads in a Teflonextraction cell.2. Cut the frozen filter sandwich in short pieces with a scalpel and add them to theextraction cell.3. Add 5 ml of the Extraction buffer, pH4.2 (see below).4. Add 5 ml of buffer-saturated phenol pH 4.2 and vibrate the mixture for 2 min witha high-speed cell disrupter (amplitude 15 mm).5. Transfer the homogenate to a 50 ml Falcon tube.6. Mix thoroughly by vortexing the capped tube and centrifuge for 20 min at 7200 x gat 4°C.7. During centrifugation, the mixture separates into a lower phenol phase with glassbeads, an interphase, and an upper aqueous phase. RNA remains exclusively in the aqueous phase, whereas DNA is contained in the two lower phases. Transfer the aqueous phase to a fresh tube (Greiner, snap caps, volume 15 ml) andproceed below (8.). For DNA isolation, add 5 ml Tris-base (pH 10.5), mix well and store DNA-containing phenolic and inter-phase at 4°C for at least 40 min but not longer than 3 h (see DNA isolation section).8. Rinse the extraction cell once with 2 ml of Extraction buffer and add it to the 50mlFalcon tube. Also add 2 ml of buffer saturated phenol to the Falcon tube for repeated phenol extraction.9. Mix thoroughly by vortexing the capped tube and centrifuge again for 20 min at7200 x g at 4°C.10. Transfer the upper aqueous phase to the Greiner tube. The volume of thecombined aqueous phase is about 4 to 5 ml.RNA isolation1. Add 0.1 volumes of 3 M sodium acetate to the Greiner tube containing the pooledaqueous phase from the extraction and phase separation procedure.2. Add 5 ml of chloroform to the Greiner tube to remove phenol from the aqueousphase. Mix vigorously by vortexing and centrifuge for 10 min at 11000 x g at 4°C to separate the phases.3. Transfer the aqueous phase to a fresh Greiner tube and repeat the chloroformstep.4. Transfer the aqueous phase to another Greiner tube and add one volume ofisopropanol to precipitate the RNA. Mix the sample by vortexing and incubate samples at -20 °C overnight. The RNA can be stored in that form for a long time and eventually be pelleted for further analysis as detailed below.DNA isolation1. Mix the sample containing the extracted DNA vigorously by vortexing andcentrifuge for 15 min at 2000 x g and 4°C.2. Transfer upper aqueous phase to a fresh Greiner tube. Add 2 ml 1M Tris-base(pH 10.5) to the lower phenolic phase and repeat vortexing and centrifugation for15 min at 2000 x g and 4°C.3. Transfer and pool the upper aqueous phases from each centrifugation. Toremove phenol and proteins, add 5 ml chloroform-isoamyl alcohol (24:1) and centrifuge for 15 min at 2000 x g and 4°C.4. Transfer upper phase to a fresh Greiner tube and repeat the chloroform step untila clear upper phase is obtained.5. Add 90 µl 3 M sodium acetate, pH 4 and 2.5 volumes of ice cold 70% ethanol tothe now clear upper phase and precipitate the DNA over night at –20°C. The DNA precipitate should be processed the next day and eventually be pelleted for further fluorometrical analysis as detailed below.Solutions- Extraction buffer (per liter) (Used for the nucleic acid extraction mixture and the buffer-satured phenol)(50 mM sodium acetate, 10 mM EDTA)4.10 g sodium acetate (Riedel de Häen #32319 Seelze, Germany)3.72 g EDTA (Idranal III, Riedel de Häen #34549 Seelze, Germany)pH 4.2 (adjust with acetic acid)- Extraction mixture0.5 % sodium lauryl sarcosine, (SLS), (Sigma # 5125, Deisenhofen, Germany) inExtraction buffer(75 parts Extraction buffer plus 4 parts 10 % SLS-solution)- buffer saturated phenolMix 400 ml of phenol (Amresco # 0981 Solon, Ohio, USA) with 400 mg of 8-hydroxyquinoline and add 400 ml of Extraction buffer. Stir for 1 h. Allow the solution to form two phases and remove the aqueous phase. Add again 400 ml of fresh Extraction buffer to the phenol phase and repeat the procedure two more times. The final pH of the buffer saturated phenol should be about pH 4.2-4.4.Supplies• High-speed cell disrupter (Microdismembrator II, Braun Corp., Melsungen, Germany; #893162/4, amplitude, 15 mm)• Teflon extraction cell (Braun Corp., Melsungen, Germany; # 854495/6; volume 20 ml)• 2 and 3 mm combusted and siliconized glass beads• Falcon tubes (volume, 50 ml)• Greiner tubes (volume, 15 ml)• Eppendorf tubes (volume, 2 ml)• Eppendorf tubes (volume, 1.5 ml)• Microcentrifuge tube filters (Anapore, Whatman Corp., Maidstone, England; # 68300203, pore size 0.2 µm)Washing and resuspension of RNA and DNA1. Pellet the precipitated nucleic acids from both the isopropanol (for RNA) and thesodium acetate-ethanol precipitation (for DNA) by centrifuging 30 min at 19000 xg and 4°C.2. Wash the pellets twice with 1 ml of ice cold 70 % ethanol. Centrifuge at 19000 x gfor 10 min at 4°C. Prior to the second centrifugation, transfer the pellet to a microtube, centrifuge again at 19000 x g for 10 min at 4°C, and discard the supernatant.3. At the end of the procedure, dry the pellet for about 10 min by centrifugationunder vacuum (speed vac or desiccator). It is important not to let the pellet dry completely as this will greatly decrease its solubility.4. Dissolve the DNA or RNA in 300 µl (or less) of freshly autoclaved molecularbiology grade water (RNase and DNase free) or preferably in Tris-Ca buffer (pH7.5) by passing the solution a few times through a pipette tip. Incubate the samplefor at least 45 min at room temperature to dissolve the nucleic acids completely.The extracts should be stored frozen at –20°C or below.Solutions- Tris-Ca buffer (used for resuspension of nucleic acids)0.1 M NaCl0.1 M Tris pH 7.50.1324 g/l CaCl2•H2OFluorometric determination of RNA and DNA concentration and purityTypically the concentration of total nucleic acids is measured with dyes such as thiazole orange or ethidium homodimer and DNA concentrations are determined with the DNA specific fluorochrome Hoechst dye 33258 (H33258); RNA is determined by subtraction of the amount of DNA from that of the total nucleic acids (double fluorochrome method) [2]. Alternatively, DNA concentrations can be estimated after enzymatic digestion of RNA (RNase digestion method) [6, 18]. RNA concentrations can also be determined after DNA digestion (DNase digestion method). However, the development of novel green-fluorescent dyes is having a significant impact on the determination of RNA and DNA concentrations [32]. These dyes are potent tools for quantifying nucleic acids due to their high fluorescence yield upon binding to nucleic acids and their high specificity for nucleic acids. Prior to fluorescence spectroscopy of the RNA concentration utilizing RiboGreen fluorescent (Molecular Probes) and of the DNA concentration using the dye PicoGreen (Molecular Probes), we recommend to DNase or RNase digest the respective extracts to avoid contaminations with unwanted nucleic acid types.1. To inactivate contaminant DNase, it is necessary to boil the RNase for 15 min at100°C prior to digestion. Perform RNase (25 µg/ml final concentration) digestion in DNA samples at 37°C for 3 h in Tris-Ca buffer. For DNA digestion in RNA samples utilize DNase in a final concentration of 10-1000 units/ml at 37°C for 3 h in Tris-Ca buffer.2. To determine the RNA concentration in the sample, add 100 µl of a 1:200 dilutionof RiboGreen (RNA quantification kit; Molecular Probes) in Tris-Ca buffer to 100 µl samples. For assessment of the DNA concentration add 100 µl of a 1:200dilution of PicoGreen (dsDNA quantification kit; Molecular Probes) in Tris-Ca to 100 µl samples.3. Measure fluorescence using low-fluorescent 96-well microtiter plates (CytoPlate,Millipore) in a microtiter plate reader set at 475 nm excitation and 530 nm emission. Set up calibration curves using E. coli 16S + 23S rRNA provided along with the RiboGreen RNA quantification kit for RNA. As DNA calibration standard we suggest to use E. coli DNA (Sigma). RNA and DNA concentrations in the samples can be calculated by comparing the fluorescence results with the respective calibration standard. Notice, that it might be advantageous to assess the nucleic acid concentrations for the standards spectrophotometrically at 260 nm.4. Purity of RNA and DNA samples can be assessed spectrophotometrically. Theratio of absorbance at 260 nm and 280 nm should be around 2.0 (1.7–2.1). Lower values indicate contamination of the nucleic acids by proteins or polysaccharides.Notes2a. Alternatively, the RNA and DNA concentrations in the samples can be determined spectrophotometrically, by measuring the absorbance or optical density (OD) at 260 nm. An OD of 1.0 equals a concentration of 40 µg/ml RNA or50 µg/ml DNA.4a: It is much safer to run a spectrum (from 230 nm to 320 nm) of RNA and DNA from environmental samples. Even if the ratio of the absorbance at 260 nm to that at 280 nm is about 2.0 the spectrum often shows contaminations at 230-250 nm.The calculated amount of RNA should be considered only as an approximate value.Supplies• Low-fluorescent 96-well microtiter plates (CytoPlate, Millipore, Eschborn, Germany)• Cytofluor 2350 Fluorescence Measurement System (Millipore, Eschborn, Germany) or Fluoroscan II System (Titertek Fluoroscan plate reader; Flow Laboratories, Meckenheim, Germany)• RNA quantification kit (Molecular Probes, #R-11490; Leiden, The Netherlands) • dsDNA quantification kit (Molecular Probes, #P-7581; Leiden, The Netherlands) • Escherichia coli DNA (Sigma, Deisenhofen, Germany; #D-0421)• Ribonuclease A type III-A (Sigma, Deisenhofen, Germany; #R-5125)• DNase I from bovine pancreas, grade II (Boehringer, Mannheim, Germany, #104 159)Application of the methodEmploying the procedure described above, we obtained high quality extracts of nucleic acids from water samples that were collected from a pond [34]. Enumeration of bacterial cells was performed by epifluorescence microscopy [29]. Bacterial cell numbers for the GBF pond were 1.4 x 1010 cells/l, with 13.7 fg of RNA or 3.9 fg of DNA per cell (giving a ratio of 3.5) (Table 1). Extraction efficiencies for both RNA and DNA using the SLS/phenol method were about 60% for the pond community, a similar value compared with efficiencies obtained for Gram-negative bacteria during calibration experiments (Table 2).Community structure of bacterioplankton of the pond water was analyzed by SSCP fingerprinting of RT-PCR and PCR amplification products of 16S rRNA and of its。