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The DNA-specific fluorochrome Hoechst 33258 (H33258) and the DNAand RNAsensitive fluorochrome ethidium homodlmer (EthDi) were used to detect ng ml-' concentrations of nuclelc acids in marine microorganisms. These fluorochromes were 4 to 5 times more sensitive to DNA than ethidium bromide, and their use significantly reduced the amount of sample necessary to measure nuclelc acids in seawater. Both fluorochromes responded in a linear fashion to standard DNA, but the response of EthDi to RNA was non-linear for the majority of RNA types tested. Chlorophyll a quenched fluorescence of both fluorochromes, especially of EthDi, at concentrations that could be encountered in sample homogenates. Two techniques were used to measure DNA and RNA on cultures of marine bacteria and phytoplankton; H33258 and EthDi together (double fluorochrome technique), and EthDi with and without RNase (RNase digestion method). In general, DNA and RNA were measured equally well using either technique; discrepancies between techniques were attributed to EthDi quenching or the specificity of H33258 for DNA rich in dA-dT base pairs. The double fluorochrome technique was used to determine depth profiles of DNA and RNA/DNA in size-fractionated natural seawater These properties showed significant changes between the surface and 100 m and may be related to changes In biomass or metabolic activity. Although consideration must be given to the RNA standard used and the presence of naturally occuring pigments, the combination of H33258 and EthDi can provide a rapid and sensitive measure of nucleic acids in seawater without the use of nucleases. INTRODUCTION tion step or in conjunction with the DNA-specific fluorochrome Hoechst 33258 (H332.58) to measure To understand plankton abundance and dynamics, DNA and RNA in marine microorganisms. The results some investigators have used fluorescent dyes to meassuggest that the combination of H33258 and EthDi ure particulate DNA (Takahashi et al. 1974, Paul & provides a sensitive method to measure nucleic acids in Carlson 1984, Paul et al. 1985). To obtain additional marine microorganisms. information, especially about metabolic activity, fluorometric and absorbance measurements of RNA have also been made (Takahashi et al. 1974, Dortch et MATERIALS AND METHODS al. 1983, Moyer & Morita 1989). However, previous methods to measure RNA were relatively insensitive, or Emission scans with DNA were measured for each too elaborate for rapid analysis (Randerath & Ranfluorochrome separately and with the 2 fluorochromes derath 1969, Thoresen et al. 1983, Iturriaga et al. 1984). combined to determine if significant interference Some fluorochromes, such as ethidium bromide occured between the fluorochromes. The DNA (EthBr), react with nucleic acids generally and thus response of H33258 and EthDi was also compared to require a digestion step to distinguish RNA from DNA. the response of EthBr, a fluorochrome often used to A dimer of EthBr, ethidium homodimer (EthDi), has measure nucleic acids. Several types and preparations been developed (Gaugain et al. 1978a) with much of RNA were measured with EthDi to determine an improved sensitivity for DNA and RNA (Markovits et appropriate RNA standard. The response of H33258 al. 1979, Reinhardt et al. 1982, Moyer et al. 1990). We and EthDi with DNA was measured in the presence of investigated the use of this fluorochrome with a digesstandard chlorophyll a (chl a), which is known to comO Inter-Research/Printed in Germany Mar. Ecol. Prog. Ser. 73: 283-293, 1991 plicate EthBr measurements (Thoresen et al. 1983). The double fluorochrome method was used to measure nucleic acids in cultures of marine bacteria at different stages of starvation-survival and in batch cultures of phytoplankton. The results were compared to measurements made using an RNase digestion method which used EthDi on subsamples with and without RNase (Thoresen et al. 1983). Natural seawater samples were also measured for DNA and RNA using the double fluorochrome method. A technique similar to the double fluorochrome method but using H33258 with EthBr to measure DNA and RNA in fish larvae has been described by Clemmesen (1988). Chemicals. The nucleic acid stains used were Hoechst 33258 (H33258-Calbiochem, San Diego, CA, USA), ethidium homodimer (EthDi-Molecular Probes, Eugene, OR, USA) and ethidium bromide (EthBr). The standard nucleic acids used were calf thymus DNA type I (Sigma, St. Louis, MO, USA), bakers yeast RNA type I11 and type XI (Sigma), Escherichia coli strain B RNA (Calbiochem), E. col1 strain W transfer RNA (tRNA) and ribosomal RNA (rRNA) (Sigma), and bovine liver rRNA (Sigma). Anacystis nidulans chl a was from Sigma, and RNase (DNase-free), heparin and Triton X-100 were from Calbiochem. Solutions and standards. All glassware used was precombusted at 480 "C to remove any nuclease contamination. The buffer solutions used were 0.05 M Tris, 0.05 M NaC1, pH 7.3 (TN buffer) or 0.05 M Tris, 0.05 M NaC1, 0.001 M EDTA, 1 % NaN3, pH 7.3 (TNEA buffer). Except where noted, TNEA buffer was used with cultures and TN buffer for natural samples. Primary stock solutions of H33258 (0.19 mM) were made in distilled, deionized water. Working stock solutions of H33258 (3.8 1tM) were made fresh daily by diluting the primary stock solution in buffer. Primary stock solutions of EthDi (47 FM) and EthBr (74 PM) were made in buffer containing 1 ' 10 methanol. Concentrations of EthDi and EthBr working stock solutions were 12 yM. All solutions were stored in the dark at 4 "C. Under these conditions, EthDi was stable for months. The concentrations of all nucleic acid standard stock solutions were determined gravimetrically, except for the rRNAs in which the concentrations were determined from the absorbance to weight conversions provided by Sigma. Stock solutions of standard DNA and Escherichia col1 strain B RNA were made in TN buffer and portions of these stock solutions were stored frozen in sterile scintillation vials. Stock solutions of other RNA standards were made in TNEA buffer and used immediately. Standards were measured by diluting the standard stock solutions with buffer to concentrations representative of the culture and sample homogenates. Each day that samples or cultures were measured for nucleic acids, the fluorescence response of 3 standards was also determined; standard DNA with H33258, standard DNA with EthDi, and E. col1 strain B RNA with EthDi. Previous experiments showed that these standards had fluorescence responses independent of sonication and of treatments with Triton X-100 or heparin. Stock solutions of Triton X-100 were 0.5 % (v/v) in buffer. RNase and heparin stock solutions were made fresh daily in buffer at 10 and 5 mg ml-' respectively. Solutions of chl a were made by dissolving and diluting chl a in methanol, and were used immediately. Cultures. A gram-negative bacterial rod (OR5-88) was isolated from a seawater sample taken off the Oregon coast (44" 39.0' N, 125" 59.6' W) in September 1988 from 5 m using the bow-pump of the RV 'Wecoma' during cruise W8808. OR5-88 was maintained in batch culture on a rotary shaker at 11 "C using SLX medium (Moyer & Morita 1989). Two chemostats were assembled according to Moyer & Morita (1989), inoculated with log phase cells, and maintained at 11 "C with SLX medium. Constant growth was assumed after 4 1 passed through the chemostats (chemostat volume = 345 ml). Doubling rates, estimated from chemostat dilution rates, were 0.5 d-' and 0.0201 h-' for replicate chemostats. Cells from each chemostat were harvested by centrifugation, rinsed 3 times in artificial seawater and starved separately in the dark at 11 "C in aged seawater. The aged seawater had been collected from 2000 m and stored in the dark for 2 yr before use. Replicate measurements of DNA and RNA were made on each starved culture on 9 different days of starvation up to 121 d. Phytoplankton cultures were provided by T. Cowles and S. Neuer (Oregon State University) and included Gymnodinium sirnplex, Thalassiosira weissflogii, Chlorella vulgaris, Asterionella japonica, Chaetoceros sp., and BBI, an unclassified algae from Station P isolated by B. Booth (University of Washington). Phytoplankton cells were grown in batch culture (IMR/4 media, Eppley et al. 1967) under continuous fluorescent Light from 8 Sylvania 60W bulbs at 12°C. Replicates of each culture were measured for DNA and RNA as discussed below Because these cultures were only used to compare techniques of measuring nucleic acids, samples were used without regard to the stage of growth of the cultures. Treatments. Cultured cells (1 to 6 ml) were pressurefiltered on 0.2 pm (bacteria cultures) or 1.0 Iim (phytoplankton cultures) Nuclepore filters at < 7 psi (4.7 X 104 Pa). All filters, including filters for 0.2 pm or 1.0 ,pm blanks, were pre-rinsed with 15 m1 of buffer. Treatments of cell homogenates and blanks were identical. The filters were placed in individual 15 m1 polypropylene centrifuge tubes on ice containing 8.0 m1 buffer and 40 p1 of Triton X-100. Cells on Nuclepore filters were disrupted on ice with pulsed sonication for 90 s using a Mordy & Carlson: Techniques for measuring DNA and RNA 285 Branson W-350 power source with a Heat Systems 1/8" (0.32 mm) microtip assembly at 5.0 output with 60 O/O duty (Paul & Myers 1982). Heparin was added (40 pl) to phytoplankton homogenates to displace nucleohistones which are known to block DNA fluorochrome binding sites in eukaryotes (Karsten & Wollenberger 1977, Bonaly et al. 1987). RNA was digested in a 2.6 m1 homogenate subsample by adding 5 p1 of RNase. Homogenates with heparin and subsamples with RNase were incubated at 40 "C for 20 min. Homogenate subsamples digested with RNase were measured with EthDi while homogenates without added RNase were measured with EthDi and H33258 as discussed below. Fluorescence measurements. Fluorescence measurements of standards, blanks, and homogenates were identical and non

An evaluation of fluorescence technigues for measuring DNA and RNA in marine microorganisms