父亲的责任
Quantification of aquatic virus by flow cytometry
Corina P . D. Brussaard 1*, Jérôme P . Payet 2, Christian Winter 3, and Markus G. Weinbauer 4
1NIOZ Royal Netherlands Institute for Sea Rearch, PO Box 59, 1790 AB Den Burg, Texel, The Netherlands 2
Department of Earth and Ocean Sciences, University of British Columbia, Vancouver, BC, Canada 3
Department of Marine Biology, University of Vienna, Vienna, Austria 4
Microbial Ecology & Biogeochemistry Group, Université Pierre et Marie Curie-Paris 6, and CNRS, Laboratoire d’Océanographie de Villefranche, Villefranche-sur-Mer, France
Abstract
For many laboratories, flow cytometry is becoming the routine method for quantifying virus in aquatic sys-tems becau of its high reproducibility, high sample throughput, and ability to distinguis
h veral subpopula-tions of virus. Comparison of viral counts between flow cytometry and epifluorescence microscopy typically shows slopes that are statistically not distinguishable from 1, thus confirming the ufulness of flow cytometry.Here we describe in detail all steps in the procedure, discuss potential problems, and offer solutions.
MANUAL
of
Materials and procedures
An outline of the FCM assay for detection and quantifica-tion of aquatic virus is prented in Fig.1.
Reagents and solutions—FCM detection and enumeration of virus requires high-quality reagents. Water samples are pre-rved using 25% electron microscopy (EM)-grade glutaralde-hyde (Sigma; storage at 4°C). The EM-grade glutaraldehyde is free of polymers and other contaminants and, hence, is opti-mal for fixing the samples. To avoid cross-contamination of samples in pipetting, it is important to prepare small aliquots of the fixative solution.
The u of ultrapure sheath fluid is esntial, since one works clo to the limits of detection of the in
strument. An improved FCM signal is obtained when using Milli-Q water (ultrapure deionized water with resistivity of 18.2 MΩcm–1) instead of the commercially available sheath fluids (e.g., F ACSF low). Working stain solution of SYBR®green I (10,000×concentrate in DMSO; Invitrogen, Molecular Probes; storage at –20°C) is usually prepared by diluting the commercial stock (1:200) in either autoclaved Milli-Q or grade molecular water. Working solution stain can be reud but it is best to limit the number of freeze–thaw cycles (two or three) to prevent the loss of staining efficiency over time. Thus, small aliquots (1 mL) of working stain solution should be prepared. The commercial stock is supplied in DMSO, but further dilution in DMSO typically increas noi levels upon addition to the samples. Occasionally, the fluorescent dye ems responsible for generating noi, and a brief spin (~20,000g) of the stock solution in a microfuge generally reduces the noi levels.
Samples are diluted in sterile TE-buffer, pH 8.0 (10 mM Tris-hydroxymethyl-aminomethane, Roche Diagnostics; 1 mM ethylenediaminetetraacetic acid, Sigma-Aldrich) to avoid elec-tronic coincidence (e.g., e below). The u of any diluents (e.g., phosphate-buffered saline, Milli-Q water, etc.) other than TE-buffer was found to negatively affect flow cytometric sig-natures of stained virus (Brussaard 2004).
TE-buffer should be autoclaved directly after preparation to maintain low background fluorescence (c
heck pH before u and adjust if needed using HCl). The quality of Tris may differ depending on the supplier, and thus, it is likely to affect the quality of TE-buffer (Brussaard unpubl. data). In principle, fil-tration before first-time u of the TE-buffer should not be nec-essary. But, once opened, small batches (i.e., ~50 mL) of the TE-buffer should be prefiltered (sterile FP30/0.2 µm; Schleicher & Schnell) just before u. Change the filter for each batch of the TE-buffer. F iltration may result in enhanced noi level, depending on the filter type. F iltration of the TE-buffer through 30-kDa molecular weight cutoff filters would be ideal but time-consuming. Instead, u a new batch of sterile TE-buffer and carefully check the noi level by running a stained blank (e“Blank and reference”) before u.
Sampling and storag e—Proper storage and prervation of aquatic samples is crucial to prevent loss of virus particles. Typically, there is no need to filter or treat the natural water samples before fixation. Filtration of the samples before fixa-tion may result in substantial loss of virus (data not shown).
Fig. 1.Different process, accompanying methodology, and critical notes for flow cytometric enumeration of aquatic virus.
Samples of 1 mL are usually taken (replicate sampling is advid), transferred into 2-mL cryovials, and fixed at a final concentration of 0.5% glutaraldehyde for 15–30 min at 4°C in the dark. After fixation, the samples are flash frozen in liquid nitrogen. F lash-freezing is very important, as fixed samples stored at 4°C show significant and rapid reductions in virus counts (Brussaard 2004; Wen et al. 2004). For the same reason, it is important to minimize the fixation period to less than an hour (15–30 min is optimal). Once frozen in liquid nitrogen, the samples should be stored at –80°C (testin
g storage for 6 months showed no detectable virus decay [Brussaard 2004]). Note that after a field expedition, frozen samples should be nt either in dry ice or in a liquid nitrogen dry-shipper to keep samples deep-frozen during transport.
FCM tup—Not all F CMs have equal nsitivity to detect and enumerate aquatic virus. Whereas some F CMs will detect only the higher green fluorescent virus subpopulations, others may not be nsitive enough to detect virus at all. The 488-nm argon lar benchtop FCMs of Becton Dickinson (e.g., BD-FACScalibur) provide high nsitivity for virus detection. The advantage of benchtop FCMs is that the machines can be easily taken on board ship.
Virus particles are too small to scatter light of standard benchtop FCMs. The u of nucleic acid–specific stains, such as SYBR Green I, is thus esntial for virus detection. FCM sig-natures of stained virus from natural samples can partially overlap with background fluorescence generated by FCM. Ulti-mately, it is important to work with a clean F CM with low background noi to obtain high-quality, reproducible data. Moreover, veral blanks should be run before analysis to check whether the FCM and the reagents are clean or not (e “Blank and reference”).
U maximum voltage for the green fluorescence photo-multiplier tube (PMT) at which no electronic
or lar noi is detected. This can be obtained by running freshly prepared Milli-Q water as sample and increasing the voltage for the green PMT until noi is detected; the maximum voltage that can be ud is just below this. In some instances, the machine may em to be clean but after running a stained blank high levels of noi are obrved. Try running TE-buffer for some time, followed by another stained blank to check. If still dirty, a uful remedy can be to purge (prime) a few times and clean once more. Once the FCM is clean and ready for u, try to analyze the samples in one ries, not interrupted by analysis of other organisms and u of other dyes. Bacterial enumera-tion can also be done from the same sample using a slightly different tting and staining protocol (Marie et al. 1999b) with no interference for virus counts. In ca of a high-event-rate sample (i.e., >1000 events s–1), rin shortly with TE-buffer or Milli-Q before the next sample.
Sample dilution—Typically, samples need to be diluted before analysis to minimize electronic coincidence of the virus particles (i.e., two or more virus particles pass the analysis window of the lar simultaneously, reducing accuracy of the analysis). For virus samples, this coincidence is minimized at event rates <1000 events s–1(Marie et al. 1999b). Salts prent in the water samples can strongly interfere with the efficiency of the stain SYBR Green I, resulting in inaccurate quantification of the virus. Conquently, the final dilution factor should be greater than 10-fold, wit
h a sample volume ≥50 µL ud for the dilution. For each sample, a rial dilution of three to four different dilutions of 500 µL final volume is usually optimal to obtain an event rate within 200–800 events s–1). Subquently, the rest of the sam-ples can be analyzed using this optimal dilution factor.
孔庙旅游攻略Sample staining—The samples are stained with SYBR Green I at a final concentration of 0.5 ×10–4of the commercial stock (i.e., add 5 µL working stain solution to 500 µL sample). The samples are then incubated at 80°C for 10 min in the dark, fol-lowed by a cooling period at room temperature in the dark for 5 min before analysis. Heating of fixed samples significantly enhances the staining efficiency of virus (Brussaard 2004; Marie et al. 1999a).
Blanks and references—Control blanks, consisting of TE-buffer with autoclaved 0.2-µm-filtered (or 30-kDa ultrafil-tered) sample at the same dilution factor as the natural sam-ples, should be ud before F CM analysis of the samples.
F iltering natural sample through a 0.02-µm pore-size filter instead of autoclaving is not advid, as this may generate sub-stantial background noi.
Blanks are diluted, stained, and procesd identically to the samples. Very low coincidence (0–15 ev
ents s–1) and background fluorescence levels should be detected before proceeding with sample analysis. Blanks ideally show a total amount of 400–1100 events in 1 min of acquisition at a flow rate of ca. 40 µL min–1. During the analysis, always add one to two blanks to every batch of samples to monitor whether the noi level stays low.
An internal reference can be ud not only to normalize the fluorescent signal of the stained virus populations, but more importantly to detect deviations of the F CM from standard behavior. Highly diluted and well-mixed fluorescent micros-pheres (F luoSpheres carboxylate modified yellow-green fluo-rescent microspheres; 1.0 µm diameter; Invitrogen, Molecular Probes; F8823; stored at 4°C) may be ud as reference. An ini-tial brief sonication of the primary stock (1% vol/vol, storage at 4˚C) is recommended to disrupt the aggregates. Working bead solutions are then prepared by diluting the primary stock in sterile Milli-Q water (i.e., add 10 µL stock in 2.5 mL Milli-Q water) every day.
Acquisition and data analysis—The appropriate ttings for detection of stained virus particles are specific for each FCM. Fluorescence and scatter signals are collected on a logarithmic scale (4-decade dynamic range) for best results. The trigger for detection is t on green fluorescence, and data are acquired on a dot plot displaying green fluorescence versus side scatter signal (Fig.2). Com
mercial benchtop FCMs come with a cer-tain minimum threshold. This standard instrument threshold level (typically 52 for BD-FACScalibur) should be ud during acquisition of the data.梦见试衣服
新鲜感
A medium flow rate between 30 and 50 µL min–1is ade-
quate to detect virus. F CMs with a sample injection port (e.g., BD-F ACScalibur) should have the outer sleeve cleaned between samples to prevent cross-contamination (wipe with Kimwipes®tissue). Samples should be mixed by hand before analysis, as vortexing may result in decay of virus (redu
ction of 15% for natural coastal awater, data not shown). Allow the flow rate to stabilize before analyzing the sample. Acquisi-tion time is typically 1 min.
植物生长的过程Data analysis of the raw data collected in list-mode files can be performed using a wide array of software (either supplied with the FCM or freeware from the internet; e.g., CytoWin or WinMDI). For optimal reproducibility and to include the very low green fluorescent virus particles in the data analysis, the gating should always be t to include all the particles (Fig. 2). Importantly, virus counts in the sample should be corrected for particles counted in the blanks (Fig.3) before calculating virus concentrations.
Asssment
Staining—FCM analysis of the stained aquatic virus gener-ally discriminates two or three viral subpopulations (V1–V3) with different green fluorescence properties (Fig.4). A fourth viral subpopulation (V4) may be obrved (Fig.5), commonly reprenting large dsDNA algal virus (Brussaard et al. 2000; Jacquet and Bratbak 2003). Although most of the bacterio-phages (i.e., virus infecting bacteria) are thought to be included in the lower fluorescent viral subpopulations (V1 and V2 windows, Fig. 4), it was recently found that some eukaryotic algal virus displayed simil新鲜蚕豆的做法
ar low fluorescence upon staining (Brussaard and Martínez Martínez 2008). Similarly, some pro-and eukaryotic algal virus were also found in the V3 window (Brussaard et al. 2000). F urthermore, the level of nucleic acid–specific fluorescence is not indicative of the viral genome size. There was no linear relationship between the viral genome size and green fluorescence properties upon staining with a nucleic acid–specific stain (Brussaard et al. 2000).
SYBR Green I has a strong affinity for dsDNA but can also stain ssDNA and RNA, according to the manufacturer (Invit-rogen). Several tests using various types of virus indicated that the ssDNA and RNA virus can be stained with SYBR Green I (Brussaard et al. 2000). Nevertheless, some RNA-virus populations may not be fully parated from the background noi fluorescence; using other acid-specific dyes such as SYBR Green II (higher quantum yield when bound to RNA than to dsDNA) or SYBR Gold did not improve the detection of the virus (Brussaard et al. 2000; Brussaard 2004).
SYBR Gold, a fluorescent dye, detects DNA and RNA and is more nsitive than SYBR Green I and can also be ud as an alternative of SYBR Green I for FCM detection of virus (Chen et al. 2001). However, FCM data revealed significantly higher counts of virus stained with SYBR Green I than with SYBR Gold (Brussaard 2004). Thus, SYBR Green I ems best for opti-Fig. 3.Cytogram of SYB
跟岗实习报告R Green I–stained blank (using autoclaved 0.2-µm pore-size or 30-kDa prefiltered awater instead of natural sample) according to protocol described herein (all events obtained plotted, i.e., a total of 840, of which 222 were in the window ud to discriminate virus). The diagonal streak of dots outside and on the right side of the virus window is due to the TE-buffer in combination with the fluorescent dye (SYBR Green I). r.u., relative units.
Fig. 2.Cytogram of SYBR Green I–stained virus in typical natural aquatic sample according to protocol described herein (10,000 events plotted). For optimal reproducibility and to include the very low green fluorescent virus particles in the data analysis, the gating should always be t to include all the particles. r.u., relative units.
mal staining and detection of virus in aquatic environ-ments. It might be uful, however, to test whether other flu-orescent stains or combination of stains can improve detec-tion when working with specific virus.
Reproducibility—A critical question for the FCM ur is how reproducible the analysis is and how rep
rentative of the “cor-rect” concentration. Usual practice is to include replicate counts in a random order. Standard deviations should be smaller than 5%. Samples should possible be run in small batches (i.e., 6–10 samples) to prevent poor reproducibility due to virus decay in thawed samples. Once thawed, the samples can be stored at 4°C for at most a few hours. Refreezing and reanalysis of samples must be avoided due to extensive loss in virus counts.
Accuracy is improved by regular calibrations of the sample flow rate. Weighing the sample before and after a known time period of running at one of the flow rates provides good esti-mates of the flow rate. However, this cannot be achieved when on board a ship. Instead, preweighed and aled tubes con-taining Milli-Q water can be ud as an alternative, and the flow rate can be determined once the tubes are weighed back in the laboratory. Another rough estimate of the flow rate while on board can be obtained using back-pipetting: a known volume is dispend in the tube, the remaining volume is back-pipetted after the run, and the actual volume taken up by the FCM can be estimated by dividing change in volume over time. Running a sample of fluorescent beads of known con-centration for determination of flow rate is not advid, since this may be unreliable due to clumping of beads.
白醋泡鸡蛋的实验Comparison of FCM versus EFM counts—A large data t (n= 259, Table1) from distinct marine envi
ronments was ud to compare viral counts obtained by FCM with EFM (using the protocol of Hennes and Suttle 1995). Overall, total virus counts ranged from <1 to 200 ×106mL–1, with highest counts in Southern North Sea (e.g., 107–108virus mL–1). Linear least-squares regression analysis indicated a strong correlation between FCM and EFM counts (FCM = 1.08 ×EFM + 0.65, r2= 0.80, n= 259). Regression slopes and intercept values were not significantly different from a 1:1 regression line with a slope of 1 and an intercept of 0 (slope: t-test = 0.143, P= 0.886; intercept: t-test = 0.069, P= 0.945).
Additionally, regression slopes ranged from 0.97 to 1.70 and were not significantly different between the environ-ments (analysis of variance [ANOVA] on ranks, P > 0.05). Highest slope values were found for North Atlantic and Curaçao samples. The deep samples (>500 m, n= 8) are likely to explain this result for North Atlantic; ratio of FCM to EFM of tho samples are high and ranged from 4 to 6 (2500–4350 m, n= 4). The high slope value for Curaçao samples is likely due to the small number of samples leading to a nonsignifi-cant regression (r2= 0.36, P < 0.28, n= 5). Coastal and offshore marine samples displayed similar regression slope values (Table 1). Moreover, the depth of sampling did not influence regression slopes (Table 1). In the Arctic, samples were col-lected over a asonal cycle at different stations, but no a-sonal and/or spatial trends were obrved in the FCM versus EFM regressions (Table2).
Fig. 5.Cytogram of SYBR Green I–stained virus in natural aquatic sam-ple according to protocol described herein (10,000 events plotted). A fourth subpopulation with enhanced side-scatter signal may be obrved. This subpopulation, V4, commonly reprents large dsDNA algal virus. r.u., relative units.
Fig. 4.Cytogram of SYBR Green I–stained virus in typical natural aquatic sample according to protocol described herein (10,000 events plotted). Virus subpopulation with lowest green fluorescence is named V1, with midlevel fluorescence V2 and highest fluorescence V3. r.u., rela-tive units.
