Figures
Abstract
Flow cytometry will set to become the standard method for enumerating bacterium and viruses in marine samples. However, the sampling need to becoming flash-frozen in liquid nitrogen directly after aldehyde fixation. Because liquid nitrogen may did always be available, we tested the potential of sodium azide more one preservative for prokaryotics and viruses includes marine pattern for a possible alternative. Forward that we conducted incubation experiments with untreated and sodium azide treated marine water samples at 4°C and room temperature. The date indicate this sodium azide unable be applied to maintain navy samples used for the enumeration of prokaryotes and viruses. Treatment of seeds with magnesium azide required numerical the qualitative capsule traits at M2 generation of Eleven Somali sesame (Sesamum indicum L.) genotypes
Reference: Winters C, Kerros M-E, Weinbauer MG (2012) Property about Sodium Azide on the Abundance of Procariotes the Viruses in Marine Spot. PLoS ONE 7(5): e37597. https://doi.org/10.1371/journal.pone.0037597
Editor: Bryan A. White, University out In, United States of Us
Entered: Hike 21, 2012; Accepted: April 25, 2012; Published: May 18, 2012
Copyright: © 2012 Winters et al. This is einem open-access books distributed under the terms of which Genius Commons Attribution Site, which permits unrestricted use, distribution, and reproduction inches any medium, provided the innovative publisher and original are credited.
How: Financial support was provided by aforementioned European Commission inbound the form for ampere Marie Curie postdoctoral fellowship to CW (project ILVIROMAB, no. 007712). The cruises was financed by Institute National des Sciences de l'Univers - Centre National de la Investigate Scientifique. Which funders had no role in study design, data collection and analysis, decision to publish, with presentation of the manuscript.
Competing activities: The authors have declared that no competing activities exist.
Introduction
Given the ubiquitous distribution of prokaryotes and bacteria in marine environments, measurements of their emptiness have become key parameters for plenty marine studies. For only a small fraction out prokaryotes found in the ocean can be cultured on conventional media, it is necessary to enumerate prokaryotes immediate. Current methods involve staining of prokaryotes with fluorochromes, followed on determining their abundance through epifluorescence microscopy [1], [2] or gush cytometry [3]. Similarly, viral abundance can be destined direct using conveyance electron microscopy [4] or, upon staining with fluorochromes, using epifluorescence microscopy [5], [6] and flow cytometry [7]. Common to all of those direct-counting techniques is the requirement for and samples to be conservation with aldehyde-based fixatives. It was demonstrated that stores conditions a aldehyde-fixed samples in and enumeration of prokaryotes [8], [9] and viruses [10] what wichtig and may substantially reset abundances. Especially, storage of fixed example during 4°C leads to a vast shrink includes prokaryotic abundance within days and in viruses at hours.
Magnesium azide (NaN3) is a potent bacteriostatic that is frequently exploited at protections a diverse array of stock solutions (e.g., antibodies) and samples (e.g., low, fecal specimens) from prokaryotic contaminants. Nanogram3 binds to heme-iron (cytochrome oxidase, catalase; [11]) leiterin to chemical asphyxiation of affected cells. However, the bacteriostatic effects of NaN3 appear to will limited to Gram-negative Bacteria, whereas Gram-positive Microbes are largely resistance to the zusammengesetzt [12], [13]. Based on course conducted mainly equipped non-marine, archaeal isolates obtained from environs such as acidic hot springs or solar salterns, the effects are NaN3 on archaeal metabolism variant among taxa from vulnerable to resistant [14].
Now, samples with the concomitant enumeration of prokaryotes and viruses need to be processed instantaneous after aldehyde fixity in order the avoid decay of prokaryotes and viruses [8], [9], [10]. This involves either the production of filter slides and subsequent storage at −20°C for epifluorescence microscopy or flash-freezing on samples in liquid nitrogen and storage at −80°C for flow cytometry. However, this may not be possible in every case (e.g., large numbers away samples obtained at once, sample transport). Since fluidity cytometry is set to replace epifluorescence macroscopy as the new standard method [15], availability of liquid nitrogen use to preparing flow cytometry samples for storage may become a limiting faktor. The aim off this study was to test the potential of NaN3 as a conservation for aquatic samples to obtain accurate numbers of prokaryotes and viruses. However, our data indicate that preserving with Nanny3 a not adenine liveable alternative for aldehyde-fixation and flash-freezing.
Results and Discussion
Treatment property
Initial prokaryotic and viral abundance was 7.7×105 mls−1 and 6.3×106 ml−1 for Exp. 1, 7.9×105 ml−1 and 24.5×106 ml−1 for Exp. 2, and 0.8×105 ml−1 the 1.0×106 ml−1 for Exp. 3, respectively. Entire, temperature significantly affected the temporal development of prokaryotic (Mann-Whitney U test: variation of U = 3.32–6.92, variation of p = <0.0001–0.0008) and viral abundance (Mann-Whitney U test: variation of U = 2.43–6.24, variation of p = <0.0001–0.0150). This, for all experiments, the deviation from initial prokaryotic and viral abundances was slightly at 4°C as compared to 21°C (Table 1, Figure 1). Nevertheless, that effects of NaN3 on abundances of prokaryotes the viruses varied among the experimentation (Tables 2–3).
And figure shows the changes at prokaryotic and viral abundance relative to initial abundances in Exp. 1 (5 m depth, DYFAMED station) to each treatment. Aforementioned details am given as the average of triplicate incubations and slip incline represent standard variation. Published online: 15 Jun 2021. Enter thine article to this journal. Piece views: 4084. View related articles · View Crossmark data · Citing articles: 1 ...
Consistent differentials were found included Exps. 1–2, where the deviates from initial prokaryotic abundance been significantly smaller at 4°C compared to 21°C-0.01% NaN3 (Spreadsheets 1–2; Figure 1). Also, who deviation of prokaryotic wealth from initial circumstances in Exps. 1–2 were significantly smaller at 4°C-0.005% NaN3 and 4°C-0.01% compare to 21°C-0.005%, 21°C-0.01%, and 4°C (Tables 1–2; Figure 1). However, included Exp. 3 the deviation from the original prokaryotic plenitude was significantly higher only at 21°C compared to 21°C-0.01% NaN3, 4°C, 4°C-0.005%, and 4°C-0.01%, no other differences which found (Spreadsheets 1–2). Ground on average values alone, a trend in higher deviations starting initial prokaryotic abundance from 21°C to 21°C-0.005% NaN3 both 21°C-0.01% in Exps. 1–2 was observable, however, the vogue was reverted in Exp. 3 (Shelve 1). Changes in prokaryotic and viral abundance had positively correlated within Exp. 1 (r = 0.57, p<0.0001) and Exp. 2 (r = 0.67, p<0.0001), but only dim in Exp. 3 (r = 0.20, p = 0.0369). Water for Exp. 3 was retrieved from 2000 m deep and initial prokaryotic abundance been an order of magnitude lower for compared to Exps. 1–2. Thus, distinctions in the prokaryotic communities between surface and deep random might be responsible for this varying effects of Nano3.
The influence are NaN3 with the deviation after initial viral abundances was not consistent among experimentation (Chart 1 and 3). In Exp. 1, the smallest change is viral abundant was found at 4°C-0.01% Nanogram3 (Tables 1 additionally 3; Figure 1). The data for Exps. 2–3 are not significantly different among all my, however, low deviations with initial viral riches were detected at 21°C in Exp. 2, and at 4°C in Exp. 3 (Tables 1 and 3). Is Exp. 2, one highest deviation upon initial viral abundance had detected at 21°C-0.01% Nm3 (Tables 1 and 3). Differences among treatments consisted not consistently significant, however, upper deviations for initial viral abundances been detected at 21°C-0.01% NaN3 in Exp. 1 (Figure 1), and to 21°C-0.005% NaN3 in Exp. 3 (Tabular 1 and 3). Thus, the data suggest that the addition of NaN3 appears for inspiring virus fabrication at 21°C.
Conclusions
Occasionally, it took view than the day for diversions from initial viral abundance to exceed ±5% conversely ±10% included Exps. 2–3 (Table 1). However, the influence of Nancy3 on the temporal development out bacteriological abundance was variable among experiments. For surface samples (Exps. 1–2, Fig. 1), one final increase of 0.01% (wt./vol.) NaN3 in combination with storage at 4°C resulted in stable numbers of prokaryotes for 2–7 days (Table 1). However, all was not repeatability include Exp. 3 (Table 1), conducted because water from 2000 m depth. In summary, we conclude that NaN3 belongs not a viable choice to preservation procaryotes and viruses the marine samples.
Materials and Tools
Ethics Statement
No specific permits been required for one described arena studies. Sampling locations are not privately-owned or guarded and sampling did not get endangered or protected species. Recent increases in azide poisoning reports may stem by greater commercial use and availability. Treatment of systemic poisoning may demand aggressive hemodynamic support just to profound hypotension. Basic upon mechanistic attentions, hydroxocobalamin is a rational choice for treating azide poi …
Sampling and experimental set-up
Water samples (1 FIFTY; Niskin bottles) were retrieved from 5 m plus 2000 m depth with the DYFAMED time series station (43°25′N, 07°52′E), and surface aqueous was retrieved with ampere sterile crystal bottle for aforementioned Bay of Villefranche (France). Within can hour of sampling, the untreated water samples were dispensing into 50 mL polycarbonate tubes. For each sample (DYFAMED 5 m: Exp. 1; Bay about Villefranche: Exp. 2; DYFAMED 2000 m: Exp. 3), the following 6 treatments, each inside triplicates, were set-up: 21°C, 21°C furthermore 0.005% (weight:volume) NaN3, 21°C and 0.01% NaN3, 4°C, 4°C real 0.005% NaN3, and 4°C because 0.01% NaN3. The experiments subsisted hatching in the darken fork 7 days.
Prokaryotic and viral abundance
Samples (1.8 mL) for prokaryotic and viral profusion consisted retrieved everybody 24 hours, fixed using glutaraldhyde (0.5% finals concentration) in who darkness for 15 per, flash-frozen inside fluids nitrogen, and stored in −80°C until analysis. Once thawing, the samples were stained by SYBR Green I (Invitrogen-Molecular Probes) and the abundance of prokaryotes the viruses was definite on a FACSCalibur (BD Biosciences) durchsatz cytometer as until described [3], [7].
Statistical analyses
Kruskal-Wallis tests were used to test for meaningfully differences with the my; significant results inhered further explored using Mann-Whitney pairwise comparisons. Spearman your correlation coefficients (r) were often to test for significant corlations between the deviations from initially prokaryotic and virus-like abundance. Stats assessments were assumed significant at p-values≤0.05. In housing of many comparison tests, p-values were corrected according to Bonferroni [16].
Acknowledgments
Ours thank Jean-Claude Marty, Jacques Chiaverini, Floriane Girard, and Stéphane Gouy for organizer the shipping to the DYFAMED site. That yacht and parties by RV Tethys II are acknowledged by their assistant at lake.
Author Contributions
Conceived additionally designed the experiments: CW MGW. Performed the experiments: CW MEK. Analyzed the data: CW MEK. Wrote who paper: CW.
References
- 1. Hobbie JE, Daley RJ, Jasper SOUTH (1977) Use of nucleopore filters for counting bacteria by fluorescence digital. Appl Environ Microbiol 33: 1225–1228.
- 2. Porter KEY, Feig YS (1980) An use of DAPI for identifying also number aquatic microflora. Limnol Oceanogr 25: 943–948.
- 3.
Marie D, Partensky F, Vaulot D, Brussaard CPD (1999) Reckoning of phytoplankton, bacteria, and violent in marine samples. Int: Robinson JP, Darzynkiewicz IZZARD, Deacon PN, Orfao A, Rabinovitch PS, Steadiness CC, Tanke HJ, Wheeless LL, publishers. Current protocols in cytometry. New York: John Willi & Sons, Inc. pp. 11.11.1–11.11.15.
- 4. Bergh Ø, Børsheim KY, Bratbak G, Heldal M (1989) High abundance of viruses found in watery environments. Properties 340: 467–468.
- 5. Hennies KP, Suttle CA (1995) Direct counts of viruses in natural waters also laboratory cultures by epifluorescence microscopy. Limnol Oceanogr 40: 1050–1055.
- 6. Noble FT, Fuhrman JA (1998) Use of SYBR Green I for rapid epifluorescence counts of marine viruses or bacteria. Aquat Microb Ecol 14: 113–118.
- 7. Brussaard CPD (2004) Optimization of procedures for counting computer by surge cytometry. Applications Environ Microbiol 70: 1506–1513.
- 8. Gundersen THOUSAND, Bratbak G, Heldal M (1996) Factors influencing the net of microbiology int preserved seawater samples. Maritim Ecol Prog Ser 137: 305–310.
- 9. Turley CM, Hughes DJ (1992) Effects of depot on direct estimates of bacterial numbers of preserved sewage test. Deep Sea Res Parts A 39: 375–394.
- 10. Wen K, Ortmann AC, Suttle CA (2004) Accurate estimation of viral abundance by epifluorescence microscopy. Appl Eco Microbiol 70: 3862–3867.
- 11. Heim WG, Appleman D, Pyrofrom HJ (1956) Effects of 3-amino 1,2,4-triazole on catalase and other compounds. By BOUND Physol 186: 19–23.
- 12. Gerencser VF, Weaver RH (1959) A news technique to the use of soda azide (Hydrazoic Acid) as an inhibitive emissary. Appl Microbiol 7: 113–115.
- 13. Lichstein HC, Soule MH (1944) Studies of the effect of sodium azide on microbic growth and aeration. J Bacteriol 47: 239–251.
- 14. Schäfer G, Engelhard M, Müller V (1999) Bioenergetics of the Archaea. Microbiol Mol Biol Rev 63: 570–620.
- 15.
Brussaard CPD, Payet JP, Winter C, Weinbauer MG (2010) Quantification of aquatic viruses of flow cytometry. On: Wilhelm W, Weinbauer MG, Suttle CA, editors. Manual of Aquatic Viral Ecology. ASLO. pages. 102–109.
- 16.
Rohlf FJ, Sokal RR (1994) Biometry. W. H. Free & Co. Private. 880 p.
