Individual strains of challenge microorganisms (Table 1 ) were grown to log-phase in Tryptone Soy Broth (TSB) and then diluted with the appropriate biofilm growth medium (BGM, Table 1 ) to approximately 1 × 105 cfu/mL. 7 mL aliquots of BGM were dispensed into each well of deep 6-well plates (BD Biosciences). Anodisc filters (25 mm dia., 0.2 μm pore size; Whatman) were carefully placed onto the support ribs within each well such that the BGM was only in contact with the downward-facing surface. Aliquots of microbial suspensions (0.1 mL aliquots of 1 × 105 cfu/mL suspension for single-species models; 45 μL of S. aureus and 5 μL of K. pneumoniae 1 × 105 cfu/mL suspensions for the polymicrobial model, Table 1 ) were pipetted onto the centre of the upper surface of each filter disc. The plate lid was replaced and the plate incubated at 35 ± 3°C. After 24 hours (Figure 1(a) ) filter discs were removed and rinsed by moving the filter backwards and forwards 10 times with forceps in 30 mL of 0.85% w/v saline to remove planktonic microorganisms and unattached matter. The filter disc-supported biofilms were then used immediately in either a simple biofilm model or a simulated wound polymicrobial biofilm model, to test various dressings and analyse their effects using multiple methodologies (Table 1 ).
Anodisc filter
Manufactured by Cytiva
Sourced in Germany, United Kingdom, United States
Anodisc filters are membrane filters designed for filtration applications in laboratories. They are made of anodized aluminum oxide and provide high porosity and flow rates. Anodisc filters are available in a range of pore sizes to accommodate different filtration requirements.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (24 mentions)
Sybr gold, by Thermo Fisher Scientific (3 mentions)
Sybr green 1, by Thermo Fisher Scientific (2 mentions)
Dmrbe trinocular, by Leica (2 mentions)
Recombinant dnase 1 rnase free, by Takara Bio (1 mentions)
Nuclepore track etch membrane, by Cytiva (1 mentions)
Dneasy powerwater kit, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Vectashield dapi, by Vector Laboratories (1 mentions)
Bz 9000, by Keyence (1 mentions)
Phosphate buffered saline (pbs), by Avantor (1 mentions)
Image pro software, by Media Cybernetics (1 mentions)
Dmrbe microscope, by Leica (1 mentions)
Vectashield antifade mounting medium h 1000, by Vector Laboratories (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Antifade solution, by Thermo Fisher Scientific (1 mentions)
Axiolab fluorescence microscope, by Zeiss (1 mentions)
Tianamp virus dna rna kit, by Tiangen Biotech (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
24 protocols using anodisc filter
1
Cultivation of Polymicrobial Biofilms
2
Enumeration of Viruses and Bacteria
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The counts of virus-like particles (VLPs) and bacterial cells were performed as previously described [20 (link),30 (link)]. Briefly, DNase-treated (RNase-free recombinant DNase I, Takara Bio, Kusatsu, Japan) and 100-fold diluted viral aliquots and bacteria aliquots were respectively passed through a 0.02-µm pore Anodisc filters (diameter, 25 mm; Whatman, Dassel, Germany) and 0.2-µm pore Nuclepore Track-Etch membranes (Whatman, Maidstone, UK). Filters were immediately stained with 25× SYBR Safe (Invitrogen, Carlsbad, CA, USA) before being incubated in the dark for 20 min. Dried filters were put onto glass slides with 30 μL anti-fade mounting medium, a mixture of 0.1% (vol/vol) p-phenylenediamine, 50% glycerin, and 50% PBS (0.13 mol/L NaCl, 7.0 mmol/L Na2HPO4, 3.0 mmol/L NaH2PO4), then VLPs or bacterial cells were enumerated using epifluorescence microscopy under blue light excitation. What needs to be stated is that specific bacterial probes have not been applied in bacterial counting. Thus, we are not 100% sure only bacteria cells were specifically counted on the track-etch membranes, which may slightly influence the accuracy of bacterial abundance (BA) and virus-to-bacterium ratio (VBR).
3
Outdoor Air Microbiome Sampling Protocol
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Outdoor air samples were collected on the rooftop of an academic building at Nanyang Technological University (Singapore), 20 m above the ground, for five consecutive days using methods described previously [14 (link)]. Briefly, air was collected using filter-based air samplers SASS3100 (Research International, Monroe, WA, USA) mounted with SASS bioaerosol electret filters (Research International). Air sampling was performed at 300 L·min−1 airflow rate at a height of 1.5 m above the floor at the rooftop balcony. The SASS filter was transferred to the lab for immediate processing or stored at −20°C prior to processing. For processing, the SASS filter was transferred to a sterile tube and washed with PBS-Triton X-100 in triplicate. Resultant washed solutions were filtered through 0.02 µm Anodisc filters (Whatman) and DNA extracted using the DNeasy PowerWater Kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. The extracted DNA including an extraction blank and sterile filter were sequenced using the HiSeq 2500 Illumina platform (Illumina, San Diego, CA, USA). All sequence data from the outdoor air study has been uploaded to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under project accession PRJNA436039. Details on the isolation and confirmatory identification of outdoor air fungi can be found in the supplementary material .
4
Sea Ice Core Sample Preparation
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To prevent contamination of samples, handling and processing the sea ice cores was conducted under a clean bench (Labogene Scanleaf Fortuna, Lynge, Denmark). Ice cores were cut individually into horizons ranging from 10 to 35 cm using a bone saw. To exclude sample contamination from sampling the surface of the ice core horizon was removed by a stainless steel grater. Afterwards, it was washed with 1 L of MilliQ to remove the particles that would eventually adhere. Each horizon was weighed before being melted in glass-preserving jars at room temperature and then concentrated onto Anodisc filters (47 mm, Whatman, Freiburg, Germany). All samples were treated with of 35% H2O2 (Roth, Karlsruhe, Germany, filtered over 0.2 µm Anodisc). After filtering the melt water the filter was overlaid with 40 mL H2O2 and incubated at room temperature overnight. Lastly, the H2O2 was drained and the filters were flushed with approx. 750 mL MilliQ water. To remove the adhering material, the filtration funnel was further flushed with 30% ethanol (VWR Chemicals, Darmstadt, Germany, filtered over 0.2 µm Anodisc) to reduce surface tension and thereby assure the concentration of all particles on the filter. The Anodisc filters were placed in glass petri dishes and dried at 30–40 °C in a drying cabinet (Memmert, Schwabach, Germany) overnight.
5
Quantifying Total Cell Numbers
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Total cell numbers were quantified via SYBR‐Gold (Invitrogen; CA, USA) staining. From each sampling site 4.75 ml of water was fixed with 0.25 ml 37% formaldehyde. One ml of an appropriate dilution of the fixed water sample (1:10 for samples from Neusiedler See; 1:100 for samples from the shallow alkaline lakes) was filtered on Anodisc filters (Ø 25 mm, pore‐size 0.2 μm, Whatman) and stained on a drop of SYBR‐Gold (Invitrogen, Lofer, Austria; 1:400× dilution of the stock) for 20 min. After drying, the filters were mounted on a microscopic slide under a drop of anti‐fading solution and analysed in a Nikon Eclipse 8000 microscope. Detailed information on the enumeration procedure can be found elsewhere (Riepl et al., 2011 ).
6
Quantification of Bacterial and Viral Abundance
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Samples for bacterial and viral abundance were transferred to 2 ml centrifuge tube, fixed immediately with glutaraldehyde (final concentration, 0.5%) and stored in darkness for 15 min. Fixed for 15 min, 0.8 ml samples were filtered onto 0.02 μm Anodisc filters (Whatman, Maidstone, United Kingdom). The vacuum pressure was about 20 kPa. The filters were placed on a drop of SYBR-Green-I solution (final concentration, 0.25%) and dyed in darkness for 15 min, according to Noble and Fuhrman (1998) (link). The slides with the fixed filters were completed in about 30 min and stored at −20°C. The counting of bacteria and viruses was made within 4 weeks. With the immersion oil (type A, Nikon), Virus and bacterial particles were counted at 1000 times magnification under 100 W mercury lamp for the epifluorescence illumination using an OLYMPUS BX41 microscope in the laboratory. At least 200 bacteria and viruses in each field were counted in at least 10 fields (Noble and Fuhrman, 1998 (link)).
7
Seawater Microbial Enumeration Protocol
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Seawater samples were fixed with 0.5% glutaraldehyde and stored at −80 °C until processed. Samples (1–2 ml) were filtered onto 0.2 μm pore-size polycarbonate filters and stained with DAPI Vectashield (Vector Labs., Burlingame, CA) for bacteria and sperm cell counts [32 (link)]. Samples (1 ml) were filtered onto 0.02 µm pore-size Anodisc filters (Whatman) then stained with SYBR Green I (Invitrogen, Carlsbad, CA) for virus-like-particle (VLP) counts [33 (link)]. Samples (5 ml) were filtered onto black 0.8 µm pore-size polycarbonate membranes and stained with DAPI Vectashield for protist enumeration. At least 200 cells from ten or more fields of view were counted by epifluorescence microscopy for bacteria and VLPs, while entire filters were examined for enumeration of protists.
8
Enumerating Bacteria and Viruses
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Representative samples for bacteria and virus counting (covering all sample types and the abundance range of the whole sample set) were additionally enumerated using epifluorescence microscopy. Samples from filter fractions >0.22 μm were centrifuged at 2,000 × g for 5 min to reduce the background fluorescence of cell debris. Virus filters were prepared after Suttle and Fuhrman (2010) . In brief, samples were diluted using 0.02 μm-filtered PBS buffer, vacuum-filtered onto 0.02 μm Anodisc filters (Whatman), stained with SYBR Green I for 15 min in the dark and mounted onto microscopic slides with 0.1% p-phenylenediamine as antifade solution. Bacterial cells were filtered onto 0.22 μm polycarbonate filters (Nucleopore, Track-Etch) as described by Lunau et al. (2005) (link) and stained with a SYBR Green I staining solution for 30 min as described by Pohlner et al. (2017) (link). A minimum of 300 viruses and 100 bacterial cells, respectively, were counted per filter in at least 15 randomly chosen counting grids at a 1,000× magnification (Leica DMRBE Trinocular, Leica Microsystems).
9
Viral Abundance Assessment by DNA Staining
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To assess viral abundance, the DNA staining method was used (Shibata et al., 2006 (link); Patel et al., 2007 (link)). Briefly, fixed subsamples (0.5–2 ml) were filtered using Anodisc filters (pore size: 0.02 μm, Whatman), and a 0.8-μm-pore size filter (mixed cellulose esters membrane filters) was used as a backing filter. Anodisc filters were air-dried and stained using SYBR Gold (Molecular Probes; stock solution diluted 1:400). After staining, the filters were air-dried again, and each filter was mounted between a slide glass and a coverslip using anti-fade mounting medium [0.1% (vol/vol) p-phenylenediamine]. Slides were kept at −30°C prior to counting. The slides were observed under an epifluorescence microscope (BZ-9000, KEYENCE) at 1,000× magnification. Ten fields and at least 200 virus-like particles (VLPs) were counted per sample. A blank was constructed with the same method but using ultrapure water to check for contamination from reagents or equipment.
10
Virus-Like Particle Quantification via Epifluorescence
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Representative samples covering all ecosystem types and the abundance range of the whole sample set were additionally counted using epifluorescence microscopy to validate VLP numbers based on flow cytometry. Filters for virus quantification were prepared following the standard protocol by Suttle and Fuhrman93 . In brief, samples were diluted using 0.02 μm-filtered phosphate-buffered saline (VWR, Darmstadt, Germany), filtered onto 0.02 μm Anodisc filters (Whatman, Maidstone, UK) by applying vacuum, stained with SYBR Green I (20 x concentration, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min. in the dark and mounted onto microscopic slides with 0.1% p-phenylenediamine (Acros Organics, Geel, Belgium) as antifade solution. A minimum of 300 VLPs were counted per filter in at least 15 randomly chosen counting grids at a 1000× magnification on a Leica DMRBE Trinocular (Leica Microsystems, Wetzlar, Germany) using the software EOS Utility v.2.10.2.0. Analysis of microscopic images was performed in ImageJ v.1.5.294 (link).
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