Samples were prepared by passing 10-L influent wastewater through a positively charged 2″ ViroCap filter (Scientific Methods, Granger, IN, USA) using a peristaltic pump. ViroCap filters have an average pore size of 2–3 µm and contain glass microfibers coated with alumina nanofibers (Karim et al. 2009 (link)). After filtration, the filters were stored at 4 °C for up to 4 h and then eluted by adding 100-mL sterile eluent (1.5% beef extract [Becton, Dickinson and Company, Franklin Lakes, NJ, USA], 0.05 M glycine [TCI, Tokyo, Japan], pH 9.5) to the filter inlet. After a 30-min filter contact time, the eluate was recovered via the filter outlet. The recovered eluate (pH 8.5–9.5) was immediately pH-adjusted to 7.0-7.5 using 1 M HCl and, if the pH was over-adjusted, 1 M NaOH. The pH-adjusted filter eluate (100 mL) was stored at − 20 °C until seeding and secondary concentration and is referred to here as the primary concentrate.
Beef extract
Manufactured by BD
Sourced in United States
Beef extract is a laboratory product used to prepare culture media for the growth and cultivation of microorganisms. It is a concentrated liquid or powder derived from the soluble components of beef. Beef extract provides a source of nutrients, including proteins, peptides, amino acids, and other essential growth factors, to support the growth of a variety of microorganisms in a controlled laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by BD (7 mentions)
Peptone, by BD (5 mentions)
Sodium chloride (nacl), by Fujifilm (2 mentions)
Casamino acid, by BD (2 mentions)
Proteose peptone, by BD (2 mentions)
Hipolypepton, by Fujifilm (1 mentions)
Agar powder, by Fujifilm (1 mentions)
Hyaluronic acid, by Fujifilm (1 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions)
Sodium ascorbate, by Fujifilm (1 mentions)
Polysorbate 80, by Fujifilm (1 mentions)
Mrs medium, by BD (1 mentions)
Srcl2, by Fujifilm (1 mentions)
K2hpo4, by Fujifilm (1 mentions)
L cysteine hydrochloride, by Fujifilm (1 mentions)
Na2so4, by Thermo Fisher Scientific (1 mentions)
Nutrient broth, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Biolife (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Nahco3, by Merck Group (1 mentions)
17 protocols using beef extract
1
Wastewater Virus Concentration Protocol
2
Concentration and Extraction of Viral Samples
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The prepared sample was processed by Method 2 (M2) as shown in Figure 1 b and adapted from Murthi et al. [32 (link)]. First, the sample was centrifuged (15 min, 6700× g, 4 °C) to separate the liquid and solid portions for separate processing (Figure 1 b). The liquid fraction was passed through a positively charged 47-mm flat disc ViroCap filter (Scientific Methods, Granger, IN, USA) via membrane filtration. The filter was eluted in a 50 mL conical with addition of 10 mL eluate (1.5% beef extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 0.05 M glycine, pH 9.5 solution) and 4.5 g sterile glass beads (3 mm diameter), followed by vortexing (5–10 min). The sample was purified with addition of 5 mL Vertrel as described previously and stored at 4 °C for plaque assay within 24 h.
The solid fraction was eluted with 100 mL of 10% beef extract, 0.05 M disodium hydrogen phosphate (Millipore Corp, Bedford, MA, USA), 0.00625 M citric acid, pH 7.0 solution, by shaking (30 min, 350 RPM). The sample was then sonicated (3 min, on ice), followed by centrifugation (15 min, 6700× g, 4 °C). The pellet was discarded, while the supernatant was secondary concentrated by skimmed-milk flocculation and extracted with 5 mL of Vertrel, as described previously. The final sample was stored at 4 °C for plaque assay within 24 h.
The solid fraction was eluted with 100 mL of 10% beef extract, 0.05 M disodium hydrogen phosphate (Millipore Corp, Bedford, MA, USA), 0.00625 M citric acid, pH 7.0 solution, by shaking (30 min, 350 RPM). The sample was then sonicated (3 min, on ice), followed by centrifugation (15 min, 6700× g, 4 °C). The pellet was discarded, while the supernatant was secondary concentrated by skimmed-milk flocculation and extracted with 5 mL of Vertrel, as described previously. The final sample was stored at 4 °C for plaque assay within 24 h.
3
Microbial Strain Cultivation and Isolation
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The NBRC100911 strain of Staphylococcus epidermidis (S. epidermidis) and the NBRC100910 strain of Staphylococcus aureus (S. aureus) were obtained from the National Institute of Technology and Evaluation (Tokyo, Japan). S. epidermidis and S. aureus strains were spread on nutrient agar (5 g/L sodium chloride [Wako Pure Chemical Corporation], 5 g/L beef extract [Becton Dickinson, Franklin Lakes, NJ, USA], 10 g/L hipolypepton [Wako Pure Chemical Corporation], and 15 g/L agar powder [Wako Pure Chemical Corporation] before autoclaving) and grown overnight at 37°C. The JCM6218 strain of D. acidovorans was obtained from the Japan Collection of Microorganisms (Ibaraki, Japan). The D. acidovorans JCM6218 strain was spread on nutrient agar and grown overnight at 27°C.
4
Degraded Hyaluronic Acid Halo Assay
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Degraded HA halo assays were performed as previously described with some modifications (Smith and Willett, 1968 (link); Oiki et al., 2019 (link)). Agar plates consisting of 0.5% peptone (Becton Dickinson, Franklin Lakes, NJ, USA), 0.3% beef extract (Becton Dickinson, Franklin Lakes, NJ, USA), 1% agar, 1% bovine serum albumin (Nacalai Tesque, Japan), and 0.4 mg/ml hyaluronic acid (FUJIFILM Wako Pure Chemical Corporation, Japan) were prepared. Bacterial strains were grown in BHI medium, washed with fresh BHI, and resuspended in fresh BHI at the indicated optical density at 600 nm (OD600). One microliter of bacterial suspensions at the indicated concentration was spotted on the peptone agar, followed by overnight incubation at 37°C in a 5% CO2 atmosphere. The plates were then flooded with 2 N acetic acid and allowed to stand for 10 min. Clear regions demarcate zones of HA degradation.
5
Biochemical Reagents and Media
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Glucose, K2HPO4, Polysorbate 80 (Tween 80; for biochemistry grade), sodium ascorbate (Wako special grade), l -cysteine hydrochloride, CsCl, H2O2, and SrCl2, were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). These reagents without grade description were of guaranteed grade. Casein peptone and yeast extract were from Nihon Pharmaceutical Co. Ltd. (Osaka, Japan). MRS medium and beef extract were from Becton, Dickinson and Co. (Franklin Lakes, NJ, USA). Bis–Tris was obtained from MP Biomedicals (Santa Ana, CA, USA).
6
Reagents and Chemicals Sourcing Protocol
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All chemicals were of reagent-grade quality or higher and used without further purification; solvents were used as received. Ethyl acetate (EtOAc), hydrogen peroxide (H2O2), dimethyl sulfoxide (DMSO), HBr, cholesterol, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), NaHCO3, KH2PO4, Na2HPO4, and other salts were purchased from Sigma Aldrich, Steinheim, Germany. Yeast extract, NaCl, and agar were purchased from Biolife, Milan, Italy. Methanol (MeOH), glycerol, Nutrient Broth (NB), and Na2SO4 were purchased from Fisher Scientific, Loughborough, UK. Tryptone, peptone, Tryptic Soy Broth (TSB) were purchased from Torlak, Belgrade, Serbia. KCl and beef extract were purchased from Becton Dickinson, New Jersey, US. Hexane was purchased from J. T. Baker, Deventer, The Netherlands. MgSO4∙7H2O was purchased from Acros Organics, Geel, Belgium. Instant Ocean® Salt was purchased from Instant Ocean, Blacksburg, US.
7
Phenotypic Characterization of E. coli O157:H7
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Curli phenotype was determined for all strains of E. coli O157:H7 and a K-12 strain. Cultures were grown in LBG or M9GT broth at 37°C for 18 h, and then plated onto Congo red indicator (CRI) agar composed of 10 g/L casamino acids (Becton Dickinson), 1 g/L yeast extract (Becton Dickinson), and 20 g/L agar (Thermo Fisher Scientific, Fair Lawn, NJ) with 20 mg/L of Congo red (Polysciences, Inc., Warrington, PA) with and without 10 mg/L Coomassie Brilliant Blue G-250 (MP Biomedicals, Inc., Solon, OH; Uhlich et al., 2006 (link)). LB agar supplemented with 200 mg/L calcoflour white stain (Fluka Analytical), a UV fluorescent stain that binds to cellulose and chitin, plates were used to identify cellulose production (Hammar et al., 1995 (link); Uhlich et al., 2006 (link)). Motility was determined using motility test medium (MTM; 3 g/L beef extract (Becton Dickinson), 10 g/L pancreatic digest of casein (Becton Dickinson), 5 g/L NaCl, and 4 g/L agar) stab cultures grown 18 h at 37°C (Atlas and Parks, 1993 ).
8
Preparation of Glucose-free MRS Medium for Panax ginseng Root Microbiome
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Panax ginseng roots were purchased from a local grocery store in Korea. Chiro-inositol and pinitol were provided by Amiocogen Co., Ltd. (Korea). Acetonotrile, methanol, and water were purchased from J. T. Baker® (USA). Ginsenoside standard Rb2, Rc, and Rd were purchased from BTGin Co., Ltd. (Korea). Yeast extract, proteose peptone, beef extract and deMan, Rogosa, Sharp (MRS) media were purchased from Becton, Dickinson and Company (BD) (USA). Glucose-free MRS was formulated according to the manual of microbiological culture media (DifcoTM and BBLTM Manual 2009 ; Ku et al. 2009 (link), 2011 (link)). The glucose-free MRS contained 10 g proteose peptone, 10 g beef extract, 5 g Yeast extract, 1 g polysorbate 80, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulfate, 0.05 g manganese sulfate, and 2 g dipotassium phosphate in 1 l of distilled water. The pH of the broth was 6.5 ± 0.2 at 25 °C and 2 % of agar was added if needed.
9
Determining Paenibacillus wasatchensis Growth Substrates
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DeMan, Rogosa, and Sharpe (MRS) broth (Hardy Diagnostics) supplemented with 1% ribose (Sigma Aldrich Inc.) was used to prepare working cultures of Pa. wasatchensis by incubation at 30°C for 4 d. A carbohydrate-restricted MRS (CR-MRS) was also prepared as described previously (McMahon et al., 2020) (link) using 10 g of protease peptone No. 3 (EMD Chemicals Inc.), 10 g of beef extract (Becton Dickinson), 5 g of yeast extract (Becton Dickinson), 5.0 g of sodium acetate (Sigma Aldrich Inc.), 1.0 g of Tween-80, 2.0 g of ammonium citrate (Sigma Aldrich Inc.), 2.0 g of dipotassium phosphate (Fisher Scientific), 0.1 g of magnesium sulfate (Fisher Scientific), and 0.05 g of manganese sulfate (Avantor Performance Materials Inc.) made up to 1 L using distilled water. This CR-MRS plus 0.5% of either sodium gluconate (Tokyo Chemical Industry Ltd.), sodium lactate (Sigma Aldrich Inc.), xylose (Sigma Aldrich Inc.), or ribose was used for determining the growth of Pa. wasatchensis strains in different substrates. For gas production studies, CR-MRS plus 1% mixtures containing gluconate and ribose at 100:0, 70:30, 50:50, and 30:70 ratios were used. For studying gas production with gluconate as the only substrate, CR-MRS plus 0.4% to 1.0% gluconate was used.
10
Bacterial Motility Assays Protocols
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The swarming assay plates contained 0.5% (w/v) glucose (Amersco), 0.5% (w/v) agar (Amersco), and 0.8% (w/v) nutrient broth containing 3.0 g/L beef extract (BD) and 5.0 g/L peptone (BD), supplemented with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Amersco) and 500 μg/ml carbenicillin (Sigma). Moreover, 3 μl of overnight liquid cultures of each test strain were inoculated on a swarming plate. After the plate was incubated at 37°C for 24 h, the longest swarming distance of the cells was measured. The swimming assay plates contained 1.0% (w/v) tryptone (Scharlab), 0.3% (w/v) agar (Amersco), and 1.0% (w/v) NaCl (J.T. Baker), supplemented with 500 μg/ml carbenicillin and 0.5 mM IPTG. Moreover, 3 μl of overnight liquid culture of each strain was inoculated on the plate and incubated at 37°C for 24 h; the diameters of the swimming zone were then measured.
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