In initial experiments, four different culture media were evaluated to identify the culture medium that best facilitates filamentous growth of biofilm as microcolonies, each at three different concentrations: 1 × YPD (10 g L−1 Yeast extract [Oxoid; LP0021], 20 g L−1 Peptone [Oxoid; LP0037], 20 g L−1 Dextrose [Difco, 215530; Becton, Dickinson and Company]), 1× Mueller Hinton Broth (LAB114; LabM), 1 × M19 without NaCl (pH 6.1, 9.4 g L−1 Peptone [Oxoid; LP0037], 4.7 g L−1 Yeast extract [Oxoid; LP0021], 2.4 g L−1 Beef extract [Difco; 212610], 10 g L−1 Glucose monohydrate [Merck; 49161]) and 0.5 × Def4 (pH 7). The 0.5 × Def4 media were based on the 0.5 × Def4 reported by Correa et al. (2012 (link)) modified as follows: 0.65 g L−1 KH2PO4, 2.75 g L−1 (NH4)2HPO4, 0.31 g L−1 MgSO4 · 7H2O, 0.45 g L−1 citric acid, 10.20 mg L−1 Fe(III) citrate hydrate, 0.525 mg L−1 H3BO3, 2.50 mg L−1 MnCl2 · 4H2O, 2.10 mg L−1 EDTA · 2H2O, 0.26 mg L−1 CuCl2 · 2H2O, 0.44 mg L−1 Na2MoO4 · 2H2O, 0.44 mg L−1 CoCl2 · 6H2O, 1.30 mg L−1 Zn acetate · 2H2O, 1.0 g L−1 NaCl, 10 g L−1 MOPS, 20.0 g L−1 fructose, supplemented with 147.0 mg L−1 CaCl2 · 2H2O. YPD, LAB114 and M19 were tested at 1×, 0.5× and 0.25× concentrations, whereas Def4 was tested at 0.5×, 0.25× and 0.125×. The media were diluted in sterile ion‐free water prior to use to obtain the desired concentration.
Lp0021
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The LP0021 is a laboratory equipment designed for general laboratory use. It serves as a basic tool for various applications in the research and scientific fields. The product specifications and technical details are not available for a concise and unbiased description without further interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Lp0042, by Thermo Fisher Scientific (3 mentions)
Cm0131, by Thermo Fisher Scientific (2 mentions)
Peptone, by Thermo Fisher Scientific (1 mentions)
Glucose monohydrate, by Merck Group (1 mentions)
Lp0037, by Thermo Fisher Scientific (1 mentions)
Tryptic soy agar, by Thermo Fisher Scientific (1 mentions)
Cm0131b, by Thermo Fisher Scientific (1 mentions)
Cm0189, by Thermo Fisher Scientific (1 mentions)
Lp0011b, by Thermo Fisher Scientific (1 mentions)
Tryptone, by Thermo Fisher Scientific (1 mentions)
Mycological peptone, by Thermo Fisher Scientific (1 mentions)
Cm0129, by Thermo Fisher Scientific (1 mentions)
Lp0013, by Thermo Fisher Scientific (1 mentions)
Lp0011, by Thermo Fisher Scientific (1 mentions)
R9504, by Merck Group (1 mentions)
C11995500bt, by Thermo Fisher Scientific (1 mentions)
Induction drive centrifugation, by Beckman Coulter (1 mentions)
Proteose peptone, by Thermo Fisher Scientific (1 mentions)
Lp0037b, by Thermo Fisher Scientific (1 mentions)
14 protocols using lp0021
1
Optimizing Filamentous Biofilm Growth
2
Mosquito Rearing and Maintenance Protocol
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Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus were obtained from Guangzhou Wolbaki Biotechnology Co., Ltd. Mosquito larvae were reared in dechlorinated tap water in plastic containers and raised with standard diet, which consisted of a 0.1% solution of three parts liver broth (CM0077, Oxoid) and two parts yeast extract (LP0021, Oxoid). Adult mosquitoes were maintained in a cage with unlimited access to water and sugar (raisin for standard diet). Mosquitoes were maintained in incubators (HWS-1000, Ningbojiangnan) with 12h:12h light:dark cycle at 27 °C with 75% humidity. Female mosquitoes aged 6–10 days post-eclosion were subjected for further investigation unless otherwise mentioned.
3
Phenotypic Differentiation of Staphylococcal and Streptococcal Species
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To differentiate staphylococcal and streptococcal species, primarily S. aureus from other staphylococci, and Viridans Streptococcus from pneumococcus, bacteria were screened for multiple phenotypes. As controls, Staphylococcous aureus ATCC 25904, Streptococcus pneumoniae strain D39; NCTC 7466 (pneumococcus control) and Streptococcus mitis NTCC10712 (viridans Streptococcus control, provided kindly by the group of Dr. Jan-Willem Veening, Lausanne, Switzerland) were used. For general overnight culture, streptococci were grown in Todd-Hewitt (CM0189, Oxoid, UK) supplemented with yeast extract (0.5 g/L, LP0021, Oxoid, UK) at 37 °C with 5% CO2, 85% relative humidity and staphylococci were grown in Tryptic Soy Agar (CM0131b, Oxoid, UK) at 37 °C.
4
Culturing and Sourcing of Terrestrial Rhabditida
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T. rostrata TR01 was obtained from the American Type Culture Collection (ATCC®PRA326™). T. rostrata TRAUS was isolated from the egg of a laboratory-reared D. reticulatum in 2015. The parents of the slug had been previously collected from Ringwood North, SE Melbourne, Australia. The isolates were routinely cultured at 20 °C in sterile PPYE medium consisting of 0.5% w/v proteose peptone (Oxoid LP0085), 0.5% w/v yeast extract (Oxoid LP0021), and 0.125% w/v glucose. The source of the isolates and relevant sequences are listed in Table 1 .
5
Bacteriophage Infection of E. coli K-12
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The bacterial strain used in this study was E. coli E. coli E. coli
Bacterial strains used in this study were grown in LB [10 g l−1 Tryptone (Oxoid: LP0042), 10 g l−1 NaCl (Oxoid: LP0005) and 5 g l−1 Yeast extract (Oxoid: LP0021)] as either liquid culture (with shaking at 100 r.p.m.) or solid culture [supplemented with 15 g l−1 agar (Oxoid: LP0011B)] at 37 °C. Phages were suspended in phage buffer (10 mM Tris.HCl pH 7.5, 8 mM MgSO4).
Bacterial strains used in this study were grown in LB [10 g l−1 Tryptone (Oxoid: LP0042), 10 g l−1 NaCl (Oxoid: LP0005) and 5 g l−1 Yeast extract (Oxoid: LP0021)] as either liquid culture (with shaking at 100 r.p.m.) or solid culture [supplemented with 15 g l−1 agar (Oxoid: LP0011B)] at 37 °C. Phages were suspended in phage buffer (10 mM Tris.HCl pH 7.5, 8 mM MgSO4).
6
Cultivation and Washing of L. varians GY32
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L. varians GY32 was isolated from heavy metals-polluted sediment and preserved in our laboratory17 (link). Strain GY32 was cultivated aerobically in Luria–Bertani broth (LB) for 12 h (30 °C, 120 rpm) in flasks. The LB (1 L) contained 5 g yeast extract (LP0021, Oxoid, United Kingdom), 10 g Tryptone (LP0042, Oxoid, United Kingdom), and 5 g NaCl. Before being used for inoculation to MFCs or further tests, the LB-cultivated bacteria cells were washed in sterilized phosphate buffered saline (PBS) or deionized water for at least three times by centrifugation at 8000×g for 5 min. PBS used here (pH 7.2, 1 L) contained 8 g NaCl (C111545, Aladdin, China), 0.2 g KCl (P112133, Aladdin, China), 3.63 g Na2HPO4·12H2O (S112623, Aladdin, China), 0.24 g KH2PO4 (P104071, Aladdin, China), the pH was adjusted by HCl (10011008, Hushi, China).
7
Preparing and Quantifying Candida albicans
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Candida albicans strain SC5314 (Gillum et al. 1984 (link)) was prepared by plating 2–10 µl of frozen glycerol stock on YPD plates [1% w/v yeast extract (Oxoid LP0021, Basingstoke, UK), 2% w/v mycological peptone (Oxoid LP0040), 2% w/vd -glucose, and 2% w/v agar No. 2 (Oxoid LP0012)] and incubating at 30°C for 48 h. A single colony was transferred from the Petri dish into NGY broth (0.1% yeast extract (Oxoid LP0021), 0.1% neopeptone (Difco, Franklin Lakes, NJ, USA), and 0.4% w/v d -glucose; MacCallum et al. 2006 (link)) and incubated at 30°C, with shaking at 200 rpm, overnight. The concentration of C. albicans cells in suspension (cells/ml) was estimated by counting using a haemocytometer. Yeast growth was assessed by measuring optical density of the cultures at a wavelength of 600 nm using a spectrophotometer. For determination of C. albicans CFUs in samples, cells were plated on Sabouraud dextrose agar (SDA; 4% w/v d -glucose, 1% w/v mycological peptone, and 2% w/v agar No. 2, pH 5.6).
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Candida albicans strain SC5314 (Gillum et al. 1984 (link)) was prepared by plating 2–10 µl of frozen glycerol stock on YPD plates [1% w/v yeast extract (Oxoid LP0021, Basingstoke, UK), 2% w/v mycological peptone (Oxoid LP0040), 2% w/v
8
Characterizing Phage Interactions with Staphylococci
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Phage host range was determined by spotting serial dilutions on double-layer agar plates with tryptone soya agar (TSA) bottom agar (CM0131, Oxoid, Basingstoke, United Kingdom) and 0.6% (w/v) top Agar No. 1 (LP0011, Oxoid) with the addition of CaCl2 to a concentration of 2 mM. The growth properties and adsorption efficiency of phages were determined in tryptone soya broth (TSB) (CM0129, Oxoid), with 2 mM CaCl2 as described previously [17 (link)] with minor modification for staphylococci [18 (link)]. The ability to grow on different media was further tested on meat peptone agar (MPA) and double-concentrated yeast extract-tryptone (2× YT) agar composed of 1.6% (w/v) tryptone (LP0042, Oxoid), 1.0% (w/v) yeast extract (LP0021, Oxoid), 0.5% (w/v) NaCl, and 1.5% (w/v) agar (LP0013, Oxoid), pH 7.0. A one-step growth curve and phage burst size were determined by the procedure described previously [17 (link)]. Free plasmacoagulase and clumping factor tests were performed as described previously [19 (link),20 (link)].
9
Bacterial Strains and Plasmid Engineering for EHEC O157:H7 Studies
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The bacterial strains and plasmids used in this study are listed in SI Appendix, Table S1 . Mutant strains of EHEC O157:H7 EDL933 were generated using the λ-Red recombination system. Complementary strains were established by cloning rbfS and rbfR and native promoters into the pACYC-184 plasmid and transformed into EHEC O157:H7 EDL933 and C. rodentium DBS100. The strain for RbfR and RbfS purification was generated by cloning rbfR and rbfS into the pET28a plasmid. Bacterial strains were grown in Luria-Bertani (LB) broth (Oxoid; LP0021 and LP0042), Dulbecco’s modified Eagle medium (DMEM, Gibco; C11995500BT), or simulated colonic environment medium (SCEM, 6.25 g/L tryptone, 2.6 g/L D-glucose, 0.88 g/L NaCl, 2.7 g/L KHCO3, 0.43 g/L KH2PO4, 1.7 g/L NaHCO3 and 4.0 g/L bile salts) (49 ) with different concentrations of riboflavin (Sigma; R9504). Bacteria were inoculated into a fresh medium and grown at 37°C under shaking. When needed, isopropyl β-d -thiogalactoside (IPTG) and antibiotics were added to the medium at the following final concentrations: 0.1 mM IPTG, 100 μg/ml ampicillin, 25 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 10 μg/ml tetracycline. The primers used for all manipulations are listed in SI Appendix, Table S1 .
10
Probiotic Strain Incorporation Protocol
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The experimental diets with the probiotic candidates were mixed weekly. The individual strains were grown in MRS broth contained 5 g/L of yeast extract (powder, Oxoid, LP0021) and 20 g/L of glucose, for overnight (at 39°C) and harvested by centrifugation at 4,420 × g for 15 min (Induction Drive Centrifugation, Beckman Model J2-21M, Beckman Instruments Inc., Palo Alto, California, USA), resuspended in PBS (pH 7.4) and mixed into a premix with the basal diet for 10 min using a miniature mixer. This pre-mixture of product with feed (1 kg) was then transferred into a larger mixer (total capacity 300 kg) where the final volume of the weekly feed batch was prepared. The mixer equipment was thoroughly cleaned between the mixing of different treatments by using a vacuum cleaner and a wash diet (basal feed).
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