All strains and plasmids used in this study are listed in Table 1 . All L. reuteri strains were derivatives of strain ATCC PTA 6475 (Biogaia) (94 (link)). Strain 6475rsiR-Stop (35 (link)) was a gift from James Versalovic (Baylor College of Medicine), and plasmid pJP042 (recT+erm+) (94 (link)) was a gift from Jan-Peter van Pijkeren (University of Wisconsin—Madison). L. reuteri was grown at 37°C in MEI broth (86 (link)) without added cysteine (MEI-C) or on solid De Man, Rogosa, and Sharpe (MRS) agar (Difco). Anaerobic cultures were incubated in an anaerobic chamber (Coy Laboratory Products) in an atmosphere of 90% nitrogen, 5% CO2, and 5% H2 or in Hungate tubes prepared, inoculated, and sealed in that chamber. Liquid media were made anaerobic before use by equilibration for at least 24 h in the anaerobic chamber. MRS plates for CFU plate counts were incubated in sealed containers made anaerobic using GasPak EZ sachets (Becton, Dickinson). Microaerobic cultures were incubated aerobically without shaking in 16- by 125-mm screw-cap test tubes containing 15 ml of MEI-C. Methylene blue (2 mg liter−1) was added when indicated (95 (link)). Aerobic cultures (5 ml in a 16-mm diameter test tube) were incubated with shaking (200 rpm). Details of H2O2 and HOCl stress treatments, transcript quantification, and phenotype analysis are described in the supplemental material.
Gaspak ez sachets
Manufactured by BD
The GasPak EZ sachets are a type of laboratory equipment used to create anaerobic (oxygen-free) environments. They generate an anaerobic atmosphere when placed in an enclosed container with samples or cultures. The sachets contain a chemical mixture that absorbs oxygen and releases carbon dioxide, creating the desired anaerobic conditions.
Automatically generated - may contain errors
6 protocols using gaspak ez sachets
1
Growth and Stress Response of L. reuteri
2
Helicobacter pylori Infection and Treatment Protocols
3
Isolation and Characterization of G. vaginalis
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The type strain of G. vaginalis (ATCC 14018) was obtained from the American Type Culture Collection. All other isolates were obtained from previous studies of women from Kenya, Canada and Belgium, as previously described [5 , 8 (link), 15 (link)]. Freezer stocks in 4% (w/v) skim milk or NYC medium (ATCC broth #1685) with 10% glycerol (v/v) were revived on Columbia 5% sheep’s blood agar (CBA; BD Biosciences, Mississauga, ON) and incubated anaerobically at 37°C using GasPak EZ sachets (BD Biosciences, Mississauga, ON) in sealed jars for 48–72h. After two passages, isolates were harvested with a sterile swab into 2 ml sterile saline (0.85% NaCl, pH 7.4) until turbidity was equivalent to McFarland standard 4. Turbidity was also assessed quantitatively by measuring optical density of 100 μl harvested cultures in duplicate wells of optically clear 96-well plates at 595 nm in a Vmax microplate reader (Molecular Devices, Inc., Sunnyvale, CA). DNA was isolated from 100 μl of harvested culture by resuspending in a 5% solution of Chelex (Bio-Rad Inc., Mississauga, ON), followed by incubation at 60°C for 30 min, 100°C for 8 min, and supernatant used for all described PCR assays.
4
Longitudinal Gut-Microbiome Co-Cultures in Leaky Gut Chip
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To perform longitudinal host–probiotic co-cultures in a Leaky Gut Chip, we used two live biotherapeutic products, Lactobacillus rhamnosus GG (ATCC 53103) and over-the-counter VSL#3 formulation (Sigma-Tau Pharmaceuticals, Inc.). For an LGG pre-culture, we grew LGG cells in a 15-mL sterilized test tube containing 3 mL autoclaved Lactobacilli MRS Broth (MRS; Difco). For a VSL#3 pre-culture, we inoculated VSL#3 power in the mixture (3 mL) of autoclaved MRS broth and Reinforced Clostridial Medium (RCM; Difco) at 1:1 (v/v) in a 15-mL sterilized tube. Next, we incubated the inoculated test tubes in a GasPak EZ container (BD Diagnostics) containing two anaerobic gas-generating GasPak EZ sachets (BD Diagnostics) without shaking for 12–18 h. After a seed culture, we centrifuged the culture broth at 10,000×g for 1 min, aspirated the supernatant, resuspended the cell pellet with an antibiotic-free, l -cysteine-containing culture medium and adjusted the final cell density at 1 × 107 CFU/mL. Using a 200P micropipette, we inoculated bacterial cells into the upper microchannel of a germ-free Leaky Gut Chip that was pre-conditioned with an antibiotic-free, AOI-created culture microenvironment, then incubated the setup without flow at 37 °C for 1 h. After incubation, we resumed the flow (50 μL/h) and physical deformations until the experiments were finished.
5
Quantifying Gut Bacterial Populations
6
Enumeration of Probiotic Bacteria in Products
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Plate count enumeration was performed in accordance with USP monographs for Bifidobacterium animalis subsp. lactis (USP-NF, 2017a ) and Lactobacillus acidophilus La-14 (USP-NF, 2017b ). For rehydration, samples of 11 g were added to 99 ml of MRS broth (BD Difco, Sparks, MD, United States) blended using Stomacher 400 Circulator at 230 RPM for 30 s or 2 min (Bi-07, La-14, respectively), held at room temperature for 30 min and then blended again as previously described. Samples were diluted 1:10,000,000,000 or 1E9 via serial dilution in 99 ml Flip-Top Dilution Bottles with Peptone Water (3M, Maplewood, MN, United States). One ml of diluted samples was transferred onto empty petri dishes in triplicate. Approximately 15 ml of 45°C MRS agar (BD) was poured into petri plates, plates were gently swirled to mix, then allowed to solidify at room temperature. Once solidified, plates were inverted, placed into anaerobic chambers with BD GasPak EZ sachets (BD), then incubated at 37°C for 48–72 h. Results were recorded as colonies visualized on plates after incubation and back calculated as colony forming units per gram of product (CFU/g).
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