Gardnerella isolates of different species (52 (link)) were obtained from the Laboratory of Bacteriology, University of Ghent, Belgium. These isolates include strains purchased from culture collections and fresh isolates from BV patients obtained from the University Clinic Bruges. Gardnerella isolates were grown on chocolate (Choc) agar plates (Becton, Dickinson) under anaerobic conditions in an anaerobic chamber equipped with anaerobic atmosphere generation bags (Sigma-Aldrich) for 48 h. All isolates were cultured in New York City broth III (NYCB), consisting of 10 mM HEPES (Sigma-Aldrich), 15 g/L proteose peptone (Sigma-Aldrich), 3.8 g/L yeast extract (Thermo Fisher Scientific), 86 mM sodium chloride (Carl Roth), and 28 mM α-d -glucose (Sigma-Aldrich), supplemented with 10% horse serum (HS) (Thermo Fisher Scientific). Table S1 lists all Gardnerella and Lactobacillus isolates studied.
Anaerobic atmosphere generation bags
Manufactured by Merck Group
Sourced in United States, United Kingdom
Anaerobic atmosphere generation bags are laboratory equipment used to create an oxygen-free environment. They facilitate the maintenance of anaerobic conditions for microbiological and other applications that require a controlled, low-oxygen atmosphere.
Automatically generated - may contain errors
Lab products found in correlation
Rcm agar, by Thermo Fisher Scientific (3 mentions)
Bhi agar, by Lab M (2 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Proteose peptone, by Merck Group (1 mentions)
Anaerobic chamber, by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
α d glucose, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Atcc 6919, by American Type Culture Collection (1 mentions)
Anaerobic jar, by Merck Group (1 mentions)
Hi fbs, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
9 protocols using anaerobic atmosphere generation bags
1
Gardnerella Isolate Cultivation Protocol
2
Antibiotic Sensitivity Assay under Aerobic and Anaerobic Conditions
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Fresh overnight cultures were serially diluted in 10 mM MgSO4. A 7-µL volume of each dilution was spotted on LB agar plates with different amounts of TMP (from 0.05 to 0.2 µg/mL). When needed, LB medium was supplemented with: 0.3 mM thymidine, 0.3 mM glycine, 0.3 mM methionine, 0.3 mM inosine, or 3,000 U/plate catalase (Sigma). After 16 h of incubation at 37 °C, growth of the spots was verified and compared with the spots on the control LB agar plates. Alternatively, plates were incubated at 37 °C in anaerobic jars with anaerobic atmosphere generation bags (Sigma-Aldrich) for 40 h. Anaerobic conditions were verified using an anaerobe indicator test (Sigma).
3
Antimicrobial Nanoparticle Efficacy Evaluation
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The eFM disks
with CTR-NPs or MTZ10-NPs were immersed in 200 μL of TSB inoculated
with 1–2 × 106 CFUs mL–1 onto
a 96-well plate and incubated at 37 °C with agitation (150 rpm)
for 24 h under anaerobic conditions. For this, an anaerobic incubation
jar (Sigma-Aldrich) with anaerobic atmosphere generation bags (Sigma-Aldrich)
and a dry anaerobic indicator strip (Millipore) was set. Afterward,
the CFUs mL–1 counted by serial dilutions, and each
bacterial suspension was spread onto TSA plates and incubated overnight
at 37 °C. In addition to the sample control constituted of the
eFM immobilizing CTR-NPs, each bacterial suspension in TSB was used
as a positive control. Additionally, the absorbance at 600 nm was
measured using a microplate reader (Synergy HT, BioTEK). For the absorbance
measurement, additional negative controls made under the same conditions
were used, i.e., TSB without bacterial inoculation and samples, and
TSB containing the eFM immobilizing CTR-NPs or MTZ10-NPs without bacterial
inoculation. Three independent assays were conducted, and each condition
was tested in triplicate.
with CTR-NPs or MTZ10-NPs were immersed in 200 μL of TSB inoculated
with 1–2 × 106 CFUs mL–1 onto
a 96-well plate and incubated at 37 °C with agitation (150 rpm)
for 24 h under anaerobic conditions. For this, an anaerobic incubation
jar (Sigma-Aldrich) with anaerobic atmosphere generation bags (Sigma-Aldrich)
and a dry anaerobic indicator strip (Millipore) was set. Afterward,
the CFUs mL–1 counted by serial dilutions, and each
bacterial suspension was spread onto TSA plates and incubated overnight
at 37 °C. In addition to the sample control constituted of the
eFM immobilizing CTR-NPs, each bacterial suspension in TSB was used
as a positive control. Additionally, the absorbance at 600 nm was
measured using a microplate reader (Synergy HT, BioTEK). For the absorbance
measurement, additional negative controls made under the same conditions
were used, i.e., TSB without bacterial inoculation and samples, and
TSB containing the eFM immobilizing CTR-NPs or MTZ10-NPs without bacterial
inoculation. Three independent assays were conducted, and each condition
was tested in triplicate.
4
Cultivation and Storage of C. acnes and S. epidermidis
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C. acnes standard strain ATCC 6919 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), while S. epidermidis DSM 28319 (equivalent to ATCC 35984) was obtained from the German Collection of Microorganisms (Braunschweig, Germany). C. acnes was cultured anaerobically on RCM agar (Oxoid Limited, Basingstoke, United Kingdom (UK) and incubated for 48 h at 37 °C under ~5% CO2 using an anaerobic jar and anaerobic atmosphere generation bags (Sigma–Aldrich, St. Louis, MO, USA). Isolated colonies of C. acnes were sub-cultured in an RCM broth for 48 h at 37 °C under anaerobic conditions. For S. epidermidis, the bacteria were cultured aerobically on brain heart infusion (BHI) agar (LAB M limited, Lancashire, UK) and incubated at 37 °C for 18 h. Isolated colonies of S. epidermidis were sub-cultured in BHI broth and incubated at 37 °C for 18 h aerobically. Glycerol stock of each bacterial strain were prepared using their appropriate media supplemented with 25% glycerol and stored in a −70 °C freezer (Thermo Fisher Scientific, Waltham, MA, USA).
5
Enumeration of Bacterial Populations
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For bacterial counts, 1 g of sample was put in a sterile bag and prepared in
triplicate (Whirl-Pak, Nasco, USA). Sample was homogenized with 9 mL of
sterilized 0.1% peptone in distilled water for 2 min using a stomacher (400,
Seward Ltd., UK). Decimal dilutions were prepared using sterilized 0.1% peptone
water. Total viable counts (TVC), LAB and coliform were enumerated using plate
count agar, De Man, Rogosa and Sharpe agar and violet red bile agar,
respectively (Difco, Becton, Dickinson and Company, USA). The LAB plates were
incubated in anaerobic chambers with anaerobic atmosphere generation bags
(Sigma-Aldrich Corp., LLC., USA). For TVC, the plates were incubated at
37°C for 48 h. While, both coliform and LAB plates were incubated at
35°C for 24 to 48 h. Microbial population was counted and expressed as
log CFU/g.
triplicate (Whirl-Pak, Nasco, USA). Sample was homogenized with 9 mL of
sterilized 0.1% peptone in distilled water for 2 min using a stomacher (400,
Seward Ltd., UK). Decimal dilutions were prepared using sterilized 0.1% peptone
water. Total viable counts (TVC), LAB and coliform were enumerated using plate
count agar, De Man, Rogosa and Sharpe agar and violet red bile agar,
respectively (Difco, Becton, Dickinson and Company, USA). The LAB plates were
incubated in anaerobic chambers with anaerobic atmosphere generation bags
(Sigma-Aldrich Corp., LLC., USA). For TVC, the plates were incubated at
37°C for 48 h. While, both coliform and LAB plates were incubated at
35°C for 24 to 48 h. Microbial population was counted and expressed as
log CFU/g.
6
Culturing Propionibacterium acnes and Staphylococcus epidermidis
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Propionibacterium acnes ATCC 6919 and Staphylococcus epidermidis ATCC 28319 were kindly provided by Dr. Mayri A. Diaz, Manchester University, UK. S. epidermidis was cultured aerobically on brain heart infusion (BHI) agar (LAB M limited, Lancashire, UK) and incubated at 37 °C for 18 h. We sub-cultured isolated colonies of S. epidermidis in BHI broth and incubated at 37 °C for 18 h aerobically. For P. acnes, we cultured the bacteria anaerobically on reinforced clostridial medium (RCM) agar (Oxoid Limited, Basingstoke, UK) for 48 h at 37 °C and ~5% CO2 using anaerobic jar and anaerobic atmosphere generation bags (Sigma-Aldrich, St. Louis, MO, USA). We sub-cultured isolated colonies of P. acnes in RCM broth for 48 h at 37 °C under anaerobic conditions.
7
Culturing Diverse Cell Lines Under Anaerobic and Hypoxic Conditions
8
Quantifying Gut Bifidobacteria in Mice
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After the 3 week supplementation with test compounds, all mice were culled and fresh faecal pellets were removed from the large intestine. Total Bifidobacteria species were evaluated with Beerens agar plates, a method that has recently been used to enumerate these bacteria in human faecal samples from infants supplemented with galacto-oligosaccharides (Stiverson et al., 2014 (link)). Briefly, faecal pellets from each animal were weighed and homogenised in PBS (1:10 w/v) and diluted in a 1:10 series in fresh PBS. An aliquot of 0.1 ml of 3 dilutions each was plated on selective Beerens agar plates in triplicate. They were then incubated at 37 °C for 48 h inside an anaerobic chamber containing anaerobic atmosphere generation bags (Sigma Aldrich, UK). The colonies formed were counted from the plates that had 30–100 colonies. The number of CFU/ml of culture for each treatment was calculated based on the number of colonies and the corresponding dilution factor. The values were then expressed as CFU/g faeces which was calculated from the original weight of the faecal matter sampled.
9
Inflammatory Murine Model for OF-gel Evaluation
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For testing the in vivo effectiveness of the OF-gel, an established inflammatory murine model was used based on Nakatsuji et al. (2008 (link)) and our previous studies (Taleb et al., 2018 ; Shamma et al., 2019 ). Standard C. acnes (ATCC 6919) strain was anaerobically cultured on reinforced clostridial medium (RCM) agar (Oxoid Limited, Basingstoke, UK) for 48 h at 37 °C with anaerobic atmosphere generation bags (Sigma–Aldrich, St. Louis, MO, USA). Isolated colonies of C. acnes were sub-cultured in RCM broth for 48 h at 37 °C under anaerobic conditions. The bacterial suspension from the liquid sub-culture optical density at 600 nm wavelength was adjusted to 1 which is equivalent to 1011 colony forming unit (CFU)/mL checked by performing viable count, and this bacterial suspension was used for preparation of the inocula of concentration 1010 CFU/mL.
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