C. difficile strains ATCC BAA-1804, ATCC BAA-1870, ATCC BAA-1803, ATCC BAA-1801, ATCC BAA-1875, ATCC BAA-1812, ATCC BAA-1814, ATCC 43598, and ATCC BAA-1382 were obtained from ATCC while C. difficile strain ATCC 9689 was acquired from Microbiologics. All strain stocks were stored at −80°C. Strains of C. difficile were grown anaerobically at 37°C in BHIS medium (37 g/liter brain heart infusion [BHI] [BD Biosciences], 5 g/liter yeast extract [Research Products International], 435 mg/liter L-cysteine hydrochloride monohydrate [RPI], 1g/liter sodium taurocholate [GoldBio]) and were maintained in cooked meat broth with iron, hemin, and vitamin K (Hardy Diagnostics). In cases where agar plating of C. difficile was necessary BHI agar with horse blood and taurocholate (Anaerobe Systems) were used. Escherichia coli strains were grown shaking (250 RPM) at 37C in Luria broth (RPI).
Hemin
Manufactured by Hardy Diagnostics
Sourced in United States
Hemin is a type of laboratory equipment used in the field of microbiology. It is a chemical compound that serves as a growth factor for certain bacterial species, particularly those that require heme for their metabolism. Hemin is used to support the growth and cultivation of these bacteria in controlled laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Vitamin k, by Hardy Diagnostics (3 mentions)
Yeast extract, by Research Products International (1 mentions)
Atcc baa 1870, by American Type Culture Collection (1 mentions)
Atcc baa 1382, by American Type Culture Collection (1 mentions)
Gaspak ez container system, by BD (1 mentions)
Bhi broth, by Thermo Fisher Scientific (1 mentions)
Vitamin k1, by Merck Group (1 mentions)
Hemin, by Merck Group (1 mentions)
Mrs broth, by Merck Group (1 mentions)
As 580, by Anaerobe Systems (1 mentions)
Anaerobe chamber, by Anaerobe Systems (1 mentions)
6 protocols using hemin
1
Culturing C. difficile and E. coli Strains
2
Anaerobic Bacterial Culture Protocols
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The following bacteria were used in this study: P. bivia (ATCC 29303) and Lactobacillus crispatus (ATCC 33197). All bacteria were grown on Brucella blood agar plates supplemented with hemin and vitamin K (Hardy Diagnostics, Santa Maria, CA, USA) at 37 °C in anaerobic conditions using the GasPak™ EZ Container System (BD Diagnostics, Sparks, MD, USA). Cultures were stored in 25% glycerol at −80 °C. Bacterial plates were restreaked once every 2–3 days as needed for experiments.
3
Anaerobic Bacterial Growth Conditions
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All bacterial growth was carried out at 37°C in an anaerobic chamber (Anaerobe Systems, AS-580) using a gas mixture of 10% hydrogen, 10% carbon dioxide, and 80% nitrogen. Culture media was always transferred to anaerobic conditions at least 24 hours before use. Brucella Blood Agar (BRU) supplemented with hemin and vitamin K (Hardy Diagnostics, A30) was used to grow bacterial strains on solid media unless otherwise specified. For all assays, bacteria were streaked out onto BRU plates from stocks stored at −80°C in 10% glycerol and grown for 48–72 hours, then re-streaked onto fresh BRU plates and grown for another 48–72 hours. Brain Heart Infusion (BHI) Broth (Oxoid, CM1135) supplemented with 5 μg/mL hemin (Sigma, 51280), 1 μg/mL vitamin K1 (Sigma, 95271), and 0.5 mg/mL L-cysteine HCl after autoclaving was used to grow bacterial strains in liquid media. For liquid assays not requiring co-culturing or subsequent growth of NM74_B14 or NM01_1-7b, Lactobacillaceae species were cultured in de Man, Rogosa, and Sharpe (MRS) Broth (Sigma-Aldrich, 69966).
4
Antimicrobial Evaluation of Coated ATO Nanotubes
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ATO nanotubes and SiO2/SiC-coated ATO nanotubes were sterilized at 120 °C in an autoclave for 60 min. After sterilization, the samples were placed into individual sterile plates. There are three samples for each group. A biofilm of Porphyromonas gingivalis (FDC 381) was grown in Brucella blood agar plate supplemented with hemin and vitamin K (Hardy Diagnostics, Santa Maria, CA, USA). The axenic nature of the bacteria was assessed by Gram staining. The number of bacterial cells in the suspension was determined using Petroff-Hausser bacterial counting chamber. Bacteria were diluted in RTF to reach the final concentrations of 1010 cells/mL. ATO nanotubes and 12 nm SiO2/SiC-coated ATO nanotubes were placed in 1 mL of bacterial suspension individually in 24-well sterile plates in anaerobic chamber. The 24-well plates were maintained in anaerobic growth chamber for 30 days with respective fresh media replenishments for every two days. After 30 days, the biofilm was removed from the ATO nanotubes and SiO2/SiC-coated ATO nanotubes using a sonication for 5 min and examined under SEM.
5
Cultivation of Bilophila and Clostridium
6
Culturing and Heat-Inactivating Anaerobic Bacteria
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M. curtisii ATCC 35241 and M. mulieris UPII 28-I were first grown on Brucella agar with 5% sheep blood (supplemented with hemin and vitamin K; Hardy Diagnostics, Santa Maria, CA) and were passaged twice before use in experiments to ensure purity. We verified motility of these strains by observing movement of cells grown in broth under wet mount and growth in motility agar (0.25% agar) (5 (link)).
A loop of bacterial cells scraped from the plate was suspended in fresh PYGmod broth and used to inoculate disposable culture tubes containing 5 ml PYGmod (40 ). Bacteria were incubated for 72 h at 37°C (without shaking) in an anaerobe chamber (Anaerobe Systems, Morgan Hill, CA), after which cells were pelleted, washed 3 times, resuspended in 3 ml PBS, and heat treated at 65°C for 30 min. Heat-inactivated cells were aliquoted and stored at −20°C until use. Each biological replicate of stimulation experiments using these heat-inactivated cells was performed using a different aliquot to avoid multiple freeze-thaw cycles.
A loop of bacterial cells scraped from the plate was suspended in fresh PYGmod broth and used to inoculate disposable culture tubes containing 5 ml PYGmod (40 ). Bacteria were incubated for 72 h at 37°C (without shaking) in an anaerobe chamber (Anaerobe Systems, Morgan Hill, CA), after which cells were pelleted, washed 3 times, resuspended in 3 ml PBS, and heat treated at 65°C for 30 min. Heat-inactivated cells were aliquoted and stored at −20°C until use. Each biological replicate of stimulation experiments using these heat-inactivated cells was performed using a different aliquot to avoid multiple freeze-thaw cycles.
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