Bacterial culturing

Bacterial Culturing

1. Using a sterile inoculating loop, streak out bacteria from a glycerol stock (stored at -80°C) onto pre-reduced BHI+ plates in an anaerobic chamber (Coy Laboratory Products) with a gas mix of 5% hydrogen and 20% carbon dioxide (balance nitrogen). BHI+ plates can be reduced by incubating in the anaerobic chamber overnight.
2. Incubate for 24-48 hours in a 37°C incubator inside the anaerobic chamber until single colonies are visible.
3. Pick a single colony with a sterile tip and inoculate into 10 mL of pre-reduced BHI+ medium in the anaerobic chamber and incubate for 48 hours at 37°C.
4. Normalize these starter cultures to an OD₆₀₀=0.1 by dilution into fresh BHI⁺ medium and culture either alone or with the corresponding bile acid substrate at 100 µM final concentration. Grow up cultures for 48 hours at 37°C.
5. Take timepoints by removing 2 mL of the culture. Extract bile acids according to the method described in the paper (“Sample Preparation for Bacterial Culture”), then quantify bile acids according to the method described in the paper (“UPLC-MS analysis”).

Preparing BHI+ plates and media (per L)

37 g      BHI [brain heart infusion agar (Bacto)] 

15 g      agar (for plates)

1. Dissolve in dH₂O, autoclave, cool to 50°C – 60°C
2. Add 1 mL hemin stock ^[1] and 500 mL vitamin K₁ stock ^[2] and 1 mL Cysteine HCL (per L) following autoclaving and while solution is stirring and has cooled to ~50°C – 60°C
3. Pour plates

Stock solutions

^[1] Hemin (Sigma)

5 mg/ml in 1N NaOH (0.5% w/v) 

filter sterilize

store at 4°C for <1 month

^[2] Vitamin K₁ (Sigma)

5 µl/ml in 100% ethanol

store at 4°C protected from light for <1 month

^[3] Cysteine HCL (Sigma) 500 mg/mL in dH₂O

filter sterilize

store at 4°C for <1 month

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