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BacterialMedia.md

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Bacterial Growth Media

Brain Heart Infusion + Arginine (BHI+)

Use: Routine growth of E. lenta and similar organisms.

Instructions: Mix Water, BHI, and agar (if solid media is desired) and autoclave on 20 minute liquid cycle. Cool media in 55˚C waterbath and add 40mL of 100g/L arginine. If arginine is added before autoclaving it will precipitate out. If making liquid media, pH to 7.6 and sterilze using 0.2µM filter.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 360 mL - - -
BHI 14.8 g BD 237500 - 37g/L
Agar 6 g Sigma A5306 - 15 g/L
Arginine 40mL Sigma A5006 100 g/L 10 g/L (1%w/v)

Brain Heart Infusion + Cysteine + Hemin + Vitamin K + Resazurin (BHI CHVR)

Use: Routine growth of many common human gut commensals.

Instructions: Mix Water, BHI, and agar (if solid media is desired) and autoclave on 20 minute liquid cycle. Cool media in 55˚C waterbath and add Cysteine, Hemin and Vitamin K3. Add Resazurin if desired.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 400 mL - - -
BHI 14.8 g BD 237500 - 37g/L
Agar 6 g Sigma A5306 - 15 g/L
Cysteine 4 mL Sigma 168149 5% w/v 0.05% w/v
Hemin 500 µL Sigma 51280 5 mg/mL 5 µg/mL
Vitamin K3 500 µL Sigma M5625 1 mg/mL 1 µg/mL
Resazurin 1.6 mL Sigma R7017 0.025% w/v 0.0001% w/v

Brain Heart Infusion + Cysteine + Hemin + Arginine + Vitamin K + Resazurin (BHI CHAVR)

Use: Routine growth of many common human gut commensals including Coriobacteriia.

Instructions: Mix Water, BHI, and agar (if solid media is desired) and autoclave on 20 minute liquid cycle. Cool media in 55˚C waterbath and add Cysteine, Hemin and Vitamin K3. Add Resazurin if desired.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 360 mL - - -
BHI 14.8 g BD 237500 - 37 g/L
Agar 6 g Sigma A5306 - 15 g/L
Cysteine 4 mL Sigma 168149 5% w/v 0.05% w/v
Hemin 500 µL Sigma 51280 5 mg/mL 5 µg/mL
Vitamin K3 500 µL Sigma M5625 1 mg/mL 1 µg/mL
Resazurin 1.6 mL Sigma R7017 0.025% w/v 0.0001% w/v
Arginine 40mL Sigma A5006 100 g/L 10 g/L (1%w/v)

Modified Gifu Anaerobic Media (mGAM)

Use: Routine growth of many common human gut commensals.

Instructions: Mix Water, BHI, and agar (if solid media is desired) and autoclave on 20 minute liquid cycle. Cool media in 55˚C waterbath and add Arginine, Hemin and Vitamin K3. Add Resazurin if desired.

Notes: The precise recipes vary a little for this formulation and it now commercially available as Hyserve 05433. Essentially the difference is that many ingredients are 2x diluted and arginine, vitamin K, hemin are added. This version just supplements the GAM available from HiMedia.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 360 mL - - -
Gifu Anaerobic Broth 23.6 g HiMedia M1801 - 59 g/L
Agar 6 g Sigma A5306 - 15 g/L
Hemin 500 µL Sigma 51280 5 mg/mL 5 µg/mL
Vitamin K3 500 µL Sigma M5625 1 mg/mL 1 µg/mL
Arginine 40mL Sigma A5006 100 g/L 10 g/L (1%w/v)

Fastidious Anaerobe Agar (FAA)

Use: Routine growth of gut anaerobes. Not preferable as it needs blood.

Instructions: Mix Water, FAA and autoclave on 20 minute liquid cycle. Cool media in 55˚C waterbath and sheeps blood. Note: it is crucial to cool the media to prevent hemolysis of blood.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 372 mL - - -
Fastidious Anaerobe Agar 18.4 g Fisher 50-201-5192 - 46 g/L
Sheeps blood 28 mL Fisher 50863756 - 7% w/v

Tryptic soy agar + sheeps blood + arginine (TSA+)

Use: Routine growth of non-Eggerthellaceae Coriobacteriia. Not preferable due to use of blood but can help with some of the more difficult to grow strains out of the freezer.

Instructions: Mix Water, TSA and autoclave on 20 minute liquid cycle. Cool media in 55˚C waterbath and add Arginine, and sheeps blood. Note: it is crucial to cool the media to prevent hemolysis of blood.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 360 mL - - -
Tryptic soy broth 12 g Millipore 1.05459.0500 - 30 g/L
Agar 6 g Sigma A5306 - 15 g/L
Sheeps blood 20 mL Fisher 50863756 - 5% w/v
Arginine 20mL Sigma A5006 100 g/L 5 g/L (0.5%w/v)

Breat Heart Infusion + Yeast Extract (BHIS)

Use: Routine growth of C. difficile.

Instructions: Mix water, BHI, Agar (if solid media desired), yeast extract, and autoclave on liquid 20 cycle. Cool and add cysteine.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 400 mL - - -
BHI 14.8 g BD 237500 - 37g/L
Agar 6 g Sigma A5306 - 15 g/L
Yeast Extract 2 g Fisher BP9727 - 5 g/L
Cysteine 4 mL Sigma 168149 5% w/v 0.05% w/v

Breat Heart Infusion + Yeast Extract + Thiamphenicol + D-Cycloserine + Cefoxitin (BHIS TCC)

Use: Selection of C. difficile transconjugants carrying thiamphenicol/chloramphenicol resistance.

Instructions: Mix water, BHI, Agar, and autoclave on liquid 20 cycle. Cool and add cysteine, thiamphenicol, D-cycloserine and cefoxitin.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 400 mL - - -
BHI 14.8 g BD 237500 - 37g/L
Agar 6 g Sigma A5306 - 15 g/L
Yeast Extract 2 g Fisher BP9727 - 5 g/L
Cysteine 4 mL Sigma 168149 5% w/v 0.05% w/v
Thiamphenicol 400 uL Sigma T0261 10 mg/mL 10 ug/mL
Cefoxitin 400 uL Fisher AAJ66800MD 8 mg/mL 8 ug/mL
D-cycloserine 4 mL Sigma C6880 25 mg/mL 250 ug/mL

Taurocholate-Cycloserine-Cefoxitin Fructose Agar (TCCFA)

Use: Isolation and quantification of C. difficile spores.

Notes for use: Consider a 30min ethanol shock before plating to prevent background growth of Enterococcus. C. difficile will appear as yellow colonies with irregular shape and will fluoresce green under UV.

Instructions: Mix water, peptone, agar, sodium phosphate, potassium phosphate, sodium chloride, magnesium sulfate, neutral red, and fructose and autoclave. Separately prepare a solution containing the sodium taurocholate, d-cyclserine and cefoxitin in 10mL water and filter sterilize. After cooling the media from the autoclave, add the filtered supplement mix.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 400 mL - - -
Proteose peptone #2 16 g Fisher 212120 - 40 g/L
Agar 6 g Sigma A5306 - 15 g/L
Na2HPO4 2 g Fisher S373 - 5 g/L
KH2PO4 0.4 g Fisher BP362 - 1 g/L
NaCl 0.8 g Fisher S27 - 2 g/L
MgSO4 (anhydrous) 40mg Fisher M65 - 0.1 g/L
Fructose 2.5 g Fisher L96 - 6 g/L
Cefoxitin 6.4 mg Fisher AAJ66800MD - 16 ug/mL
Neutral Red 12 mg Sigma N7005 - 30 µg/mL
Sodium Taurocholate 400mg Sigma T4009 - 1mg/mL (0.1% w/v)
D-cycloserine 100 mg Sigma C6880 - 250 ug/mL

10X PBS

Use: Serial Dilutions of cultures

Instructions: Mix 800mL water with the items below, pH to 7.4 with HCl/NaOH as needed, bring up to 1L and autoclave. To make 1X, dilute 10x in sterile water, or mix with water and autoclave again.

Ingredient Volume or Weight Ordering Information
Water Up to 1L -
NaCl 80 g EMSX0420-3
KCl 2 g Sigma 97061-566
Na2HPO4 14.4 g Sigma S9763-100G
KH2PO4 2.4 g Sigma P5655-100G

MRS-Vancomycin Agar

Use: Selection of Lactobacillus

Instructions: Mix water, MRS, Agar, and autoclave on liquid 20 cycle. Cool and add vancomycin filter sterilized.

Ingredient Volume or Weight Ordering Information Stock Concentration Final Concentration
Water Up to 1L - - -
MRS 33 g RPI 31FZ13 - 33g/L
Agar 15 g Sigma A5306 - 15 g/L
Vancomycin 2 mL VWR 97062-548 25 mg/mL 50 ug/mL

Supplements

Arginine (100 g/L)

Dissolve 50 g L-arginine (Sigma A5006) in 500 mL water. Autoclave for 20min or 0.2 µM filter sterilize. Store at 4˚C.

Cysteine (5% w/v)

Dissolve 2.5g (Sigma 168149) in 50mL water. Filter-sterilize (0.2µM) and store at RT to prevent crashing out. Heat in waterbath as necessary to (re)dissolve.

Vitamin K (1 mg/mL)

Dissolve 50mg vitamin k3 (menadione; Sigma M5625) in 50mL 100% ethanol. Store at 4˚C protected from light.

Hemin (5 mg/mL)

Dissolve 50mg hemin (Sigma 51280) in 10mL 1N NaOH. Sterilize with 0.2 µM filter. Store at 4˚C.

Resazurin (0.25 % w/v)

Dissolve 25 mg resazurin (Sigma R7017) in 100 mL water and filter sterilize with 0.2 µM filter. Store at 4˚C.

Sheeps Blood

Sheeps blood must be purchased fresh and used generally within 1 month. Avoid use unless necessary.

Thiamphenicol (10 mg/mL)

Dissolve 100 mg (Sigma T0261) in 10 mL 50/50 water/ethanol. Store at -20 ˚C.

Cefoxitin (8 mg/mL)

Dissolve 80 mg cefoxitin (Fisher AAJ66800MD) in 10 mL water. Filter sterilize with 0.45 µM filter, aliquot, and store at -20˚C.

Tetracycline (50 mg/mL)

Dissolve 100 mg tetracycline (Sigma 87128) in 2 mL Ethanol. Aliquot, and store at -20˚C.

Kanamycin (50 mg/mL)

Dissolve 100mg Kanamycin sulfate (Sigma 60615) in 2 mL water. Filter sterilize with 0.45 µM filter, aliquot, and store at -20˚C.

Ampicillin (50 mg/mL)

Dissolve 100 mg ampicillin (Sigma A9393) in 2 mL water. Filter sterilize with 0.45 µM filter, aliquot, and store at -20˚C.

Chloramphenicol (20 mg/mL)

Dissolve 60 mg chloramphenicol (Sigma C0378) in 3 mL Ethanol. Aliquot and store at -20˚C.

Erythromycin (10 mg/mL)

Dissolve 20 mg erythromycin (Sigma E5389) in 2mL Ethanol. Aliquot and store at -20˚C

D-Cycloserine (25 mg/mL)

Dissolve 100 mg d-cycloserine (Sigma C6880) in 4 ml water. Filter sterilize using a 0.45-µm filter. DO NOT STORE, MAKE FRESH!

Sodium Taurocholate (10% w/v)

Dissolve 400 mg in 4 mL water. Filter sterilize using a 0.45-µm filter and freeze for storage.

Vancomycin (25 mg/mL)

Dissolve 250 mg in 10 mL water. Store at -20˚C.