BISC209/S11: Recipes

Media Recipies

Nutrient Agar: A general purpose solid medium
Beef extract 3.0g/L, Peptone 5.0g/L, Agar 15.0g/L; Deionized water to 1 L at pH 6.6- 7.0 at 25°C. This medium is commercially available.

Nutrient Broth: A general purpose liquid medium.
Beef extract 3.0g/L, Peptone 5.0g/L. Commercially available and identical to Nutrient agar without the 15.0 g of solidifying Agar.

Gram positive spore forming enrichment:

• Glycerol Yeast Extract Agar (general enrichment medium for Gram positive spore forming bacteria) Glycerol 5.0 ml, Yeast Extract 2.0g/L, Dipotassium phosphate 1.0g/L, Agar 15.0 g/L, deionized water to 1 Liter.

Denitrifying Methylotrophs(Hyphomicrobium) Medium with or without methanol (DMM) (Marine Biology Laboratory, Woods Hole, MA recipe)

• 100X FWB (fresh water base) 10.0 ml; 1 M MOPS, pH 7.2 20.0 ml; Trace element solution 1.0 ml; 1 M Na₂SO₄ 0.2ml; 150 mM Potassium phosphate (pH 7.2) 1.0 ml; 0.5 M NH₄Cl 10 ml; KNO₃ 5.0 g; Bring to 1 liter with deionized water. pH 7. Autoclave then add: 2.5 ml FRESH methanol (oxidized methanol becomes toxic formaldehyde over time). vitamin mix 10 ml . Dispense into sterile full screw cap tubes.
• For solid medium add agar 15 g before autoclaving.

• Maintenance medium (we may not use this): PyCM agar (0.25% peptone, 0.05% yeast extract, 1 mM CaCl₂, 2 mM MgSO₄, 1% agar)
• Freshwater base medium (FWB)
  Order from WARD in 2011? We made it from greenhouse compost in 2010.
  peptone, 5 g; yeast extract, 1 g; FeP04.4H20,O.Ol; NaC1, 5 g; and distilled water, 1,OOO ml (pH 7.6).
  
  

Nitrogen Cyclers:
Azotobacteria enrichment medium (selects for bacteria able to use mannitol as their sole carbon source)

• Azotobacter N-Free Media (1 L)
  Solution A K2HPO4 : 1.6 g KH2PO4: 0.4 g
  Add distilled water to make 0.5 L
  Solution B MgSO4: 0.4 g CaSO4: 0.2 g FeSO4/7H2O: 0.006 g MoO3: 0.002 g sucrose: 10 g
  Add distilled water to make 0.5 L
  Aseptically combine 1A:1B after autoclaving; for plate version, add 15 g agar to solution B prior to autoclaving. After autoclaving, media will contain some solid material that should be swirled prior to pouring plates
  

Miscellaneous media and reagents

Hydrocarbon minimal salts broth (source: Scott, Christina, C.L., and W.R. Finnerty. 1975. A comparative Analysis of the Ultrastructure of Hydrocarbon-oxidizing Micro-organisms. J. of GEn. Micro. 94, 342-350.)

• 2g (NH₄)₂SO₄, 4g KH₂PO₄, 4g Na₂HPO₄ 0.2g MgSo₄7H₂, 0.001gCaCl₂, 0.001g FeSO₄7H₂O, pH 7.8.
• Supplement this medium with the desired hydrocarbons. e.g. 0.5% (v/v) hexadecane for Acinetobacter sp., 1%(v/v) hexadecane Arthrobacter sp, or Corynebacterium.

Cellulitic enrichment

• Oatmeal agar: (to enrich for Streptomycetes)
  Oatmeal extract: 20g oatmeal (from grocery store) 1 liter distilled water. Autoclave 30min (put flask in a tray with with couple inches water), strain through cheesecloth. Place 250 mls of extract into 500 ml flasks and reautoclave and store RT until needed: Use 500 ml oatmeal extract and 500 ml distilled water, add 15 g agar. Autoclave and cool. Add 10 cycloheximide (to inhibit fungi) if desired, swirl to mix gently. makes 65 or so 15 ml plates.

• Emerson agar: (to enrich for Streptomycetes)
  yeast extract, 1.0 g; beef extract, 4.0 g; Bacto-peptone, 4.0 g; NaCI, 2.5 g; cerelose, 10 g; agar 20 g. (add Cycloheximide)

STOCK: Cycloheximide solution (1000 ug/ml in 70% ethanol).
cycloheximide inhibits the growth of eukaryotic cells (e.g. fungi). It inhibits protein biosynthesis by interfering with peptidyl transferase activity of the 60S ribosome, preventing protein elongation. Use caution when working with cycloheximide.

• Add 74ml 95% ethanol and 26 ml sterile distilled water to a Sterile 250 ml flask. Add 1gram cycloheximide powder. Store at 4 degrees. Dilute to 50mg/ml stock as needed.
  Unless otherwise noted add 1 ml of 50 mg/ml stock to 1 liter of medium = 50ug/ml final concentration in medium.

1x PBS:
8 g/l NaCl, 0.2 g/l KCl, 1.43 g/l Na2HPO4, 0.24 g/l KH2PO4

Pourite: An antifoaming agent used to prevent bubbles in agar containing media that is not commercially available This agent helps reduce foaming and bubbles when pouring agar plates. On drop for volumes up to 800 ml and 2 drops up to 1 liter. Purchase this from American Scientific Products.

Additional misc test reagent media

Luria Bertoni Broth

• Add the following to 800ml H2O; Bacto-tryptone 10 g; yeast extract 5 g; NaCl 10 g. Adjust pH to 7.5 with NaOH. Adjust volume to 1L with dH2O. Sterilize by autoclaving.

Sulfur Reduction/Indole Production/Motility media (SIM)

• Approximate Formula* Per Liter Pancreatic Digest of Casein 20.0 g; Peptic Digest of Animal Tissue 6.1 g; Ferrous Ammonium Sulfate 0.2 g; Sodium Thiosulfate 0.2 g; Agar 3.5 g. reference: MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,

MOPS

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12