BISC209/S11: Recipes

Media Recipies

Nutrient Agar: A general purpose solid medium
0.3% Beef extract, 0.5% Peptone, 1.5% agar, pH 6.6- 7.0 at 25°C. This medium is commercially available.

Nutrient Broth: A general purpose liquid medium.
0.3% Beef extract, 0.5% Peptone- Commercially available and identical to Nutrient agar without the 1.5% solidifying Agar.

Gram positive spore forming enrichment:

• Glycerol Yeast Extract Agar (general enrichment medium for Gram positive spore forming bacteria) 0.5% (v/v) Glycerol, 0.2% Yeast Extract, 0.1%Dipotassium phosphate, 1.5% Agar

Denitrifying Methylotrophs(Hyphomicrobium) Medium with methanol (DMMM) (Marine Biology Laboratory, Woods Hole, MA recipe)

• DMMM medium (1% Freshwater Base (FWB: 10% NaCl, 4% MgCl2*6H20; 1% CaCl2*2H2O; 2% KH2Po4 (acidic); 5% KCl); 0.02M 3-(N-morpholino)propanesulfonic acid (MOPS C7H15NO4S pH 7.2), 0.2mM Na2SO4,; 0.15mM K3PO4 pH 7.2; 5.0 mM NH4Cl, 0.5% KNO3; pH 7); 1% vitamin mix (Sigma product number M7150 Murashige and Skoog Vitamin Powder); 0.25% methanol
  
• DMM medium: DMMM without 0.25% methanol.
• DMM solid medium: DMM with 1.5% agar grown in a methanol gas enriched atmosphere chamber.
  
  

100X FWB (fresh water base) 10.0 ml; 1 M MOPS (3-(N-morpholino)propanesulfonic acid), pH 7.2 20.0 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: PyCM agar (0.25% peptone, 0.05% yeast extract, 1 mM CaCl₂, 2 mM MgSO₄, 1% agar)
• Freshwater base medium (FWB)
  recipe2011 : 1 liter water, 100g NaCl, 40 g MgCl2*6H20; 10g CaCl2*2H2O; 20 g KH2Po4 (acidic); 50g KCl . Order from WARD in future- could not find online?
  
  
  

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: 0.16% K₂HPO₄:0.04% KH₂PO₄
  
  Solution B: 0.04% MgSO4; 0.02% CaSO4; 0.0006% FeSO4/7H2O; 0.0002% MoO3; 1.0% mannitol.
  
  Aseptically combine 1 part of A with 1 part of B (SolutionA:SolutionB=1:1) after autoclaving. Add 0.25 ml filtered multivitamin mix. After autoclaving media will contain some solid material that should be swirled prior to pouring plates.
  For solid medium add 2% agar to solution B prior to autoclaving.

Effective concentrations: 0.08%K₂HPO₄; 0.02%KH₂PO₄, 0.02% MgSO₄ 0.01% CaSO₄; 0.0015% FeSO₄/7H₂O; 0.00025% g MoO₃; 0.5% sucrose (2010,2011).

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.)

• 0.2% (NH₄)₂SO₄, 0.4% KH₂PO₄, 0.4% Na₂HPO₄ 0.02% MgSo₄7H₂, 0.0001%CaCl₂, 0.0001% 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.

Cellulose degradation medium

• Acid washed cellulose in Nutrient Agar (0.3%Beef extract , 0.5% Peptone, 1.5% Agar; at pH 6.6- 7.0 at 25°C.

STOCK: Cycloheximide solution (50 mg/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. Unless otherwise indicated,dilute stock to 1mg/ml final concentration in medium.

1x PBS:
0.8% NaCl, 0.2% KCl, 0.143% Na2HPO4, 0.024% 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. One 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

• Bacto-tryptone 1%; yeast extract 0.5%; NaCl 1.0% at pH 7.5

Sulfur Reduction/Indole Production/Motility media (SIM)

• Approximate Formula* Per Liter Pancreatic Digest of Casein 2%; Peptic Digest of Animal Tissue 0.61%; Ferrous Ammonium Sulfate 0.02%; Sodium Thiosulfate 0.02%; Agar 0.35% reference: MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,

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