BISC209/S12: Recipes

Media Recipies
Nutrient Agar: A general purpose solid medium  0.3% Beef extract, 0.5% Peptone, 1.5% agar, pH 6.8 at 25°C. This medium is commercially available from BD Difco.

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. 

Dilute Nutrient Agar: dilute full strength NA 1:10- 1:300 depending on how much growth reduction you want to achieve. In our experiments we will use a 1:10 dilution of beef extract and peptone= 0.03% beef extract and 0.05% peptone.

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 

Nitrogen Cyclers: Azotobacteria enrichment medium (selects for bacteria able to fix nitrogen (ammonifiers) and to use mannitol as their sole carbon source) Azotobacter N-Free Media (1 L) Solution A: 0.16% K2HPO4:0.04% KH2PO4 <BR> <BR> Solution B: 0.04% MgSO4; 0.02% CaSO4; 0.0006% FeSO4/7H2O; 0.0002% MoO3;  1.0% mannitol. <BR> <BR> 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.<BR> For solid medium add 2% agar to solution B prior to autoclaving.<BR> Effective concentrations: 0.08%K2HPO4; 0.02%KH2PO4, 0.02% MgSO4 0.01% CaSO4; 0.0015% FeSO4/7H2O; 0.00025% g MoO3; 0.5% mannitol.<BR><BR>

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'''Simmons Citrate Medium selects for bacteria (nitrifiers) able extract nitrogen from ammonium (in this case ammonium phosphate) while using citrate as their sole carbon source. The indicator shows a color change from green to blue when an alkaline pH of 7.6 is reached as the ammonium is converted to ammonia.'''<BR> 0.02% MgSO4(Magnesium Sulfate), 0.1% NH4H2PO4(monoammonium phosphate), 0.1% K2HPO4(dipotassium phosphate), 0.2% C6H5Na3O7(sodium citrate), 0.5% NaCl(sodium chloride), 2.5% agar, 0.008% C27H27Br2O5SNa(bromothymol blue) at pH6.9 +/-0.2 at 25°C.

Miscellaneous media and reagents<BR><BR> 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. </LI></UL>

<BR> Cellulose degradation medium <ul><li> 0.188% ashed, acid washed cellulose powder and 0.02% Congo red in Nutrient Agar (0.3%Beef extract, 0.5% Peptone, 1.5% Agar; at pH 6.6- 7.0 at 25°C.

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<BR><BR> 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. <BR>

1x PBS:<BR> 0.8% NaCl, 0.2% KCl, 0.143% Na2HPO4, 0.024% KH2PO4<BR><BR>

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.<BR><BR>

Additional Misc. Reagents & Media Recipes
Luria Bertoni Broth <BR>  Bacto-tryptone 1%; yeast extract 0.5%; NaCl 1.0% at pH 7.5 </LI></UL>

Sulfur Reduction/Indole Production/Motility media (SIM)<BR>  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, </LI></UL>

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