BISC209/F13: 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 Agar + Starch: A general purpose solid medium
Nutrient Agar as above with 2.5% (wt/vol) soluble starch in Nutrient Agar
Reference: Beishir, Lois. 1996. Microbiology in Practice 6th ed. HarperCollins Publishers Inc. New York. Module 33: 301-306.
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.

Medium for 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

Media to Detect and Enrich for Nitrogen Cyclers:
Azotobacteria enrichment medium (selects for bacteria able to fix nitrogen (ammonifiers) and to use mannitol as their sole carbon source)
Azotobacter Nitrogen-Free Media (1 L)
Recipe: 0.08% K₂HPO₄; 0.02% KH₂PO₄, 0.02% MgSO₄ 0.01% CaSO_4; 0.0015% FeSO4/7H₂O; 0.00025% g NaMoO₃; 0.5% sucrose (or substitute mannitol for the sucrose if you want to select for Azotobactersonly ---we will use sucrose not mannitol to be less selective).
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.

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.
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

Pidovskaya Medium Modified (Nautiyal, 1999)
1% glucose; 0.5% Calcium Phosphate [Ca₃(PO₄)₂; 1% MgCl₂.6H₂O; 0.25% Magnesium Sulfate (MgSO₄.7H20);0.2% (NH4)2SO4; 0.25% KCL; 0.0025% BromoPhenol Blue; 1.5-2% agar
References: Pikovskaya, R.I. 1948. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Microbiologia 17, 362-370>
Pranjal Baruah (2007)Isolation of phosphate solubilizing bacteria from soil and its activity. Available at: Biotechindia.files.wordpress.com/2007/12/isolation.pdf.

Cellulose Congo Red medium
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.

EMB Eosin Methylene Blue medium
Recipe:1% peptone, 1% Lactose, 0.2% dipotassium phosphate, 0.04% eosin Y, 0.0065% methylene blue 1.5% Agar. Final pH 6.9-7.3

Phenylethyl alcohol (PEA) media
Recipe: 1.80% Bacto Agar, 1.50% Tryptone, 0.50% Phytone, 0.50% Sodium Chloride, 0.25% Phenylethyl Alcohol (PEA).

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.

Luria Bertoni Broth
Bacto-tryptone 1%; yeast extract 0.5%; NaCl 1.0% at pH 7.5

Mannitol Nitrate Motility Soft Agar (MNM)
Casein Peptone (1% wt/vol), Mannitol (0.75%), Potassium Nitrate (KNO³) (0.1%), Phenol Red (0.004%), Agar (.035%) Final ph 7.6 +/-0.2 at 25C

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.

Phosphate Buffered Saline (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.