BISC209/S11: Recipes: Difference between revisions

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


'''Nutrient Broth''':  A general purpose liquid medium.<BR>
'''Nutrient Broth''':  A general purpose liquid medium.<BR>
Beef extract 3.0g/L, Peptone 5.0g/L. Commercially available and identical to Nutrient agar without the 15.0 g of solidifying Agar.   
0.3% Beef extract, 0.5% Peptone- Commercially available and identical to Nutrient agar without the 1.5% solidifying Agar.   




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<UL><LI>'''Glycerol Yeast Extract Agar''' (general enrichment medium for ''Gram positive spore forming bacteria'')
<UL><LI>'''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.
0.5% (v/v) Glycerol, 0.2% Yeast Extract, 0.1%Dipotassium phosphate, 1.5% Agar
</LI></UL><BR><BR>
</LI></UL><BR><BR>


'''Denitrifying Methylotrophs(Hyphomicrobium) Medium with or without methanol''' (DMM)  (Marine Biology Laboratory, Woods Hole, MA recipe)<UL><LI>
'''Denitrifying Methylotrophs(Hyphomicrobium) Medium with methanol''' (DMMM)  (Marine Biology Laboratory, Woods Hole, MA recipe)<UL><LI>


DMM 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)<BR>
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<BR>


DMM solid medium: DMM with 1.5% agar grown in a methanol gas enriched atmosphere chamber.<BR>


DMMM medium: DMM with 0.25% methanol.
<LI> DMM medium:  DMMM without 0.25% methanol.
 
<LI> DMM solid medium: DMM with 1.5% agar grown in a methanol gas enriched atmosphere chamber.<BR>
 
  <BR></li></UL>
 


100X FWB (fresh water base) 10.0 ml; 1 M MOPS (3-(N-morpholino)propanesulfonic acid), pH 7.2 20.0 ml; 1 M Na<sub>2</sub>SO<sub>4</sub> 0.2ml; 150 mM Potassium phosphate (pH 7.2) 1.0 ml; 0.5 M NH<sub>4</sub>Cl 10 ml; KNO<sub>3</sub> 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.  <LI>
100X FWB (fresh water base) 10.0 ml; 1 M MOPS (3-(N-morpholino)propanesulfonic acid), pH 7.2 20.0 ml; 1 M Na<sub>2</sub>SO<sub>4</sub> 0.2ml; 150 mM Potassium phosphate (pH 7.2) 1.0 ml; 0.5 M NH<sub>4</sub>Cl 10 ml; KNO<sub>3</sub> 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.  <LI>
For solid medium add agar 15 g before autoclaving. </LI></UL>
For solid medium add agar 15 g before autoclaving.  
<UL><LI><BR>
<UL>
 
<LI>'''Maintenance medium:  PyCM agar''' (0.25% peptone, 0.05% yeast extract, 1 mM CaCl<sub>2</sub>, 2 mM MgSO<sub>4</sub>, 1% agar)
Maintenance medium (we may not use this):  PyCM agar (0.25% peptone, 0.05% yeast extract, 1 mM CaCl<sub>2</sub>, 2 mM MgSO<sub>4</sub>, 1% agar)
<LI>'''Freshwater base medium (FWB)'''<br>
<LI>'''Freshwater base medium (FWB)'''<br>
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?  <br>
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?  <br>
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'''Azotobacteria enrichment medium (selects for bacteria able to use mannitol as their sole carbon source)'''<BR>
'''Azotobacteria enrichment medium (selects for bacteria able to use mannitol as their sole carbon source)'''<BR>
<UL><LI>Azotobacter N-Free Media (1 L)<BR>
<UL><LI>Azotobacter N-Free Media (1 L)<BR>
Solution A K2HPO4 : 1.6 g KH2PO4: 0.4 g<BR>
Solution A: 0.16% K<sub>2</sub>HPO<sub>4</sub>:0.04% KH<sub>2</sub>PO<sub>4</sub> <BR>
Add distilled water to make 0.5 L<BR>
<BR>
Solution B MgSO4: 0.4 g CaSO4: 0.2 g FeSO4/7H2O: 0.006 g MoO3: 0.002 g sucrose: 10 g<BR>
Solution B: 0.04% MgSO4; 0.02% CaSO4;  0.0006% FeSO4/7H2O; 0.0002% MoO3;  1.0% mannitol. <BR>
Add distilled water to make 0.5 L<BR>
<BR>
Aseptically combine 1A:1B after autoclaving; for plate version, add 0.25 multivitamin mix, and for solid medium 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<BR></ul>
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.  </ul>


 
Effective concentrations:  0.08%K<sub>2</sub>HPO<sub>4</sub>; 0.02%KH<sub>2</sub>PO<sub>4</sub>, 0.02% MgSO<sub>4</sub>
<BR><BR>
0.01% CaSO<sub>4</sub>; 0.0015% FeSO<sub>4</sub>/7H<sub>2</sub>O; 0.00025% g MoO<sub>3</sub>;  0.5% sucrose (2010,2011).          <BR><BR>
'''Miscellaneous media and reagents'''<BR><BR>
'''Miscellaneous media and reagents'''<BR><BR>


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(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.)
(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.)
<UL><LI>
<UL><LI>
2g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 4g KH<sub>2</sub>PO<sub>4</sub>, 4g Na<sub>2</sub>HPO<sub>4</sub>
0.2% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 0.4% KH<sub>2</sub>PO<sub>4</sub>, 0.4% Na<sub>2</sub>HPO<sub>4</sub>
0.2g MgSo<sub>4</sub>7H<sub>2</sub>, 0.001gCaCl<sub>2</sub>, 0.001g FeSO<sub>4</sub>7H<sub>2</sub>O, pH 7.8.  <LI>
0.02% MgSo<sub>4</sub>7H<sub>2</sub>, 0.0001%CaCl<sub>2</sub>, 0.0001% FeSO<sub>4</sub>7H<sub>2</sub>O, pH 7.8.  <LI>
Supplement this medium with the desired hydrocarbons.
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>
e.g. 0.5% (v/v) hexadecane for ''Acinetobacter sp''., 1%(v/v) hexadecane ''Arthrobacter sp'', or ''Corynebacterium''. </LI></UL>
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'''Cellulose degradation medium'''
'''Cellulose degradation medium'''
<ul><li>
<ul><li>
acid washed cellulose....
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.




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<BR><BR>
<BR><BR>
'''STOCK: Cycloheximide solution (1000 ug/ml in 70% ethanol).''' <br> 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.  
'''STOCK: Cycloheximide solution (50 mg/ml in 70% ethanol).''' <br> 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.  
<UL><LI>
Unless otherwise indicated,dilute stock to 1mg/ml final concentration in medium.
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.<BR>
Unless otherwise noted add 1 ml of 50 mg/ml stock to 1 liter of medium = 50ug/ml final concentration in medium.</LI></UL>
<BR>
<BR>




'''1x PBS:'''<BR>
'''1x PBS:'''<BR>
8 g/l NaCl, 0.2 g/l KCl, 1.43 g/l Na2HPO4, 0.24 g/l KH2PO4<BR><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
'''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.<BR><BR>
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 test reagent media==
==Additional misc test reagent media==
'''Luria Bertoni Broth'''  <BR>
'''Luria Bertoni Broth'''  <BR>
<UL><LI> 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. </LI></UL>
<UL><LI> Bacto-tryptone 1%; yeast extract 0.5%; NaCl 1.0% at pH 7.5 </LI></UL>


'''Sulfur Reduction/Indole Production/Motility media (SIM)'''<BR>
'''Sulfur Reduction/Indole Production/Motility media (SIM)'''<BR>
<UL><LI>  
<UL><LI>  
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,  
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>
</LI></UL>
'''MOPS'''<BR>
[[image:mopsmmRECIPE-1a.jpg]]


==Links to Labs==
==Links to Labs==

Latest revision as of 13:49, 20 May 2011

Wellesley College-BISC 209 Microbiology -Spring 2011


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


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