Growth media

01/08/10

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Growth media

 

On the following pages you can find recipes for standard culture media which are generally used for growing Fusarium species and for standard molecular biological procedures

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IMAS and IM

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Defined Fusarium Medium (DFM)

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Complete medium (CM)

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Yeast Peptone Dextrose (YPD)

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Bells medium for increased production of aurofusarin related pigments

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Fries medium

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YES medium

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RA medium for production of macroconidia (asexual spores)

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Carrot agar for inducing the sexual state

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LB medium used for E. coli and A. tumefaciens

 

We normally make sterile stock solutions of the individual components and then mix them under sterile conditions. For solid media we typically start with a water agar solution which has been melted and then cooled to 55 oC. Sterilization: autoclavation (A) or sterile filtration (SF)

 

IMAS-medium (liquid and solid):

CAS 100 ml 500 ml
  2.5x Salt solution (SF or A)   40.0 ml 200.0 ml
  20 % (w/v) Glucose (SF or A)   0.90 ml 4.5 ml
  20 % (v/v) Glycerol (SF or A)   2.50 ml 12.5 ml
  (Agar)   (2 g) (10.0 g)
  H2O (A)   fill to 94 ml fill to 470 ml
  MES-solution (1 M) (SF)   4.0 ml 20.0 ml
  Acetosyringon (10 mM) (SF) (FRESH!!)   2.0 ml 10.0 ml

 

  2.5X Salt solution (1 l): CAS  
    KH2PO4   3.625 g
    K2HPO4    5.125 g
    NaCl    0.375 g
    MgSO4, 7H2O   1.250 g
    CaCl2, 2H2O   0.165 g
    FeSO4, 7H2O   0.0062 g
    (NH4)2SO4   1.250 g
    Add MilliQ water to   1000 ml
         
    Dissolve each salt ONE AT A TIME    
    Filter-sterilize (0.2 mm filter)    
    Store at room temperature.    

                 

  10 mM Acetosyringone (AS) CAS  
    Acetosyringone   0.01962 g
    MilliQ-water   10 ml
         
    Adjust the pH to 8 with 5 M KOH (7.013 g KOH in 25 ml water)    
    Filter-sterilize (0.2 mm filter)    
    Store in 10 ml aliquots at -20 oC    
         
  1 M MES CAS  
    MES hydrate 4432-31-9 19.52 g
    MilliQ-water   80 ml
         
    Adjust pH to 5.3 with 5 M KOH.    
    Bring the volume to 100 ml with MilliQ-water    
    Filter-sterilize (0.2 mm filter)    
    Store in 20 ml aliquots at -20 oC    

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Defined Fusarium Medium (DFM)

CAS   500 ml
  20 % Glucose (SF)     31.25 ml
  50 mM Aspargin (SF)     100.0 ml
  0.21 M MgSO4 (26 g/ 500 ml) (A)      5.0 ml
  1.12 M KH2PO4 + 0.7 M KCl (pH 6) (A)     5.0 ml
  1000 x Trace elements (A)     0.5 ml
  Agar     10.0 g
  MilliQ     348.3 ml

 

  1000 x Trace elements for DFM CAS 1000 ml
    Na2B4O7 * 10H2O, (Borax = Natriumtetraborat)   40 mg
    CuSO4 * 5H2O   400 mg
    FeSO4 * 7H2O   1,2 g
    MnSO4 * 1H2O   700 mg
    NaMoO2 * 2H2O   800 mg
    ZnSO4 * 7H2O   10 g
    Add MilliQ water to   1000 ml
         
         
    Dissolve each salt ONE AT A TIME!    
    Autoclave the solution (note the the iron will oxidate and turn the solution red)    

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Bells (Bell, A.A. Pest Manag Sci 2003)

CAS   1000 ml
  (- all components are mixed from salts)      
  Maltose     20 g
  Urea     0.140 g
  KH2PO4     1 g
  MgSO4 *6H2O       0.5 g
  KCl     0.5 g
  CaCO3     0,5 g
  MES     1 ml
  MilliQ water     fill to 990 ml
         
  Adjust pH to 5.1 with 1 M K2HPO4      
  Add water to 1000 ml and autoclave      

 

  MES = Minor Elements Solution CAS 1000 ml
    FeSO4 *7H2O   10 g
    ZnSO4 *7H2O       50 g
    CuSO4 *5H2O   2.5 g
    MnSO4 *H2O   0.5 g
    NaB4O7   0.5 g
    NaMoO4 *2H2O   0.5 g
    Citric acid   50 g
    Add MilliQ water to    1000 ml
         
    Dissolve each salt ONE AT A TIME!    
Autoclave the solution (note the the iron will oxidate and turn the solution red)

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Yeast extract – sucrose (YES)

  CAS 1000 ml
  (- all components are mixed from salts)      
  Yeast extract     20 g
  Sucrose     150 g
  MgSO4 *7H2O     0.5 g
  Agar     20 g
Trace solution 1.0 ml
  MilliQ water     fill to 990 ml
         
  Adjust pH to 6.5      
  Add water to 1000 ml and autoclave      

                                                 

  Trace solution CAS 100 ml
    ZnSO4 * 7 H2O   10 g
    CuSO4 * 5 H2O       50 g
MilliQ water fill to 100 ml

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Carrot-agar  (Klittitch 1988)

Chop 400 g baby carrots in food processor
   Put carrot in 2 litre glass beaker, add 400 ml deionised water, cover loosely with Saran wrap and microwave 2 x 10 min.
  Let cool 1 – 2 hrs     
  Puree in food processor
  Return to beaker, add 500 ml water, mix well
Dispense evenly into 5 x 500 ml Pyrex bottles (ca. 225 ml per bottle) containing 4 g agar per bottle (~ 1.8 % agar)
  Autoclave 20 min
   
   
   

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Fries medium (Pringle and Braun (1957), Phytopathology (47) p. 369-371)

CAS   1000 ml
Ammonium tartrate 5.0 g
NH4NO3 1.0 g
KH2PO4 1.0 g
MgSO4 *6H2O 0.46 g
NaCl 0.1 g
CaCl2 *2 H2O 0.13 g
  Sucrose     30.0 g
  MnSO4 *H2O     0.76 mg
  Boric acid     1.0 mg
  CuSO4 *5H2O     0.36 mg
ZnSO4 *7H2O 0.1 mg
FeSO4 *7H2O 20 mg
  Bacto Agar     20 g
MilliQ water fill to 1000 ml
  Measured pH in final media it should be 5.6      
Autoclavate

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YPD medium

  1000 ml
Yeast extract 10 g
Peptone 20 g
MilliQ water Fill to 950 ml
Autoclave
  Add 50 ml 50 % glucose (SF) before use    

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Complete medium (CM)

  1000 ml
Succrose 30 g
KH2PO4                                1 g
MgSO4 x 7 H2O 0,5 g
KCl                                        0,5 g
TES** 0,2 ml
NaNO3                              2 g
  Bacto Casamino   2,5 g
  Yeast extract   1 g
  Vitamin solution   10 ml
  MilliQ water   fill to 1000 ml

 

Vitamin solution
Thiamine HCl  100 mg
Riboflavin                                     30 mg
Pyridoxine HCl 75 mg
  D-pantothenate Ca   200 mg
  P-aminobenzoic acid   5 mg
  Nicotinamide                                  75 mg
  Choline Cl   200 mg
Folic acid 5 mg
D-biotin 5 mg
Myo-inositol 4 g
50% ethanol fill to 1000 ml

            

TES**
Citric acid 5 g
Zinc sulfate x H2O 5 g
Fe(NH4)2(SO4)2 x H2O 1 g
  CuSO4 x H2O   0,25 g
  MnSO4 x H2O   50 mg
  H3BO4   50 mg
  NaMoO4 x H2O   50 mg
MilliQ water 95 ml

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RA medium

  1000 ml
Succinic acid (C4H4O4Na2 * 6H2O)  50 g
NaNO3   12.1 g
Glucose     1 g
Vogels salte 50x (-N, -C) 20 ml
MQ water fill to 1000 ml

 

  50 x Vogels stalte     
    Na3-citrat * 2H2O   125 g
    KH2PO4   250 g
    MgSO4 * 7H2O   10 g
    CaCl2 *2H2O      5 g
    200x Trace opløsning   5 ml
MQ water fill to 1000 m

 

  200 x Trace    
    Citric acid *H2O     5 g
    ZnSO4 *7H2O   5 g
    Fe(NH4)2(SO4)2 *6H2O   1 g
    CuSO4 *5H2O   0.25 g
    MgSO4 *H2O   0.05 g
H3BO3   0.05 g
Na2MoO4 * 2H2 0.05 g
MQ water fill to 1000 ml

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LB medium

  1000 ml
Bacto-tryptone 10 g
Bacto-yeast extract 5 g
NaCl (solid) 10 g
MilliQ water add to 950 ml
Adjust pH with 5 M NaOH
  E. coli: 7.4 to 7.5
  A. tumefaciens: 7.7 - 7.8
MilliQ add to 1000 ml
For solid medium add 10 g/L of agar

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Dette sted blev sidst opdateret 01. August 2010