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Alternaria Broth

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					                                  Alternaria Broth
                        For toxin production and DNA extraction


ddH2O                           1 liter                2 liters              3 liters
DL Asparagine                    1.2 g                  2.4 g                 3.6 g
NaCl                             0.1 g                  0.2 g                 0.3 g
K2HPO4_3H2O             1.2 g                  2.4 g                 3.6 g
MgSO4 (anhydrous) **            0.25 g                  0.5 g                0.75 g
Yeast Extract                    0.5 g                  1.0 g                 1.5 g
CaCl2 (not necessary)           0.13 g
D-Glucose (anhydrous)           20.7 g                 41.4 g                62.1 g
(=Dextrose)
Heat at moderate temperature to get into solution
Dispense 100ml into 250ml flasks, cap flasks with a cotton foil lid and autoclave.




      Grow fungi on PDA until plate is populated sufficiently, app. 4-5 days.
      Using a sterile 10 ml pipette, aseptically cover the culture with 5ml sterile ddH2O.
      Gently rub the mycelia with the tip of the pipette to loosen the spores. Tip the
       petri dish to stir up the spores.
      Using good sterile technique, transfer 2mls of the ddH2O-fungal suspension into
       the 250 ml. flasks.
      Shake at 115 rpm at RT for 5-10 days.




** MgSO4*7H2O           0.5 g
PDA
     39g      Potato Dextrose Agar
     1000 ml ddH2O
     Autoclave

Acidified PDA
     Make the PDA medium
     Autoclave, let cool to 60OC
     Add 1.25 ml of 20% lactic acid per liter (wear gloves)

Weak PDA
     1.2g     Potato Dextrose Broth
     16 g     Bacto-Agar
     1000ml ddH2O
     Autoclave

Skim Milk
     15g      Skim Milk
     1000ml ddH2O
     Dispense 4ml to each test tube. Autoclave.
     Cool, store in the media refrigerator.


LB Media (Luria-Bertani Medium)
     10g.      Bacto-Tryptone
     5g.       Bacto-Yeast Extract
     10g       NaCl
     950 ml ddH2O
     Stir until the solutes dissolve. Adjust the pH to 7.0 with 5 N NaOH (app. 0.2 ml).
      Adjust the volume of the solution to 1 liter with ddH2O.
     Autoclave

LB Media
     10g.      Bacto-Tryptone
     5g.       Bacto-Yeast Extract
     5g        NaCl
     950 ml ddH2O
     Stir until the solutes dissolve. Adjust the pH to 7.0 with 5 N NaOH (app. 0.2 ml).
      Adjust the volume of the solution to 1 liter with ddH2O.
     Autoclave
2 % Water Agar
       20g.      Difco Agar, granulated
       1000ml    ddH2O
       Autoclave

0.2 % Water Agar, for Dilution Plating
       2g.         Difco Agar, granulated
       1000ml      ddH2O
       Boil to get into solution. Dispense at 40ml/milk dilution bottle. Cap.
       Autoclave

V-8 Juice Medium
       87.5ml      V-8 juice
       1.5g.       CaCO3
       10g.        Bacto Agar
       500ml.      ddH2O
       Autoclave

Recipe for V-8 Slants
    For 1000 ml.                         For 500 ml.
       175 ml. V-8                      88 ml.   V-8
       3g.       CaCO3                  1.5g.    CaCO3
       20g.       Bacto Agar            10g.     Bacto Agar
       800ml.    ddH2O                  400ml.   ddH2O


Heat until agar dissolves. Use aspirating syringe to dispense into test tubes: use
6mls/tube. Rinse syringe with hot soapy water as soon as you are finished dispensing,
then distilled water. Autoclave. When the tubes come out of the autoclave, lean tubes
against a crisper lid to create the slant.




Potato Carrot Agar
       20g.        Carrot
      20g.       Potato (exclude the skin)
      20g.       Bacto Agar
Dice the vegetables into small pieces. Place pieces into a beaker with aprox. 200ml
ddH2O. Boil on stir/hot plate until carrot is soft, aprox. 50-60 min.on low heat (#3-4).
Force slurry through a fine sieve (available in Mary Olson’s lab, Marley 719). Add
ddH2O to make l liter, then add agar. Mix and autoclave. While pouring plates,
occasionally swirl to keep carrot/potato mix suspended.




                                   ARSA MEDIA
This medium was developed specifically for the isolation and
enumeration of A. radicina from carrot seed and field soil.

Step I
ARSA is prepared in two parts:
Part A
16.0g        Bacto Agar
1.0g         KH2PO4
1.0g         KNO3
0.5g         KCl
0.5g         MgSO4
500ml        ddH2O

Part B
5.0 g Glucose (normally prepared with Sodium polypectate
               (Sigma P-1879)
500 ml ddH2O
(Mix to get into solution, then remove the stir bar from Part B!!!)

Autoclave Parts A and B separately, cool to 50 C, and combine. Use good
sterile technique.

Step II

1)        Prepare Antibiotics:

 50mg        chlortetracycline HCl (Sigma C-4881)
 50mg        streptomycin sulfate (Sigma S-6501)

Place each into a sterile 15 ml centrifuge tube; add 10 ml sterile ddH2O
and vortex to get into solution.

2) Prepare Fungicides:

 A) Mertect 340-F (contains 106 mg thiabendazole, Merick & Co.)
        -Mertect has the consistency of sludge: you need to stir
     vigorously to get it into solution. Use a sterile 10ml pipette,
     and stir for app. 4-5 min. until you get a consistent mixture.
        -Make a 10X stock solution by placing 1 ml Mertect with 9 ml
     sterile ddH2O into a sterile 15 ml centrifuge tube and vortex.

 B)        2.5 ml 10X   Mertect
                5mg     Botran 75WP (contains 4 mg dicloran, Upjohn Co.)
              200mg     Bayleton 50WP (contains 100 mg triadimefon, Miles)
             7.5mls     sterile ddH2O

Place each into a 15 ml centrifuge tube and vortex to get into
solution.

Step III
Add the antibiotics and the fungicides to the combined parts A and B.
Pour plates.



KMB (Kings Medium B Agar)
          20g.   Proteose Peptone No. 3 (Difco)
          1.5g.  K2HPO4
          0.738g MgSO4 (Anhydrous) or 1.5g MgSO4*7H2O
      10ml     Glycerol
      990ml ddH2O
      15g      Bacto Agar
      Mix Proteose Peptone, K2HPO4, MgSO4, and ddH2O till in solution. Add
       glycerol with a pipette. Rinse the pipette in the solution to remove residual
       glycerol from the inside of the pipette. Adjust pH to 7.4. Add agar and autoclave.



YDC (Yeast Dextrose Carbonate Agar)
Solution I
    20g.       Dextrose
    800 ml ddH2O
Solution II
    10g.      Yeast Extract
    20g.       CaCO3
    10 ml      Glycerol
    800ml ddH2O
    15g        Bacto Agar
    Make Solution I and Solution II, remove the stir bar from Solution I only.
       Autoclave separately. Combine both solutions using good sterile technique. The
       CaCO3 can easily precipitate, so gently mix media periodically while pouring the
       media into plates or tubes.




Komada’s Medium (1975)
      1g.       K2HPO4 (or 1.3g K2HPO4*3 H2O)
      0.5g.     KCl
      0.25g     MgSO4 Anhydrous (or 0.5g MgSO4*7H2O)
      2g        L-asparagine
      20g      D-galactose
      10mg     Fe-Na-EDTA
      20g      Bacto-Agar
      1000ml ddH2O
      Autoclave, then add to the warm media (cool to 55*C first):
      1g       Quintozene (PCNB)
      0.5g     Oxgall
      1g       Na2B4O7*10H2O
      300mg Streptomycin sulfate
      Adjust pH to 3.8+0.2 with a solution of 10% phosphoric acid.


Isolation from soil: dilution plate method. Keep the dishes in diffuse light for incubation.
Under these conditions, the colonies of F. oxysporum are pigmented with a reddish
purple color and surmounted by a pinkish white aerial mycelium.


Nash and Snyder’s Medium (1962)
      15g.     Peptone
      1g.      K2HPO4
      0.25g    MgSO4 Anhydrous (or 0.5g MgSO4*7H2O)
      20g      Bacto-Agar
      1000ml ddH2O
      Autoclave, then add to the warm media (cool to 55*C first):
      1g       Quintozene (PCNB)
      100mg Penicillin
      300mg Streptomycin sulfate


Prepare the petri dishes 5-7 days before proceeding with the isolations. Incubate in dark
@24 *C. Light will kill Fusarium. Too moist plates will favor bacteria.

				
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