Catalase Agar

  1. Prepare and autoclave whatever type of medium is required. Place on stir plate to cool, ~1 h.
  2. Weigh out ~50,000 Units of catalase per L of medium. Each catalase prep will say on the label “X Units/mg solid.” This is how you calculate concentration in Units.
  3. Pour ~ 10 mL of 37º C catalase buffer (phosphate buffer, pH 7, see below) into a clean 15 mL conical tube. Exact volume isn’t important.
  4. Add catalase and mix by inversion. Don’t shake. You will see some undissolved “floaters” in the prep; this is unavoidable, but the rougher you are with the prep the more there will be.
  5. When medium is cool enough to pour, place in hood and flame opening. Using a 10 mL syringe fitted with a 0.22 μm Millex-GV syringe filter (the yellow ones, made with a PVDF low-protein binding membrane), filter the catalase prep directly into the medium. Mix thoroughly.
  6. Pour plates. Mark with a silver stripe to indicate catalase addition.

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Catalase buffer

1.7 g KH2PO4*3H2O
~ 14 mL 0.1 N NaOH
to 250 mL H2O, pH 7 @ 37C

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Catalase, 0.1%

  1. Weigh 10 mg powedered catalase and add to ~ 10 mL catalase buffer (see below).
  2. Filter sterilize using 0.2 um PVDF filter (Millex GV, yellow syringe filter)
  3. Store in the dark at room temperature for ~ 1 week.
  4. This solution may be used as a 100X solution to treat liquid growth media. Add 10 uL per 1 mL of culture.

 

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LB+SUCROSE AGAR

For selecting against sacB-containing strains

Per L:
In one flask mix:
Tryptone 10 g
Yeast Extract 5 g
Agar, 10 g
750 mL water

In a second flask mix:
50 g sucrose
250 mL water

Autoclave separately and mix while still hot.

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MINIMAL GLYCEROL Medium

Per L, mix and autoclave:
K2HPO4*3H2O 7 g
KH2PO4 2 g
(NH4)2SO4 1 g
Sodium citrate 0.5 g
80% glycerol 2 mL

After removing from the autoclave add:
20% L-arabinose 1 mL
1M MgSO4 1 mL
0.2% Vitamin B1 1 mL

 

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ODA Agar

For detecting peroxidase activity

Per L, mix and autoclave:
Tryptone 10 g
Yeast extract 5 g
NaCl 10 g
Agar 15 g

After medium is cool enough to handle, add:
20% L-arabinose 5 mL
60 mM o-dianisidine 10 mL

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o-dianisidine, 60 mM

Add 3.675 g o-dianisidine powder to ~ 200 mL milli-Q H2O in flask with stir bar. While stirring, gradually add 1N HCl just until powder goes into solution. Filter sterilize, cover in foil, and store in the refrigerator.

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PLAG

For growing marine bacteria and testing for contamination in phytoplankton cultures

Per L, mix and autoclave:
960 mL milli-Q H2O
Sigma Sea Salts 27 g

After cooling, add:
0.8 M NH4Cl 1 mL
0.05M NaH2PO4 1 mL
Pro99 Trace Metals (Andersen)  1 mL
F/2 Vitamins  1 mL
5% Sodium pyruvate  10 mL
5% Sodium lactate  10 mL
5% Potassium acetate  10 mL
5% Glycerol  10 mL

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YTSS

For growing marine bacteria and testing for contamination in phytoplankton cultures

Per L, mix and autoclave:
Tryptone 4 g
Yeast extract 2.5 g
Sigma Sea Salts 15 g

For purity tubes, distribute 3 mL each to glass test tubes using pump.