Mesoplama florum:Culture media

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Current revision (16:11, 17 December 2013) (view source)
 
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Autoclave at 121C for 20 minutes and cool to room temperture (broth) or 55C (Agar) on a 55C water bath.
Autoclave at 121C for 20 minutes and cool to room temperture (broth) or 55C (Agar) on a 55C water bath.
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Centrifuge 2 x 45 ml of Gibco 18180-059 liquid yeast extract at high speed to remove sediment. Pour only 45 ml into centrifuge bottles, eliminating as much as possible of the sediment in the bottom of the Gibco stock.
+
While autoclaving, thaw 200 ml of Sigma H-1139 heat inactivated horse serum.
 +
If preparing agar, warm the horse serum to 45C (55C will precipitate protein). Also warm 100 ml of Gibco 18180-059 autolyzed yeast extract 55C without shaking.
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Thaw Sigma H-1138 heat inactivated horse serum.  For agar, bring both horse serum and centrifuged yeast extract to 37C. Horse serum at 55C will sediment protein.
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Add the sterile horse serum and yeast extract to the autoclaved medium. Avoid the yeast extract precipitate if possible.
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Assemble 500  ml bottle top 0.22μ filter on the 1 liter bottle.  For agar, it is helpful to put the bottle on a foam insulating pad to delay cooling.  Add by vacuum filtration:
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Assemble a 150 ml bottle top 0.22μ filter on the 1 liter bottle, or use a 0.22μ syringe filter.  For agar, it is helpful to put the bottle on a foam insulating pad to delay cooling.  Add by vacuum filtration:
{|
{|
|-
|-
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| Horse serum (Sigma H1138)
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| Penicillin G 200,000 U/ml
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| 200 ml
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| 1 ml
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|-
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| Yeast extract
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| 2% phenol red 2 ml or 1% cresol red
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| 90 ml
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| 5 ml
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|-
+
-
| Penicillin G 500,000 U/ml
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-
| 400 μl
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-
|-
+
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| 2% phenol red or 1% cresol red
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| 2 ml (5 ml for cresol red)
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|}
|}
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 +
Mix gently by rolling the bottle to minimize bubble formation. Agar can be placed back in the 55C water bath to allow the bubbles to subside. Poured plates can be swirled to move bubbles to the side of the plate.
Adjust pH to 7.4 with 1 M HCl or 1 M NaOH.  Measured pH of as-made broth is 7.46, probably close enough.  For cresol red containing solution, adjust pH to 7.8
Adjust pH to 7.4 with 1 M HCl or 1 M NaOH.  Measured pH of as-made broth is 7.46, probably close enough.  For cresol red containing solution, adjust pH to 7.8
-
Phenol red 2% solution must be neutralized with sufficient NaOH to convert it to the sodium salt.  The phenol or cresol red amount can be adjusted to control the depth of color to taste.
+
When preparing a phenol red 2% solution it must be neutralized with sufficient NaOH to convert it to the sodium salt.  The phenol or cresol red amount can be adjusted to control the depth of color to taste.
====SP-4 (ATCC 988) Medium====
====SP-4 (ATCC 988) Medium====

Current revision

ATCC 1161 Medium

Mix a base broth in a 1 liter 45mm cap bottle the following chemicals:

Heart Infusion Broth (Difco 0038) 17.5 g
Sucrose 40 g
Agar Noble (Difco 0142) if necessary 12 g
DI Water 710 ml

Autoclave at 121C for 20 minutes and cool to room temperture (broth) or 55C (Agar) on a 55C water bath.

While autoclaving, thaw 200 ml of Sigma H-1139 heat inactivated horse serum. If preparing agar, warm the horse serum to 45C (55C will precipitate protein). Also warm 100 ml of Gibco 18180-059 autolyzed yeast extract 55C without shaking.

Add the sterile horse serum and yeast extract to the autoclaved medium. Avoid the yeast extract precipitate if possible.

Assemble a 150 ml bottle top 0.22μ filter on the 1 liter bottle, or use a 0.22μ syringe filter. For agar, it is helpful to put the bottle on a foam insulating pad to delay cooling. Add by vacuum filtration:

Penicillin G 200,000 U/ml 1 ml
2% phenol red 2 ml or 1% cresol red 5 ml

Mix gently by rolling the bottle to minimize bubble formation. Agar can be placed back in the 55C water bath to allow the bubbles to subside. Poured plates can be swirled to move bubbles to the side of the plate.

Adjust pH to 7.4 with 1 M HCl or 1 M NaOH. Measured pH of as-made broth is 7.46, probably close enough. For cresol red containing solution, adjust pH to 7.8

When preparing a phenol red 2% solution it must be neutralized with sufficient NaOH to convert it to the sodium salt. The phenol or cresol red amount can be adjusted to control the depth of color to taste.

SP-4 (ATCC 988) Medium

Mix a base broth containing:

Mycoplasma broth base (BBL 111458) 3.5 g
Tryptone 10 g
Peptone 5.3 g
Glucose 5.0 g
DI water 615 ml

Adjust pH to 7.5 and autoclave at 121C for 20 minutes.

Centrifuge 2 x 17.5 ml of liquid yeast extract (Gibco 18180-059) at high speed to remove sediment.

Mix 2 g of Yeastolate (Difco 5577) in 100 ml DI water.

Sterile filter into the cooled medium:

penicillin G 500,000 U/ml 400 μl
2% phenol red 2 ml
2% Yeastolate (Difco 5577) 100 ml
Newborn calf serum 170 ml
liquid yeast extract 35 ml
10x CMRL-1066 solution 50 ml
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