Purified rice starch is important for all these media. To prepare (7), place 500 mg of powdered rice starch into each of several 16x125mm culture tubes and heat at 150 ° C, with loose caps, in a dry oven for 2.5 hours with the starch distributed along the length of the horizontal tubes. (Autoclaving is unacceptable as the starch will swell with the moisture.) After cooling, tighten the caps and store at room temperature. To prepare for use, add 9.5 ml of sterile distilled water or phosphate buffered saline (PBS) to one tube and vortex to resuspend. Distribute 1 ml of the resuspended starch to each of 10 tubes containing 9 ml of sterile water or PBS and refrigerate. The final concentration of diluted rice starch is 5 mg/ml. Before use, resuspend the rice by vortexing or vigorous shaking and pipet the desired volume into culture tubes with medium making sure that the stock rice stays in suspension. Different isolates require varying amounts of rice starch but 0.2 ml (1 mg) is often a suitable amount to add.

Diphasic Media:

Locke-egg (LE) medium (NIH modification of Boeck and Drbohlav's medium (21))

Prepare Locke's solution: Dissolve:

• 8.0 g Sodium Chloride
• 0.2 g Calcium Chloride
• 0.2 g Potassium Chloride
• 0.01 g Magnesium Chloride
• 2.0 g Sodium Phosphate, dibasic
• 0.4 g Sodium Bicarbonate
• 0.3 g Potassium Phosphate, monobasic

Prepare egg slant:

Surface sterilize fresh hens eggs by flaming in 70% ethanol and break into a graduated cylinder. Add 12.5 ml Locke's solution per 45 ml of egg. Emulsify in a Waring-type blender and filter through gauze into a flask. Place under vacuum to draw out all air bubbles. Add 5 ml amounts of the emulsified egg to standard 16x125 mm culture tubes and autoclave at 100° C for 10 minutes with the tubes at an angle that produces a 12-15 mm (ca. 0.5 inch) butt. The resulting egg slants should be free of bubbles. Cool to room temperature and overlay slants with 6 ml Locke's solution and autoclave 15 minutes, 121° C, 15 lbs. pressure. After cooling to room temperature tighten the caps and refrigerate for up to 6 months,

Robinson's medium (18).

This is a complex medium that has nevertheless found widespread use for the isolation of enteric amebae.

Prepare the following stock solutions:

1. 0.5% Erythromycin in distilled water and filter sterilize. Refrigerate.

2. 20% Bactopeptone in distilled water. Autoclave and refrigerate.

3. 10X Phthalate solution stock:
   • 102 g Potassium Hydrogen Phthalate
   • 50 ml of 40% Sodium Hydroxide

   Bring to 1 liter at pH 6.3. Autoclave 15 minutes, 121° C, 15 lbs. pressure and store at room temperature.

   Dilute 1:10 with sterile water before use. A stock of Phthalate/Bactopeptone can be made by adding 1.25 ml of 20% Bactopeptone per 100 ml 1X Phthalate solution. Store refrigerated.

4. 10X R Medium stock: Dissolve in distilled water:
   • 25.0 g Sodium Chloride
   • 10.0 g Citric Acid
   • 2.5 g Potassium Phosphate, monobasic
   • 5.0 g Ammonium Sulfate
   • 0.25 g Magnesium Sulfate.7H2O
   • 20 ml 85% Lactic Acid solution.

     Bring to 500 ml.

     Dilute stock 1:10 adjusting pH to 7.0 and autoclave 15 minutes, 121° C, 15 lbs. pressure in 20 ml amounts.

   
   
5. BR medium: Inoculate 1X R medium with a standard Escherichia coli strain such as 0111. Incubate at 37° C for 48 hours and store at room temperature (good for several months).

6. BRS medium: Add an equal volume of heat inactivated bovine serum to BR medium and incubate at 37° C for 24 hours. Store at room temperature (good for several months).

Prepare agar slants.

Many people use 1/2 oz.Quorpak bottles but we have also used standard culture tubes with good success. Autoclave a solution of 1.5% Noble Agar in 0.7% Sodium Chloride/distilled water for 15 minutes at 121° C with 15 lbs. pressure. Dispense in 5 ml (tube) or 7 ml (bottle) amounts, re-autoclave and slant until cool and set. For slants in tubes use an angle that produces a 12-15 mm (ca. 0.5 inch) butt. When cool, tighten lids and store at room temperature or refrigerated.

To one tube slant (bottles proportionately more) add: 3 ml 1X Phthalate/ Bactopeptone; 1 ml BRS; 50 µl Erythromycin. This must be done shortly before inoculation.

Monophasic:

TYSGM-9 (8):

Dissolve:

• 2.8 g Potassium Phosphate, dibasic
• 0.4 g Potassium Phosphate, monobasic
• 7.5 g Sodium Chloride
• 2.0 g Casein Digest Peptone
• 1.0 g Yeast Extract

Bring to 950 ml with distilled water. Dispense in 95 ml amounts and add 0.2 g bovine gastric mucin to each bottle. Autoclave for 15 minutes at 121° C with 15 lbs. pressure and store refrigerated. Before use, add 0.1 ml of a filter sterilized 5% stock of Tween 80 in distilled water and 5 ml of heat inactivated adult bovine serum. Dispense in 8 ml amounts into 16x125mm culture tubes.

LYSGM is a modification of TYSGM-9 that removes the need for Casein Digest Peptone. Derived from LYI-S-2 in the same way that TYSGM-9 is derived from TYI-S-33, LYSGM has the identical recipe and preparation to TYSGM-9, except that Casein Digest Peptone is replaced by 0.5g of Neutralized Liver Digest (Oxoid L027) and the amount of Yeast Extract is increased to 2.5g. I also use 0.1g porcine gastric mucin Type II (M2378 from Sigma) per 100ml bottle and it seems to work equally well.

Robinson's medium (18) can also be used in a monophasic form. Established cultures can be transferred to and subcultured in tubes containing the overlay only (with the volume adjusted appropriately).

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