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Note The shelf life of solutions 1, 2, and 3 is 22 months.
TYI-S-33 (Keister’s Modification) for Giardia lamblia
For TYI-S-33 (Keister’s modification [2]), prepare TYI broth exactly as for E. histolytica with the following changes.
(i) Increase the amount of L-cysteine hydrochloride to 2.0 g/liter.
(ii) Add 500 mg of dehydrated bovine bile per liter.
(iii) Adjust the pH to 7.0 to 7.1.
(iv) Sterilize by filtration through a 0.22-µm-pore-size filter. Do not autoclave! The complete medium is made by addition of bovine serum to 10%.
TYSGM-9 Medium for Entamoeba histolytica
Nutrient Broth
The nutrient broth may be stored for several months at –2°C.
5% Tween 80 Solution
1. Vigorously stir, with a magnetic stirrer, 95 ml of glass-distilled water in a bottle.
2. Add 5 g of Tween 80 (very thick solution; must be weighed), and keep stirring for a few minutes.
3. Filter sterilize through a 0.22-µm-pore-size membrane.
4. Aseptically dispense into a number of sterile screw-cap test tubes, 10 ml per tube.
5. Label as 5% Tween 80 solution, with the preparation date and an expiration date of no longer than 1 month. Store at 4°C.
Phosphate-Buffered Saline (PBS no. 8), pH 7.2
1. Dissolve the salts in the distilled water with a magnetic stirrer.
2. Autoclave for 15 min at 121°C.
3. When cool, label as PBS no. 8 with the preparation date and an expiration date of 3 months.
Rice Starch
For best results use rice starch obtained from BDH, Ltd. (Merck, Ltd.) or Gailard Schlesinger, Inc.
1. Dispense 500 mg of rice starch into each of several 16- by 125-mm screw-cap tubes; do not tighten the caps.
2. Place the tubes horizontally in a dry-heat sterilizer or an oven. Make sure that the rice starch is uniformly distributed loosely over the undersurface of the tubes.
3. Heat the tubes for 2.5 h at 150°C.
4. When cool, tighten caps and label as rice starch with the date of preparation and an expiration date of 3 months.
Rice Starch Suspension
1. Add 9.5 ml of sterile PBS no. 8 to each tube of rice starch.
2. Shake vigorously or use a vortex machine to uniformly suspend the rice starch at the time of use.
Stock Antibiotic Solution
1. Using a 6-ml syringe and 20-gauge needle, add 5 ml of sterile distilled water to a vial of penicillin G sodium (106 U).
2. Using a 6-ml syringe and 20-gauge needle, add 5 ml of sterile distilled water to a vial of streptomycin sulfate (106 µg/ml).
3. Shake gently, and let stand for 30 min to dissolve the antibiotics completely in the distilled water.
4. Mix the two antibiotics in a graduated flask or cylinder, and bring the volume to 125 ml with distilled water. The stock concentration of antibiotics is 8,000 U of penicillin/ml and 8,000 µg of streptomycin/ml.
5. Filter sterilize the antibiotic solution through a 0.22-µm-pore-size membrane filter, dispense the filtrate into a number of sterile screw-cap vials or sterile cryovials (1 ml per vial), and label as stock antibiotic solution with the preparation date and an expiration date of 6 months. Store at 120°C in a cryovial box.
Buffered Methylene Blue Solution
Solution A, 0.2 M acetic acid
Add the acetic acid to the water, mix, and store in a glass-stoppered bottle. Label with the date of preparation and an expiration date of 1 year.
Solution B, 0.2 M sodium acetate
Dissolve the sodium acetate in 400 ml of distilled water in a volumetric flask, bring the volume to the 1,000-ml mark, mix well, and store in a glass-stoppered bottle. Label with the date of preparation and an expiration date of 1 year.
Acetate buffer, pH 3.6
Mix solutions A and B in a volumetric flask, and bring the volume to 100.0 ml with distilled water. The pH should be 3.6. Store in a glass-stoppered bottle. Label with the date of preparation and an expiration date of 1 year.
Methylene blue stain
Dissolve the dye in the buffer, and store in a glass-stoppered bottle. Label with the date of preparation and an expiration date of 1 year.
Complete Medium (TYSGM-9 Medium)
1. Place 200 mg of gastric mucin (U.S. Biochemical Corp., catalog no. 16025) in a 125-ml screw-cap bottle or Erlenmeyer flask.
2. Add 97 ml of nutrient broth; using a magnetic stirrer, stir vigorously for at least 1 h or until the medium becomes clear.
3. Autoclave for 15 min at 121°C; cool to room temperature.
4. Add aseptically, in a biological safety cabinet, 5.0 ml of heat-inactivated bovine serum.
5. Add 0.1 ml of the 5% Tween 80 solution.
6. Dispense aseptically, in a biological safety cabinet, into a number of sterile 16- by 125-mm screw-cap tubes, 8 ml per tube.
7. Add 0.25 ml of rice starch solution after vigorously shaking the tube.
8. Store the tubes at 4°C for not more than 1 month.
9. The final pH of the medium should be 7.2.
Robinson’s Culture
Robinson’s medium is a complex medium that has nevertheless found widespread use for the isolation of enteric amebae. To prepare Robinson’s medium, prepare the six following stock solutions (2).
Solution 1 (0.5% Erythromycin) (Filter Sterilized)
Prepare 0.5% erythromycin in distilled water and filter sterilize. Refrigerate.
Solution 2 (20% Bacto Peptone) (Autoclave)
Prepare 20% Bacto Peptone in distilled water. Autoclave and refrigerate.
Solution 3 (10× Phthalate Solution, Stock) (Autoclave)
Bring to 1 liter at pH 6.3. Autoclave for 15 min at 121°C under a pressure of 15 lb/in.2. Store at room temperature. Dilute 1:10 with sterile water before use.
A stock solution of phthalate-Bacto Peptone can be made by adding 1.25 ml of 20% Bacto Peptone per 100 ml of 1× phthalate solution. Store refrigerated.
Solution 4 (10× R Medium Stock) (Autoclave)