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)
Bring to 500 ml. Dilute stock 1:10, adjusting pH to 7.0. Autoclave for 15 min at 121°C under a pressure of 15 lb/in.2 in 20-ml amounts.
Solution 5 (BR Medium)
To prepare BR medium, inoculate 1× R medium with a standard Escherichia coli strain such as O111. Incubate at 37°C for 48 h, and store at room temperature (good for several months).
Solution 6 (BRS Medium)
To prepare BRS medium, add an equal volume of heat-inactivated bovine serum to BR medium and incubate at 37°C for 24 h. Store at room temperature (good for several months).
1. To prepare agar slants, many people use screw-cap glass bijou bottles (total volume, 7 ml), but standard culture tubes also work well.
2. Autoclave a solution of 1.5% Noble agar in 0.7% NaCl-distilled water for 15 min at 121°C under a pressure of 15 lb/in.2.
3. Dispense in 5-ml (tube) or 3-ml (bottle) amounts, reautoclave, and slant until cool and set. For slants in tubes, use an angle that produces a 12- to 15-mm (ca. 0.5-in.) butt.
4. When cool, tighten lids and store at room temperature or refrigerated.
5. To one tube or bottle, add the following: 3 ml of 1× phthalate-Bacto Peptone, 1 ml of BRS medium, and 50 µl of erythromycin. This must be done on the same day as the inoculation. Note that although erythromycin is added to Robinson’s medium at every subculture, this does not lead to a monoxenic culture as occasionally stated. Additional antibiotic treatment would be needed for this to be the case.
Xenic Culture
When initiating xenic cultures, the stool samples should be inoculated into at least two tubes, one with and the other without antibiotics. In some cases, some component of the natural bacterial flora may be helpful or even necessary for the amebae to become established.
1. Warm several tubes of TYSGM-9 medium in the incubator (35°C for 1 to 2 h).
2. Add 0.1 ml of stock antibiotics to each tube of medium. The final concentration of antibiotics is 100 U of penicillin per ml and 100 µg of streptomycin per ml.
3. After vortexing or vigorously shaking the tube, use a Pasteur pipette to add 3 drops of the starch suspension to each tube of the medium.
4. Place a pea-sized portion of fresh stool sample into the bottom of the tube, and break up the stool gently with the pipette.
5. Tightly cap the tubes, and incubate at a 45 to 50° angle at 35°C for 48 h.
6. Examine the tubes with an inverted microscope and the 10× objective for the presence of amebae. Amebae, if present, are usually seen attached to the underside of the tubes, interspersed with the fecal material and rice starch. Sometimes it is necessary to gently invert the tubes in order to disperse the stool material and rice starch to uncover the amebae. If you do not have an inverted microscope, proceed to step 13.
7. If amebae are not seen, stand the tubes upright for about 30 min at 35°C.
