Balantidium coli.
Boeck and Drbohlav’s Locke-Egg-Serum (LES) Medium for Amebae
Boeck and Drbohlav’s LES medium is another culture medium used to diagnose the presence of amebae.
Boeck and Drbohlav’s LES Medium
Locke’s solution
Autoclave before storage.
Preparation of Complete Medium
1. Wash four eggs, wipe the shells with 70% alcohol, and break the eggs into a sterile flask containing glass beads.
2. Add 50 ml of Locke’s solution, and shake until homogenous.
3. Dispense the medium so that a slant of 1 to 1.5 in. (1 in. = 2.54 cm) is produced in the bottom of the tube. (Tube size is not critical.)
4. Plug the tubes, and place them in a slant position in an inspissator at 70°C until the slant solidifies. Inspissator conditions may be achieved in the autoclave by leaving the door ajar (nonpressurized system).
5. Autoclave the tubes at 121°C for 20 min. Discard any damaged slants.
6. Prepare a mixture of 8 parts sterile Locke’s solution to 1 part sterile inactivated human serum. Sterilize the mixture by filtration, and incubate at 37°C for 24 to 48 h as a sterility check before use. Cover the slants to a depth of <1 cm with the sterile solution, and inoculate in the same manner as for Balamuth’s medium. LES medium should have a loopful of sterile rice powder added before inoculation.
TY1-S-33 Medium for Entamoeba histolytica (17)
TYI Broth
Note Biosate may be replaced with 20 g of Trypticase (BBL) and 10 g of yeast extract (BBL). Some lots of Biosate, Trypticase, yeast extract, or serum may inhibit growth.
1. Adjust pH to 6.8 with 1 N NaOH, and filter through Whatman no. 1 paper. Autoclave at 121°C for 15 min.
2. Cool, and add:
3. Aseptically dispense 13 ml of complete medium into 16- by 125-mm screw-cap tubes. To prepare Special 107 Vitamin Mix, aseptically combine the following:
Total volume of complete Special 107 Vitamin Mix is 120 ml.
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
