in this chapter.
Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa. Unfortunately, the digest used almost exclusively in the development of these media has not been available since the early 1980s. Many digest products have been tried in the interim with marginal results in supporting the growth of E. histolytica. Diamond et al. (14) have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture, and serum. This may serve as a replacement for TYI-S-33, widely used for the axenic culture of E. histolytica and other intestinal protozoa.
Specimens include stool material, mucus, or a combination of the two. The clinical specimen(s) should be no more than 24 h old; a maximum of 2 to 3 h is recommended.
Balamuth’s Aqueous Egg Yolk Infusion Medium for Amebae
Balamuth’s aqueous egg yolk infusion medium is used to detect the presence of amebae. The specific solutions required are phosphate buffer and whole-liver concentrate solution.
Balamuth’s Aqueous Egg Yolk Infusion Medium
Phosphate buffer
Mix the solution in the ratio of 3 parts tribasic (A) to 2 parts monobasic (B) to make 1 M phosphate buffer stock. Dilute the stock buffer to 0.067 M before use (add 492 ml of distilled H2O to 1 liter of 1 M phosphate buffer).
Whole-liver concentrate solution
Suspend the powder in cold water, and autoclave. Filter through a Büchner funnel to remove sediment, dispense in 10-ml quantities, and reautoclave.
Preparation of Complete Medium (15)
1. Using a blender, blend 12 fresh hard-boiled egg yolks with 375 ml of 0.8% sodium chloride.
2. Autoclave at 7 lb/in.2 pressure for 10 min, repressurize slowly, and stir.
3. Autoclave again for 45 min at 7 lb/in.2 pressure.
4. Allow to cool slightly, and add distilled water to replace evaporation loss. Transfer the material to a muslin bag, and express the liquid portion, saving all the fluid. Return the volume to 375 ml with 0.8% sodium chloride.
5. Autoclave for 20 min at 121°C; cool to 5°C. Do not agitate the fluid at this point or during filtration.
6. Decant the fluid carefully through gauze into a Büchner funnel with Whatman no. 3 filter paper. Filter papers can be replaced as necessary.
7. Measure the filtrate, add an equal volume of 0.067 M phosphate buffer, and autoclave for 20 min at 121°C. After cooling, add stock liver concentrate (1 part stock liver to 9 parts medium).
8. Material can then be decanted into sterile flasks, which are stored until the medium is dispensed.
Procedure
Tubes should contain 6 to 8 ml of fluid and should be incubated for 4 days at 37°C as a sterility check. Before inoculation, a loopful of sterile rice powder or starch (can be autoclaved in a screw-cap tube) is added to each tube. To each tube, add stool material, mucus, or a combination of the two (about the size of a small pea), break it up thoroughly in the medium, and incubate at 37°C. The cultures should be checked at 2, 3, and 4 days by examining 0.1 ml of sediment under the microscope (low-intensity light) for characteristic motility. Although the initial culture may appear to be negative, subcultures may reveal organisms (Fig. 8.1).
Figure 8.1 Protozoa from culture systems. (Upper left) Entamoeba histolytica/E. dispar trophozoite from liquid medium containing rice starch (note that there are no definitive erythrocytes within the cytoplasm, so that it is not possible to differentiate the true pathogen, E. histolytica, from the nonpathogen, E. dispar). (Upper right) Naegleria fowleri trophozoite from nonnutrient agar culture with bacterial overlay (note that this trophozoite has been stained). (Lower left) Acanthamoeba spp. trophozoite from nonnutrient agar culture with bacterial overlay (note the spiky acanthapodia). (Lower right) Acanthamoeba spp. cysts from nonnutrient agar culture with bacterial overlay (note the double hexagonal wall appearance. doi:10.1128/9781555819002.ch8.f1
According to Dolkart and Halpern (16), the addition of gastric mucin to the egg component is reported to improve the performance of Balamuth’s medium.
The addition of rice flour (prerinsed with distilled water) to Balamuth’s medium is reported to support abundant growth of 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.