4. Material from the surface of a positive agar plate can be removed, fixed, and stained with trichrome for microscopic examination at a higher magnification (×1,000). Results obtained with wet mounts should always be confirmed by performing permanent stained smears (trichrome, iron hematoxylin) for nuclear characteristics to differentiate the amebae from host cells. Organisms may not be recovered if appropriate centrifugation speeds and times are not used.
Liquid Culture Media for Pathogenic Free-Living Amebae
Although Acanthamoeba spp. can be grown in serum-free liquid medium, N. fowleri requires the addition of fetal calf serum or brain extract to grow in a liquid medium. Peptone-yeast extract-glucose medium for Acanthamoeba spp. and Nelson’s medium for N. fowleri are described below.
Peptone-Yeast Extract-Glucose (PYG) Medium for Acanthamoeba spp.
Final pH is 6.5 ± 0.2.
1. Dissolve all ingredients except CaCl2 in about 900 ml of distilled water in a bottle or flask with a magnetic stirrer.
2. Add CaCl2 while stirring.
3. Bring the volume to 1,000 ml with distilled water.
4. Dispense into 16- by 125-mm screw-cap tubes, 5 ml per tube.
5. Autoclave at 121°C for 15 min.
6. When tubes have cooled, label the tubes as Acanthamoeba medium with the preparation date and an expiration date of 3 months.
7. Store at 4°C.
Nelson’s Medium for N. fowleri
1. Add ameba saline to distilled water to make 1× ameba saline.
2. Dissolve the ingredients in ameba saline with a magnetic stirrer.
3. Dispense into 16- by 125-mm screw-cap tubes, 10 ml per tube.
4. Autoclave for 15 min at 15 lb/in.2.
5. Cool, and label as Nelson’s medium with the preparation date and an expiration date of 3 months.
6. Store at 4°C.
7. Add 0.2 ml of heat-inactivated fetal calf serum to each tube before inoculating with the amebae.
Cell Culture
Acanthamoeba, Balamuthia, Naegleria spp., and Sappinia spp. can be inoculated onto a number of different mammalian cell lines. The organisms grow very well and demonstrate cytopathic effects, similar to those seen in routine viral cultures. However, most laboratories do not offer these methods on a routine basis.
Standard Commercial Media for Growth and Isolation of Acanthamoeba spp.
Several studies have reported on the use of standard commercial media used for growth and isolation of Acanthamoeba spp. Buffered charcoal-yeast extract agar (BCYE) is an excellent commercially available culture medium for the recovery of Acanthamoeba spp. Good trophozoite recovery was obtained using BBL Trypticase soy agar (TSA) with 5% rabbit blood, TSA with 5% horse blood, and Remel TSA with 5% sheep blood. BBL TSA with 5% horse blood or 5% rabbit blood yielded good recovery of cysts. Nonnutrient agar with either live or dead bacteria yielded good recovery of trophozoites; however, live bacteria may be required for good cyst recovery.
Blastocystis spp. (Blastocystis hominis)
Blastocystis is a single-celled enteric protozoan that has a worldwide distribution. Blastocystis belongs to the phylum Stramenopila, is found in humans and animals, and is the most common parasite isolated from human stool samples throughout the world. Rates of infection vary from <5% in developed countries to >50% in developing countries. 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. Xenic or monoxenic laboratory cultures of Blastocystis isolates, which are cultures of Blastocystis cells grown in association with nonstandardized or single known species of microorganisms, respectively, can be maintained in TYGM-9 medium or Boeck and Drbohlav’s inspissated egg medium (see below) (12).
Tryptone/Yeast Extract/Glucose/Methionine) TYGM-9 Xenic Medium
See TYSGM-9 Medium for Entamoeba histolytica earlier in this chapter.
Diphasic LE Medium (Blastocystis spp.)
LE medium is the NIH modification of Boeck and Drbohlav’s medium.
Locke’s Solution
Autoclave for 15 min at 121°C under a pressure of 15 lb/in.2. Cool to room temperature, and remove any precipitate by filtration (Whatman no. 1 paper). Reautoclave to sterilize.
Note The overlay can also be prepared using 90% PBS, 9% sterile horse serum, 1% or 20% [wt/vol] bacteriologic peptone, 1 mg of rice starch, and 500 µl of penicillin-streptomycin solution (12).
Preparation of Complete Medium
1. To prepare the egg slant, surface sterilize fresh hen eggs by flaming in 70% ethanol and break into a graduated cylinder.
2. Add 12.5 ml of Locke’s solution per 45 ml of egg.
3. Emulsify in a Waring-type blender, and filter through gauze into a flask.
4. Place under vacuum to draw out all air bubbles.
5. Add 5-ml amounts of the emulsified egg to standard culture tubes (16 by 125 mm), and autoclave at 100°C for 10 min with the tubes at an angle that produces a 12- to 15-mm (ca. 0.5-in.) butt. The resulting egg slants should be free of bubbles.
6. Cool to room temperature, overlay slants with 6 ml of Locke’s solution, and autoclave for 15 min at 121°C under a pressure of 15 lb/in.2.
7. Let the slants cool to room temperature, tighten the caps, and refrigerate the slants for up to 6 months.
The culture should be examined every 2 days for 1 week (phase-contrast microscopy is recommended) (12). Another option is to use reduced light with the condenser lowered to provide more contrast. If parasites are seen in the sediment, the material can be placed in a fecal preservative for staining and examination using the oil immersion objective (100× oil immersion objective).
Trichomonas vaginalis
Specimens from women may consist of vaginal exudate collected from the posterior fornix on cotton-tipped applicator sticks or genital secretions collected on polyester sponges. Specimens from men can include semen, urethral samples
