Label as TYM medium with the preparation date and an expiration date of 10 days.
9. Store at 4°C.
Hollander’s Modification of TYM Complete Medium
Hollander’s modification of TYM complete medium differs from TYM medium by the replacement of cysteine with additional ascorbic acid and the addition of potassium chloride, potassium carbonate, and ferrous sulfate (2).
Proceed as described above for Diamond’s TYM complete medium.
Note Hollander also includes agar to 0.05%.
Diamond’s Complete Medium (Modified by Klass)
1. Dissolve the ingredients one at a time in the order given.
2. Adjust the pH to 6.0 with 1 N HCl or 1 N NaOH.
3. Dispense in 12.5-ml aliquots to 16- by 125-mm screw-cap tubes.
4. Autoclave at 121°C for 15 min.
5. When cool (50°C), add 1 ml of sterile inactivated horse serum and 0.5 ml of antibiotic mixture to each tube.
6. Label as modified TYM medium with the date of preparation and an expiration date of 3 weeks.
Antibiotic Mixture
1. Mix thoroughly.
2. Dispense 1 ml of the antibiotic mixture into sterile screw-cap vials or sterile cryovials.
3. Label as antibiotic solution with the date of preparation and an expiration date of 1 year.
4. Store at 220°C in cryoboxes.
Serum Substitutions
Bovine, sheep, or horse serum can be substituted in the step involving CPLM complete medium.
Axenic Culture
1. Inoculation of culture medium.
A. Remove tubes containing culture medium from 4°C, and incubate at 37°C for 1 to 2 h.
B. Vigorously shake the cotton-tipped portion of the applicator stick containing the patient specimen in the medium, and then break off the tip with sterile forceps and drop it into the medium.
C. If the material is collected on polyester sponges, drop the sponges into the medium and shake the tube.
D. Centrifuge urine samples for 10 min at 250 × g, aspirate the supernatant, and inoculate the sediment into the medium.
E. Examine the tubes daily for several days, and subculture if necessary. To subculture, first shake the tube to disperse the organisms uniformly, remove about 1 to 2 ml, and inoculate into a warmed, fresh tube.
F. Incubate the tubes in a slanted position (45° angle).
G. Incubate the control tubes and those containing patient material for at least 72 to 96 h.
H. Examine the entire length of the tube. If the specimen is positive, T. vaginalis will be found freely swimming or attached to the tube walls.
I. Do not report negative results until 96 h.
2. To maintain stock cultures:
A.With an inverted microscope, examine stock culture tubes of T. vaginalis for any signs of bacterial contamination. Select one or more tubes showing good growth. Since the tubes are incubated in a slanted position, usually at an angle of 45°, a thick button of organisms is seen at the bottom of the tube. Gently invert the tube once or twice to disperse the trichomonads uniformly, and examine the tubes again. A large number of the organisms should be freely swimming, and a few will be attached to the tube walls.
B. Immerse the tubes in a bucket of ice-cold water for about 5 to 10 min to dislodge the trichomonads from the tube walls. Invert the tubes several times to distribute the organisms.
C. With a sterile Pasteur pipette, remove about 1.0 to 1.5 ml; inoculate 0.5 to 1.0 ml into a fresh tube. Inoculate the rest of the fluid into nutrient agar, brain heart infusion, and thioglycolate broth for routine monitoring of bacterial contamination. Inoculate several additional tubes this way, and incubate the cultures slanted at a 45° angle at 37°C as before.
D. If growth is poor and only few organisms are present along with a lot of debris, centrifuge the tube at 250 × g for 10 min, aspirate the supernatant, transfer the sediment to a fresh tube, and incubate as before.
Quality Control for Pathogenic Flagellates (T. vaginalis)
ATCC 30001 (T. vaginalis) should be available as a control strain when these cultures are used for clinical specimens.
1. Check all reagents and media (at least once a week). All media, including Ringer’s solution, should be free of any signs of precipitation and bacterial and/or fungal contamination.
2. Depending on use, the microscope(s) may have had to be calibrated within the last 12 months, and the original optics used for the calibration should be in place on the microscope(s). The calibration factors for all objectives should be posted on the microscope for easy access.
3. Maintain stock cultures of ATCC 30001 (T. vaginalis).
A. Transfer stock cultures weekly.
a. Stock organisms should always be cultured at the same time a patient specimen is inoculated into culture medium.
b. If the stock organisms multiply and remain viable during the 96 h, patient results can be reported.
B. Stain
a. A slide prepared from a stock strain of T. vaginalis is run in parallel with the patient slide.
b. Staining results are acceptable when the control organisms stain well.
4. Control organisms must be cultured each time a patient specimen is inoculated into the culture medium.
Note If no trophozoites are seen after 4 days of incubation, discard the tubes and report the culture as negative. Results of patient specimens should not be reported as positive unless control cultures are positive. Cultivation is the most sensitive method for the diagnosis of trichomoniasis. Every effort, therefore, must be made to inoculate patient materials into culture medium. However, since this method may take as long as 3 to 4 days and the patient materials occasionally contain nonviable organisms, it is imperative that microscopic examination of wet smears and/or stained smears (Giemsa) also be performed.
Axenic Cultivation of T. vaginalis in a Serum-Free
