or urine. Urine samples collected from the patient should be the first voided specimen in the morning. It is critical that clinical specimens be inoculated into culture medium as soon as possible after collection (23–26). Although collection swabs can be used, there are often problems with specimens drying prior to culture; immediate processing is mandatory for maximum organism recovery. The culture method is considered to be the most sensitive for the diagnosis of trichomoniasis; however, because of the time and effort involved, many laboratories have changed to the monoclonal antibody detection kits or more automated molecular methods. Another approach would be to use the plastic envelope method for Trichomonas vaginalis (InPouch TV [BIOMED Diagnostics, San Jose, CA]), a simplified technique for the transport and culture that is illustrated in Fig. 8.3 and 8.4. Because of its long shelf life, relatively low expense, and high sensitivity, studies confirm that the pouch system provides a good diagnostic culture method for T. vaginalis (27, 28). It has been found to be more sensitive than either Diamond’s or Trichosel medium (27).
Figure 8.3 Illustration of the InPouch TV culture system for Trichomonas vaginalis. From top to bottom: (1) introduction of the specimen into the upper chamber containing a small amount of medium; (2) application of a plastic holder for microscope viewing prior to expressing medium into the lower chamber (optional); (3) transfer of a small amount of medium in the upper chamber to the lower chamber; (4) rolling down the upper chamber and sealing it with the tape; (5) plastic viewing frame used to immobilize the medium in the pouch for examination under the microscope. doi:10.1128/9781555819002.ch8.f3
Figure 8.4 InPouch TV culture system for Trichomonas vaginalis. Note the pouch, swabs, and plastic pouch holder for microscopic examination of the pouch contents. doi:10.1128/9781555819002.ch8.f4
Lash’s Casein Hydrolysate-Serum Medium
Lash’s casein hydrolysate-serum medium is a culture medium used to diagnose T. vaginalis.
Lash’s Casein Hydrolysate-Serum Medium
Dissolve in 500 ml of distilled water. To this solution, add:
1. Adjust the pH to 6.0 with concentrated phosphoric acid. Dispense in 5-ml aliquots, plug the tubes, and autoclave at 121°C for 15 min.
2. Prepare serum solution:
3. Sterilize the serum solution by filtration.
4. The complete medium contains 5 ml of serum solution and 5 ml of basic solution for each tube.
5. Incubate the cultures at 37.5°C, and examine 24 and 48 h after inoculation.
T. vaginalis has been studied for a number of years with agar plate cultures for both cloning and diagnosis (2–4). Although broth and agar culture for T. vaginalis are successful, many laboratories have transitioned to molecular based methods. Many media are available for the isolation of T. vaginalis; some of these can be purchased commercially and have a relatively long shelf life.
CPLM (Cysteine-Peptone-Liver-Maltose) Medium
Ringer’s solution
Dissolve the ingredients in the order listed, and bring the volume up to 100.0 ml with distilled water.
Liver infusion
1. Place the distilled water in a large beaker.
2. Add the liver infusion powder.
3. Infuse for 1 h at 50°C.
4. Raise the temperature to 80°C for 5 min to coagulate the protein.
5. Filter through a Whatman no. 1 paper with a Büchner funnel.
Methylene blue solution
Mix well until dissolved.
CPLM (Cysteine-Peptone-Liver-Maltose)
Complete Medium
1. With a magnetic stirrer, mix Ringer’s solution and the liver infusion in a large beaker.
2. Add peptone, maltose, cysteine HCl, and agar in that order, and heat the mixture until dissolved.
3. Add 0.7 ml of aqueous methylene blue.
4. Adjust the pH to 5.8 to 6.0 with 1 N NaOH or 1 N HCl.
5. Dispense 8-ml volumes into culture tubes.
6. Autoclave at 121°C for 15 min.
7. Aseptically add 2 ml of human serum, heat inactivated at 56°C for 30 min and cooled, per tube. Horse serum is recommended as a replacement for human serum, particularly when considering safety issues such as handling human blood and blood products.
8. Label as CPLM medium with the preparation date.
9. Store at room temperature. Use as long as the amber zone indicating an anaerobic condition persists.
Diamond’s TYM (Trypticase-Yeast Extract-Maltose) Complete Medium
1. Dissolve the buffer salts in the distilled water with a magnetic stirrer.
2. Add the remaining ingredients except the agar, in the order given, one at a time until dissolved.
3. Adjust the pH to 6.0 with 1 N HCl.
4. Add agar, and heat to dissolve.
5. Autoclave at 121°C for 15 min.
6. Cool to 45°C, and add 100 ml of bovine, sheep, or horse serum that has been inactivated for 30 min at 56°C.
7. Aseptically dispense 10-ml volumes to 16- by 125-mm screw-cap tubes.
