MODIFIED DIAMONDS MEDIUM

Cat. no. K02 Modified Diamonds Medium, 16x100mm Tube, 6ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Modified Diamonds Medium is recommended as an enriched and selective medium for the isolation and cultivation of Trichomonas species from clinical specimens, especially T. vaginalis.

SUMMARY

The cultivation of T. vaginalis is the most sensitive method for the diagnosis of this sexually transmitted organism. Modified Diamonds Medium has been found to be an effective medium for the culture of this organism. The medium is enriched with yeast extract and supplemented with inactivated horse serum, amphotericin B, penicillin G, and gentamicin. Modified Diamonds Medium is formulated to allow trichomonads to grow, while suppressing bacterial growth. The addition of small amounts of agar reduces the oxygen tension, resulting in more prolific growth of trichomonads, which optimally grow and reproduce under anaerobic conditions.(8) Culture medium techniques allow for a more accurate method for detecting Trichomonas species, and are an effective means of determining treatment efficacy.

FORMULA

Ingredients per 880ml of deionized water:*

Pancreatic Digest of Casein 15.0gm
Yeast Extract 12.0gm
Glucose 5.5gm
Sodium Chloride 2.5gm
L-Cystine 0.5gm
Sodium Thioglycollate 0.5gm
Gentamicin 80.0mg
Amphotericin B 2.0mg
Penicillin G 1,000,000U
Horse Serum 120.0ml
Agar 0.75gm

Final pH 7.0 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (evaporation or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated into an appropriate transport medium, and kept at 30-35ºC. until inoculation.

Method of Use: Allow the Modified Diamonds Medium to come to room temperature before inoculation.

Vaginal or urethral discharges and prostatic secretions collected at the time of examination may be used to inoculate the medium, which is then taken directly to the laboratory for incubation. Urine specimens or other inoculum with suspected low numbers should be centrifuged and the sediment used as inoculum. (Additionally, at the time of collection, a fresh wet smear should be examined microscopically for the presence of trophozoites). Consult appropriate references for further inoculation methods.(2-4)

Modified Diamonds Medium is inoculated by immersing the specimen in the medium, and gently twirling. Breaking the swab to leave in the medium is not necessary. Incubate with tight cap at 30-35ºC. for 1-3 days. Examine the culture after 24 hours of incubation microscopically for the presence of trophozoites.* Aseptically remove a drop of the culture and place it on a slide and cover with a glass coverslip. Examine under 100x-400x magnification. If the organism is not seen, incubate the culture again for up to three days, examining daily or every other day in the same manner. If after three days no trichomonads are seen, the specimen is considered negative.

*Specimens collected at an off site clinic, or delayed in transport to the laboratory can be examined after 2 days of incubation to increase sensitivity.

It is suggested to culture the patient one week after therapy is completed. If the culture is negative, a second culture is taken in two to four weeks. If this third culture is negative for the presence of trophozoites, treatment was effective.

INTERPRETATION OF RESULTS

The wet mount is examined under 100x-400x magnification, with phase contrast and/or differential interface contrast optics preferred. If motile trophozoites are observed between 7-23mm in size, the test is considered positive for Trichomonas vaginalis. If no trophozoites are seen after three days of incubation, discard and report as negative.

LIMITATIONS

Modified Diamonds Medium, is a selective medium and may, to some extent, inhibit the specific strains it is designed to grow. Other microorganisms may grow in this medium as microbial resistance to the antibiotics contained within this medium can occur.

Due to the fastidious nature of T. vaginalis, the culture will remain viable for a short period of time after reaching the stationary phase. The use of a daily wet mount preparation cannot be over emphasized to achieve consistently accurate results.

It is recommended to perform quality control on the media each week of testing. Positive controls should be subcultured every three to four days to maintain viability.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, pasteur pipets, slides, coverslips, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

The microscope should be calibrated (within the last 12 months), and the objectives and oculars used for the calibration procedure should be in place on the microscope when objects are measured.(5)

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Trichomonas vaginalis
Clinical strain
A 24-72hr 35°C Aerobic Growth; twitching motility seen microscopically at
10x-40x - wet mount
Candida albicans
ATCC ® 10231
B 72hr 35°C Aerobic Inhibited
Escherichia coli
ATCC ® 25922
B 72hr 35°C Aerobic Inhibited

User Quality Control

PHYSICAL APPEARANCE

Modified Diamonds Medium should appear clear, and light amber in color.

T. vaginalis growing in Modified Diamonds Medium

Trichomonas vaginalis (Clinical strain) growing in Modified Diamonds Medium (Cat. no. K02). Incubated aerobically for 96 hours at 35ºC. Showing turbidity in lower portion of tube.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Garcia, L.S. and D.A. Bruckner. 2007. Diagnostic Medical Parasitology, 5th ed. American Society for Microbiology, Washington, D.C.

5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

6. Lossick, L.G. 1988. The diagnosis of vaginal trichomoniasis. J. Am. Med. Assoc.; 259:1230

7. Schmid, G.P., et al. 1989. Evaluation of six media for the growth of Trichomonas vaginalis from vaginal secretions. J. Clin. Microbiol.; 27:1230-1233.

8. Poch, F., et al. 1996. Journal of Clinical Microbiology; Vol. 34, No. 10, p. 2630-2631.

9. Gelbart, S.M., et al. 1990. Journal of Clinical Microbiology; Vol. 28, No. 5, p. 962-964.

ATCC is a registered trademark of the American Type Culture Collection.

032816gr