Mycoplasma Isolation and Identification Kit (Modified Broth Dilution Method)

Mycoplasma Isolation and Identification Kit (Modified Broth Dilution Method)
Instruction Manual
[Product Name] Mycoplasma Isolation and Identification Kit (Modified Broth Dilution Method)
[Package Specification] 20 servings/box
[Intended Use] Suitable for the culture of Ureaplasma urealyticum and Mycoplasma hominis in clinical settings.
[Principle of Assay] This kit is designed based on the modified broth dilution method, integrating the isolation and cultivation of Uu and Mh from the urogenital tract, their identification and enumeration, as well as susceptibility testing against ten antibiotics. It is easy to use and delivers reliable results.
[Main Components] Composed of culture medium, microtiter plate, sterile pipette tips, and sterile mineral oil.
Kit components:
1. Uu–Mh medium, 20 vials, 3.5 ml per vial 2. Microplate for pore size testing, 20 plates
3. Sterile mineral oil, 2 vials, 20 ml per vial 4. Sterile pipette tips, 20 pieces
5. Report signature 20 copies 6. Instruction manual 1 copy
[Storage Conditions and Shelf Life] Store below 0°C; shelf life is 12 months. See the outer carton or label for the date of manufacture.
[Specimen Requirements] Specimens should be sent for testing immediately after collection; they may be stored at room temperature for no more than 2 hours, or at 2–8°C for no more than 5 hours.
Male: Use a sterile male swab to gently rotate and collect secretions from 2–4 cm inside the urethral opening; prostatic fluid or semen may also be collected for culture. If urine must be used for mycoplasma culture, collect approximately 10–20 mL of the midstream portion of the first-morning voided sample, centrifuge at 2,000 rpm for 10 minutes, and inoculate the pellet; however, the positive rate for mycoplasma in urine is significantly lower than that in urethral secretions.
Women: After using a vaginal speculum to dilate the vagina, gently rotate a female swab at the cervical os, 1–2 cm inside, to collect cervical secretions; amniotic fluid culture may also be used. Urine is not recommended.
[Testing Method]
1. Reagent equilibration: Remove the appropriate number of culture media and test plates based on the number of samples, allow them to reach room temperature, and thoroughly mix the culture media.
2. Blank negative control: Unscrew the vial cap, use a sterile pipette tip to draw 100 µL of culture medium, and dispense it into well A1 of the test plate (the blank control well).
3. Inoculation of the specimen: Insert the swab into the culture bottle, rotate it and squeeze several times to allow the specimen to elute from the swab, then discard the swab; alternatively, directly add 50 µL of a positive culture (obtained from a standard mycoplasma medium); or inoculate the culture medium with 0.1 mL of urine sediment and mix thoroughly.
4. Add samples to the test plate: Pipette 100 µL of sample-containing culture medium into each well (except well A1).
5. Mineral oil–sealed incubation: Add 1–2 drops of sterile mineral oil to each well, cover with the test plate lid, and incubate in a constant‑temperature incubator at 37°C ± 1°C. Observe the results after 24–48 hours and complete the report form as specified.
[Interpretation of Test Results] Uu counts should be reported within 24 hours, Mh counts within 48 hours, and other results within 48 hours, with a maximum delay of 72 hours. If the culture medium in each well remains clear and changes color from yellow to red, this indicates positive mycoplasma growth (+); no color change indicates negative mycoplasma growth (−). If the culture medium in any well becomes turbid, suggesting bacterial or yeast growth, the result should not be recorded; instead, a smear should be prepared for staining and microscopic examination.
As shown in the figure, the microplate strip is divided into two functional zones: culture and identification, and susceptibility testing.
→High concentration
→Low concentration
Types of antibiotics:
Minocycline (MIN) Doxycycline (DOX) Ciprofloxacin (CIP) Sparfloxacin (SPA)
Ofloxacin (OFL) Azithromycin (AZI) Roxithromycin (ROX) Josamycin (JOS)
Clarithromycin (CLA) Spectinomycin (SPE)
Example of interpreting identification and counting results:
    1   2           1   2            1   2            1   2             1    2
A ○ ○ A ○ ○ A ○ A ○ ○ A ○
－/＋ Uu/Mh －/＋ Uu/Mh －/＋ Uu/Mh －/＋ Uu/Mh －/＋ Uu/Mh
B ○ ○ B ○ B ○ B B
No mycoplasma growth Mycoplasma growth, <10^4 Uu growth, ≥10^4 Mh growth, ≥10^4 Both Uu and Mh grow, ≥10^4
Example of interpreting sensitivity test results:
    3     4                      3      4                    3     4
○ ○ ○ ○
MIN DOX MIN DOX MIN DOX
○ ○

Minocycline: Resistant Minocycline: Moderately Sensitive Minocycline: Sensitive
Doxycycline: Sensitive Doxycycline: Resistant Doxycycline: Moderately Sensitive
(Note: The color turns red, while the ○ remains unchanged.)

[Limitations of the Analytical Method]
1. Before inoculating the culture medium into the microtiter plate, ensure thorough mixing; interpret the results based on the readings from the wells. If the medium turns red but remains turbid, do not report a positive result—repeat the test.
2. When the specimen contains excessive secretions, it is recommended to filter the sample before inoculation.
3. Proper specimen collection and timely inoculation are key to improving the positive rate and accuracy.
4. Contraindicated in women with threatened abortion; avoid use after vaginal administration.
5. After repeated freeze–thaw cycles, a small amount of precipitate may appear; however, once the medium is thoroughly mixed, it will not affect the experimental results.
[Product Performance Specifications]
1. Appearance
The culture medium is transparent, appearing as a clear pale yellow liquid; at 25°C, its pH is 6.0 ± 0.5.
2. Analytical Sensitivity
The culture medium exhibits a detection sensitivity of CCU = 10⁻⁴ for the standard strain of Ureaplasma urealyticum ATCC 27618 and the standard strain of Mycoplasma hominis ATCC 14027.
3. Specificity Analysis
The culture medium yielded negative results for Gram-positive bacteria (Staphylococcus aureus ATCC 25923), Gram-negative bacteria (Escherichia coli ATCC 25922), and fungi (Candida albicans ATCC 10231).
4. Compliance Rate
The micropore test plate achieved a 100% concordance rate in the isolation and identification of the standard strain Ureaplasma urealyticum ATCC 27618 and the standard strain Mycoplasma hominis ATCC 14027.
The micropore test plate demonstrated a drug-susceptibility concordance rate exceeding 90% for the standard strain of Ureaplasma urealyticum, ATCC 27618, and the standard strain of Mycoplasma hominis, ATCC 14027.
[Precautions]
1. This product is intended for in vitro use only and must not be ingested. It is for single-use only; do not use if the packaging is damaged.
2. Before use, carefully read the instruction manual and use the product within its expiration date.
3. Use protective equipment to prevent direct contact with all potentially infectious materials. Thoroughly wash your hands after each experiment.
4. After use, dispose of waste in accordance with the requirements of the hospital or the environmental protection authorities.
[Basic Information]
Registrant (or Filing Entity)/Manufacturer Name: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Address: No. 18, Innovation Fourth Road, Gangwan Avenue, Tangjiawan Town, Zhuhai City, Guangdong Province Postal Code: 519085
Contact Information: Phone: 0756-3890858 Fax: 0756-3890848
Name of the after-sales service provider: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Contact Information: Customer Technical Service Phone: 0756-3890835
E-mail: haitai@zhhaitai.com
Company website: www.zhhaitai.com
Production Address: No. 18, Innovation Road No. 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City, Guangdong Province
Production License Number or Production Filing Certificate Number: Yue Food and Drug Administration Medical Device Production License No. 20040941
[Medical Device Registration Certificate Number/Product Technical Requirement Number]
[Date of Approval and Amendment of the Instruction Manual]