Trans-Isolate (T-I) Medium: Inoculation and Transport

Tran-isolate (T-I) is a biphasic medium used to inoculate and transport CSF samples of patients suspected of having bacterial meningitis. If the CSF cannot be transported to a microbiology laboratory immediately (within 1 hour from the time of collection) for culture, a T-I medium is used.

Uses

Trans-Isolate medium is a biphasic medium consisting of a solid phase, slant containing activated charcoal, soluble starch, and agar, and a liquid phase consisting of soybean-casein digest-gelatin broth buffered at pH 7.2 with 0.1 M 3-(N-morpholino) propanesulfonic acid buffer. These supplements and nutrients support the growth of pathogens causing bacterial meningitis.

T-I medium supports the growth and survival of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. In the case of stock culture, T-I medium supported the growth for at least three months.

NOTE: National Reference Laboratory (NRL) can procure T-I media from the World Health Organization (WHO).

Quality Control

Upon receipt, visually inspect all media for signs of contamination and physical defects. Examination of contamination includes turbid liquid, color change, or growth of bacteria or mold on the slant. Material defects include cracked glass, broken seal, leaking, reduced volume, or absent liquid phase. If you see any of the above signs, discard the T-I bottle.

After visual inspection, store all eligible T-I media at 4°C.

Sterility Testing

• Incubate one T-I bottle vented and one non-vented for 48 hours at 37°C.
• Using a sterile syringe, withdraw 50 μL of the liquid phase of T-I and plate it onto a chocolate agar plate.
• Incubate the plate at 37°C with 5% CO₂ for 48 hours. Any growth on the media is indicative of contamination.

Growth Promotion

1. Using sterile technique, inoculate one T-I bottle with 100 μL of an inoculum containing 10³ CFU/ml for each of the following pathogens: N. meningitidis, H. influenzae, and S. pneumoniae.
2. Incubate the plate at 37°C with 5% CO₂ for 48 hours.
3. Observe for growth of bacterial colonies.

T-I Inoculation

Remove T-I bottles from the refrigerator at least 30 minutes before inoculating them with the CSF sample and allow them to warm at room temperature.

1. Lift the small metal cap on top of the T-I bottle using sterile forceps. (Do not completely remove the aluminum cover).
2. Wipe the rubber stopper with a 70% alcohol swab (do not use povidone-iodine as it may be carried into the medium by the passing needle, thus inhibiting the growth of bacteria.)
3. Aseptically inoculate 0.5-1.0 ml of the CSF into the T-I medium using a 21G (0.88 mm) sterile syringe through the rubber stopper of the lid.
4. Invert the T-I bottle several times and incubate the inoculated T-I medium at 35-37°C with ~5% CO₂ (or in a candle jar) overnight or until transport is possible.
5. Label the T-I bottle clearly with the patient’s date and name. Include identification number and other necessary information.

Transport of T-I media

1. If T-I media cannot be shipped within 24 hours, ventilate with cotton plugged needle inserted through the rubber stopper without touching the media and incubate at 37°C. Before transporting the T-I media, remove the venting needle and disinfect the rubber stopper. Then transport in triple packaging following guidance for transport of infectious biological material. Transport T-I media at ambient temperature. If the T-I medium can be transported to a microbiology laboratory on the same day of inoculation, do not vent the T-I bottle until it arrives in the receiving laboratory.
2. While shipping the sample to a national or reference laboratory, include a case report along with T-I media.
3. Upon arrival, wipe the rubber stopper with 70% alcohol, insert a venting needle into the T-I bottle, incubate at 35-37°C with ~5% CO2 (or in a candle-jar), and observe daily for turbidity in the liquid phase for up to 7 days.
4. Before subculture, remove the venting needle and wipe the rubber stopper with 70% alcohol.
5. Use a sterile needle and syringe to transfer 50-100 µl of the liquid portion of the T-I medium onto both a blood agar plate (BAP) and chocolate agar (CAP) for primary culture.
6. Approximately 50-100 µl is used to streak each plate. To streak two plates, draw approximately 100-200 µl with the syringe at one time to minimize the possibility of contaminating the T-I medium.
7. Streak the BAP and CAP for isolation, incubate the plates at 35-37°C with ~5% CO2 (or in a candle jar), and examine the plates daily for up to 72 hours.
8. If no growth is observed, subculture the T-I medium again on day 4 and day 7.
9. Dispose of all inoculated T-I media in the same manner as infectious bacterial cultures.

Isolates should always be inspected for purity of growth by looking at colony morphology before any testing is performed. Cultures should be re-streaked if contamination is seen to ensure purity before testing.

References

1. Ajello, G. W., Feeley, J. C., Hayes, P. S., Reingold, A. L., Bolan, G., Broome, C. V., & Phillips, C. J. (1984). Trans-isolate medium: a new medium for primary culturing and transport of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Journal of clinical microbiology, 20(1), 55–58. https://doi.org/10.1128/jcm.20.1.55-58.1984 
2. Wasas, A. D., Huebner, R. E., & Klugman, K. P. (1999). Use of Dorset egg medium for maintenance and transport of Neisseria meningitidis and Haemophilus influenzae type b. Journal of clinical microbiology, 37(6), 2045–2046. https://doi.org/10.1128/JCM.37.6.2045-2046.1999