Procedure for primary culture from Trans-Isolate (T-I) medium Remove the aluminum cap of Trans-isolate medium with forceps 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.) Aseptically inoculate 0.5-1.0 ml of the CSF into the T-I medium with the use of syringe Incubate the inoculated T-I medium at 35-37°C with ~5% CO2 (or in a candle-jar) overnight or until transport is possible If the T-I medium can be transported to a microbiology laboratory the same day of inoculation, do not vent the T-I bottle until it arrives in the receiving laboratory. 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. Prior to subculture, remove the venting needle and wipe the rubber stopper with 70% alcohol. 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. 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. 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. If no growth is observed, subculture the T-I medium again on day 4 and day 7. Isolates should always be inspected for purity of growth by looking at colony morphology before any testing is performed. If any contamination is seen, cultures should be re-streaked to ensure purity prior to testing.