Primary Culture From Trans-Isolate (T-I) Medium
1. If the CSF cannot be transported to a microbiology laboratory immediately (within 1 hour from the time of collection) for culture and analysis, a bottle of T-I medium should be inoculated. To do this remove the aluminum cap with forceps, wipe the rubber stopper with a 70% alcohol swab (do not use povidone-iodine) and use a syringe to aseptically inoculate 0.5-1.0 ml of the CSF into the T-I medium for transport and growth of bacteria.
a. If the T-I medium cannot be transported to a microbiology laboratory the same day of inoculation, insert a venting needle (sterile cotton-plugged hypodermic needle) through the rubber stopper of the T-I bottle, which will encourage growth and survival of the bacteria.
-Be sure that the venting needle does not touch the broth.
b. 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 transportation is delayed more the 4 days, remove the vented T-I bottle from the incubator or candle jar and place it at room temperature (25°C) until shipment.
c. Remove the venting needle and wipe the rubber stopper with 70% alcohol before shipment. It is essential to avoid contamination when sampling the bottles to obtain specimens aseptically.
d. Once the T-I medium arrives in the microbiological laboratory, it can be cultured immediately.
2. 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.
-Do not use povidone-iodine on the rubber stopper as it may be carried into the medium by the passing needle, thus inhibiting the growth of bacteria.
3. After 18-24 hours of incubation at 35-37°C with ~5% CO2 (or in a candle-jar) with a venting needle, use a sterile needle and syringe to transfer 50-100 μl of the liquid portion of the T-I medium onto both a BAP and 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.
4. 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.
5. If no growth is observed, subculture the T-I medium again on day 4 and day 7.
6. 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.
7. If the T-I medium appears to be contaminated, selective media may be used.
Read Also : RDT for Pneumococcal Meningitis and QC of Primary Culture Media
IV. Processing blood specimens
Laboratory personnel handling blood culture specimens must be able to isolate bacteria on appropriate primary culture media and properly subculture isolates to obtain pure cultures for
testing.
A. Primary culture from a blood culture bottle
The blood culture bottle should be immediately inoculated (within 1 minute) after venipuncture to prevent the blood from clotting in the syringe. The inoculated blood culture bottle should be transported to a microbiology laboratory as soon as possible for incubation and subculture.
1. If transport to a microbiology laboratory is not possible the same day, place the blood culture bottle in an incubator at 35-37°C with ~5% CO2 (or in a candle-jar) until transport is possible. Inoculated blood culture bottles should not be placed in the refrigerator.
2. If transport to a microbiology laboratory is feasible the same day, incubate the blood culture bottle at 35-37°C with ~5% CO2 (or in a candle-jar).
3. Examine the blood culture bottle for turbidity at 14-17 hours and then every day for up to 7 days. Any turbidity or lysis of erythrocytes may be indicative of growth, and subcultures onto primary culture media should be made immediately.
-Because N. meningitidis, S. pneumoniae, and H. influenzae are fragile organisms, subcultures should be performed on day 4 and day 7 regardless of turbidity, as the absence of turbidity does not always correlate with the absence of bacterial growth.
4. Before subculture, swirl the blood culture bottle several times to mix the contents.
5. Disinfect the rubber septum of the blood culture bottle with a 70% alcohol swab.
-Do not use povidone-iodine on the rubber septum as it may be carried into the medium by the passing needle, thus inhibiting the growth of bacteria.
-Alternatively, if the blood culture bottle has a screw-cap, open the bottle and remove the fluid using sterile technique (i.e., flaming the bottle mouth upon opening and closing the cap).
6. Aspirate 1 ml with a sterile syringe and needle from the blood culture bottle and transfer 0.5 ml to a BAP and 0.5 ml to a CAP.
7. Streak the plates for isolation, incubate at 35-37°C with ~5% CO2 (or in a candle-jar), and examine daily for up to 72 hours.
8. 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.
9. Once pure bacterial growth has been confirmed by subculture from the blood culture bottle, the bottle should be disposed of according to proper safety procedures.
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