Procedure for Culture Specimen Processing: Pulmonary specimens

These are the steps to be followed when processing pulmonary specimens in TB culture laboratories:

Beginning the Specimen Processing Procedure:

1. Process only one specimen at a time, and do not leave open containers or open centrifuge tubes in the Bio Safety Cabinet (BSC).
2. Process the available specimen in a 50 ml sterile, plastic, screw-capped centrifuge tube (Figure).

Figure: Capped 50 ml sterile, plastic, screw-capped centrifuge tubes

NALC - NaOH Procedure:

1. Always open the cap of the specimen container slowly to minimize aerosol production.
2. Aliquot reagent in a separate tube for each specimen to avoid contamination of reagent stocks. A freshly prepared single-use aliquot is preferred.
3. Note the volume of the specimen. Add an equal volume of N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) solution and tighten the cap.
4. In order to avoid cross-contamination, do not allow the NALC-NAOH solution container to touch the specimen tubes.
5. After the addition of the decontaminant and or digestant tighten the caps and vortex for not more than 20 seconds at a moderate speed.
6. Invert each tube 5 times, making the figure 8 with your wrist, to ensure that the NALC-NaOH solution contacts the entire inner surface of the tube and cap.
7. Avoid extreme agitation or shaking which can cause inactivation of the NALC.
8. Let the tubes stand at room temperature for 20-25°C, for 15 minutes and mix by gently inverting the tube.
   • NaOH exposure time must be strictly limited to 15 minutes to prevent the over-killing of the TB bacilli.
   • If stronger decontamination is needed, the starting concentration of NaOH may be increased to 5-6%, but the time of exposure should not be extended.
9. Neutralize the specimen with a phosphate buffer of pH 6.8 to the 45 ml mark. Do not exceed the 45 ml mark.
10. To avoid cross-contamination do not allow the diluent (phosphate buffer) container to touch the mouth of the specimen tubes.
11. Single-use aliquots of phosphate buffer or a dispenser are preferred to avoid cross-contamination during the procedure.
12. After centrifugation, open the safety bucket in the BSC and carefully pour off the supernatant into a splash-proof discard container with a suitable disinfectant (5% phenol).
    • If required, swab the tube with a disinfectant-soaked gauze (use individual pieces) and recap carefully.
    • While wiping, do not allow the disinfectant to flow into the tube.
13. Re-suspend the sediment in 1–2 ml of sterile phosphate buffer (pH 6.8) using a new transfer pipette.

Please click here to see a full video on sputum specimen processing for culture.

Resources

• MGIT Procedure Manual, Mycobacteria Growth Indicator Tube (MGIT) Culture and Drug Susceptibility Demonstration Projects, FIND Training Manual.
• GLI Training Module on Specimen Processing, STOP TB Partnership.

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