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Procedure for Culture Specimen Processing: Pulmonary specimens
Learning ObjectivesLearn about Procedure for Culture Specimen Processing: Pulmonary specimens.
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These are the steps to be followed when processing pulmonary specimens in TB culture laboratories:
Beginning the Specimen Processing Procedure:
- Process only one specimen at a time, and do not leave open containers or open centrifuge tubes in the Bio Safety Cabinet (BSC).
- 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:
- Always open the cap of the specimen container slowly to minimize aerosol production.
- Aliquot reagent in a separate tube for each specimen to avoid contamination of reagent stocks. A freshly prepared single-use aliquot is preferred.
- Note the volume of the specimen. Add an equal volume of N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) solution and tighten the cap.
- In order to avoid cross-contamination, do not allow the NALC-NAOH solution container to touch the specimen tubes.
- After the addition of the decontaminant and or digestant tighten the caps and vortex for not more than 20 seconds at a moderate speed.
- 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.
- Avoid extreme agitation or shaking which can cause inactivation of the NALC.
- 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.
- Neutralize the specimen with a phosphate buffer of pH 6.8 to the 45 ml mark. Do not exceed the 45 ml mark.
- To avoid cross-contamination do not allow the diluent (phosphate buffer) container to touch the mouth of the specimen tubes.
- Single-use aliquots of phosphate buffer or a dispenser are preferred to avoid cross-contamination during the procedure.
- 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.
- 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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