Inoculation of PZA Growth Control/Drug Containing Tube

Inoculation of PZA Growth Control/Drug-containing Tube

1. Label two MGIT PZA tubes, one as GC (growth control) and one as PZA (drug-containing). Using a pipette, aseptically add 0.8 ml of PZA supplement to each of the two tubes.

2. Aseptically add 0.1 ml (100 µL) of the reconstituted drug into the PZA tube. If  possible, use a micropipette. Try to be as accurate as possible in adding the drug. This will give you 100 µg PZA per ml of the medium. Do not add drugs to the GC  tube.

3. Inoculate 0.5 ml of the culture suspension to the PZA tube using a sterile pipette.

4. For growth control inoculation, first, dilute the inoculum 1:10 by adding 0.5 ml of  the culture suspension (the one used for the drug tube) to 4.5 ml of sterile saline. Mix well by tightening the cap and inverting it at least 5-6 times. Add 0.5 ml of this  diluted suspension into the tube labelled GC.

   Note: For the PZA susceptibility test, the inoculum for the control is diluted 1:10 and not 1:100 as in SIRE AST.

5. Tighten the caps and gently invert both MGIT tubes several times to mix. Place them in two AST Set Carriers with the sequence of the first GC and the PZA.

6. Enter the PZA set into the instrument using AST set entry feature. Make sure the GC is placed first and PZA second in the AST Set Carrier. Select PZA as the drug in the second tube AST set carrier definition when performing the AST set entry.

7. Check the purity of the inoculum by streaking a loopful of the culture suspension onto a blood agar plate. If a blood agar plate is not available, use Chocolate agar or BHI agar. Incubate and check for growth after 48 hours. If growth appears on the streaked plate, discontinue the susceptibility test and do not use the results of this susceptibility test. Repeat testing after obtaining a pure culture.

Precautions: All the additions and handling of cultures should be done only inside a biosafety cabinet. To avoid contamination, use properly sterilized tubes, reagents and other items. Proper reconstitution of the PZA drug and accurate addition of the drug to the medium is essential for getting correct results. Preparation of inoculum is critical. It should be as homogeneous as possible with the least amount of mycobacterial clumps.

Dilution (1:10) of the culture suspension for growth control and mixing is critical. Use only “PZA Supplement” and PZA medium for the PZA susceptibility test. Make sure the tubes in the AST set carrier are placed in the proper sequence (i.e. GC, PZA).

Resource

Mycobacteriology Laboratory Manual 

Assessment