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Section III: Drug Susceptibility Testing
The PZA supplement should be stored at 2-8ºC upon receipt. Avoid freezing or overheating
and use prior to the expiration date. Minimize exposure to light.
4. Procedures
a. Reconstitution of lyophilized PZA drug
Reconstitute each of the PZA drug vials with 2.5 ml of sterile distilled/deionized water. Mix
well. The reconstituted drug solution will contain 8000 µg/ml of PZA.
b. Preparation of the inoculum
The PZA susceptibility test is recommended for a pure culture of M. tuberculosis complex.
The test culture should be thoroughly checked for its purity and a confirmed identification of
M. tuberculosis.
Preparation from a positive MGIT tube: Use a freshly positive MGIT tube as described in
the section for SIRE testing. Please refer to Section III-A-4).
Day 0 – the day a MGIT tube is positive by the instrument. Re-incubate.
Day 1 or 2 – one or two days after instrument positive. Use undiluted for the susceptibility
testing inoculation.
Day 3, 4 or 5 – mix well and dilute 1:5 by adding 1.0 ml of positive broth in 4.0 ml of sterile
saline. Mix well. Use this for the susceptibility testing inoculation.
Day 6 and onward – subculture in a fresh MGIT tube and follow the above guidelines.
Caution: Avoid mycobacterial clumps by mixing the growth well (vortex) and let it stand
for 5-10 minutes. Take the supernatant broth for inoculation preparation.
Preparation from growth on a solid medium: Follow the same procedure as described for
SIRE susceptibility testing. Scrape off as many colonies as possible from the surface of the
solid medium using a sterile loop or wooden applicator stick. Transfer into a sterilized tube
containing 4-5 ml of sterile 7H9 broth with 8-10 glass beads. Tighten the screw cap and
vortex the broth for 1-2 minutes. Leave the culture suspension undisturbed for 20 minutes.
Carefully remove the supernatant fluid and transfer to a fresh sterile tube. Vortex again and
leave undisturbed for 15 minutes. Transfer the supernatant fluid into a third sterile tube.
Adjust the turbidity of the suspension to McFarland #0.5 standard by gradually adding sterile
saline.
For susceptibility test inoculation, dilute this suspension 1:5 by adding 1.0 ml of the
suspension to 4.0 ml of sterile saline. Use this diluted suspension for setting up the
susceptibility.
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