PZA Inoculum Preparation

The PZA susceptibility test is recommended for a pure culture of the M. tuberculosis complex. The test culture should be thoroughly checked for its purity and confirmed identification of M. tuberculosis.

Preparation from a positive MGIT tube: 

Use a freshly positive MGIT tube.

1. Day 0 – the day an MGIT tube is positive by the instrument. Re-incubate.

2. Day 1 or 2 – one or two days after instrument positive. Use undiluted for the susceptibility testing inoculation.

3. 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.

4. 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 letting it stand for 5-10 minutes. Take the supernatant broth for inoculation preparation.

Preparation from growth on a solid medium: 

1. Scrape off as many colonies as possible from the surface of the solid medium using a sterile loop or wooden applicator stick. 

2. 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. 

3. Leave the culture suspension undisturbed for 20 minutes. Then, carefully remove the supernatant fluid and transfer it to a fresh sterile tube. 

4. Vortex again and leave undisturbed for 15 minutes. Then, transfer the supernatant fluid into a third sterile tube. 

5. Adjust the turbidity of the suspension to McFarland #0.5 standard by gradually adding sterile saline.

6. 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.

Resource

Mycobacteriology Laboratory Manual 

Assessment