Quality Control of Selective Middlebrook- 7H9 Liquid Medium

Quality control (QC) Testing of 7H9 Liquid Medium

Every new lot of MGIT medium and every new lot of the enrichment should be quality control tested by the user upon receipt and before it is used routinely.

A. QC strains

Cultures: The following three mycobacterial cultures are recommended for quality control testing.

1.M. tuberculosis, H37Rv ATCC 27294
2.M. kansasii ATCC 12478
3.M. fortuitum ATCC 6841

 If the ATCC reference strains of M. kansasii or M. fortuitum cannot be obtained, then a laboratory isolate which is well-characterized may be used as a quality control strain. Well-characterized strains will be available from the mycobacterial strain bank of TDR/WHO. ⁷³

B. Preparation of culture suspension

1.  Subculture the above mycobacteria on several LJ slants.
      • Incubate at 37ºC + 1ºC.
      • Observe growth visually.
      • As soon as there is good, confluent and pure growth, use this growth for making suspension. Growth should appear within 10-15 days of subculturing and should be used within this period. Aged cultures would not give reliable results.
2. Remove growth from the slant by carefully scraping the colonies off the slant with a sterile loop or sterile spatula made from wooden applicator sticks. Take extreme precautions not to scrape off any culture medium (which gives false turbidity measurement).
3.  Transfer growth into a screw cap tube containing 4 ml of sterile 7H9 broth and glass beads (6-10 beads, 1-2 mm diameter), which helps to break up clumps (Tube A).
4.   Vortex the tube for at least 1-2 minutes. Ensure the suspension is well dispensed and turbid (greater than McFarland #1 turbidity).
      Let the suspension stand undisturbed for 20 minutes.
5.    Using a transfer pipette, carefully transfer the supernatant to another sterile screw cap glass tube (Tube B). Avoid picking up any sediment.
      Let this stand undisturbed for 15 minutes.
6. Carefully transfer the supernatant into another screw cap glass tube (Tube C) without taking any sediment. Next, adjust the turbidity of suspension in Tube C to McFarland #0.5 turbidity standard by adding more 7H9 broth or sterile saline/deionized water and mixing well. If the suspension is too turbid, transfer some of the suspension to another sterile tube and adjust the turbidity to McFarland #0.5 standard.   This is the working suspension for QC testing. This suspension may be frozen in small aliquots (1-2 ml) in appropriate tubes/vials at -70ºC + 10ºC. The frozen suspension may be used for up to 6 months. Once thawed, do not refreeze.

 C.Preparation of dilutions

1. Dilute the working suspension McFarland #0.5, freshly prepared or frozen) 1:5 by taking 1.0 ml of suspension and adding into 4.0 ml of sterile water or saline. Mix well (Tube 1).
2. Dilute two more times 1:10 by adding 0.5 ml of suspension Tube 1 into 4.5 ml of sterile water or saline (Tube 2). Mix well and then again add 0.5 ml from Tube 2 to 4.5 ml of sterile saline or distilled/deionized water (Tube 3). Mix well. Final dilution 1:500 (Tube 3). Stop here for M. tuberculosis and use Tube 3 for QC testing.
   • For M. fortuitum, further dilute Tube 3 -1:10. Take 0.5 ml of suspension from Tube 3 and add to 4.5 ml of sterile water or saline and mix well. Final dilution 1:5000 (Tube 4). Use Tube 4 for QC testing.
   • For M. kansasii, dilute Tube 4 once again 1:10 by adding 0.5 ml from Tube 4 to 4.5 ml of sterile saline/water, mix well. Final dilution 1:50,000 (Tube    Use Tube 5 for QC testing.

 D. Inoculation/incubation
1. Supplement MGIT medium with Growth Supplement and PANTA as recommended.
2. Inoculate 0.5 ml from Tube 3 to each of the two MGIT tubes for M. tuberculosis. Similarly, inoculate 0.5 ml from Tube 4 for M. fortuitum and Tube 5 for M. kansasii into each of the two labelled MGIT tubes. Mix.
3. Enter the inoculated tubes in the MGIT 960 instrument. Take the tubes out when indicated positive by the instrument. Retrieve data for time to positive.

E. Expected results

 M. tuberculosis Tube fluorescence positive in 6 to 10 days
 M. kansasii Tube fluorescence positive in 7 to 11 days
 M. fortuitum Tube fluorescence positive in 1 to 3 days

If the above criteria are not met, repeat the test. If the QC test still does not give satisfactory results, check the viability of the inoculum, the age of the culture if stored frozen and other procedures. If everything meets the established specifications, contact Technical Services at BD Diagnostic Systems.

F. Precautions.

1. Use freshly prepared suspension adjusted to McFarland #0.5 standard. If frozen (-70ºC +/- 5ºC), thaw prior to use for each quality control testing. Do not store or refreeze once thawed.
2. All work should be carried out in a proper biological safety cabinet.
3. All materials should be sterilized by autoclaving prior to disposal.
4. Follow all the recommended safety precautions.

Resource

Mycobacteriology Laboratory Manual 

Assessment