CDST_LT: Inoculum preparation and incubation for MGIT DST

1. Label the required number of 7 mL MGIT tubes with lab ID. In addition, label tubes with one of each of the following: GC (Growth Control), drug 1, drug 2 etc.

2. Place the tubes in the suitable AST set carrier (5 tubes or 8 tubes), from left to right: GC, drug 1, drug 2 etc. (as per the number of drugs to be tested).

3. Aseptically add 800 μl of BACTEC MGIT OADC Supplement to each MGIT tube. Do not use MGIT 960 growth supplement or PZA supplement.

4. 4 Aseptically pipette 100 μl of the reconstituted drug into the corresponding labelled MGIT tube; e.g., add 100 μl AMK (amikacin) solution to the MGIT tube labelled as “AMK”, etc.

5. It is important to add the correct drug to the corresponding tube.

6. Do not add drugs to the MGIT GC tube.

Inoculation Preparation for Drug-containing Tube (1-2 days)

1. DST must not be set up on the same day an MGIT tube signals positive (0 Day).

2. If the culture is worked up one or two days after signalling positive, it can be used directly to inoculate the MGIT tubes for DST.

3. Mix well (vortex) the confirmed MTB pure culture isolate to break up the clumps. Leave the tube undisturbed for about 5-10 minutes to let big clumps settle on the bottom.

4. Use the undiluted supernatant for inoculation of the drug tube set.

5. Aseptically add 0.5 ml of the well-mixed culture suspension (inoculum) into each drug-containing tube using a pipette. Do not add to the growth control (GC).

 Inoculation Preparation for Growth Control (GC) Tube (1-2 days)

For the growth control (GC) tube, first, dilute the test culture suspension 1:100 by adding 0.1 ml of the test culture suspension to 10.0 ml of sterile saline. Mix well by inverting the tube 5-6 times. Use this diluted suspension to add 0.5 ml into the growth control tube.

  Inoculation Preparation for Drug-containing Tube (3-5 days)

1. If the culture is used to set up DST between three and five days after signalling positive.

2. Mix well (vortex) the confirmed MTB pure culture isolates to break up the clumps. Leave the tube undisturbed for about 5-10 minutes to let big clumps settle on the bottom.

3. Use the undiluted supernatant for preparation of 1:5 dilution, 1 ml of MGIT broth in 4 ml of sterile saline (1:5 dilution).

4. Use this well-mixed diluted culture for inoculation of the drug tube set.

5. Aseptically add 0.5 ml of the well-mixed culture suspension (inoculum) into each drug-containing tube using a pipette. Do not add to the growth control (GC).

 Inoculation Preparation for Growth Control (GC) Tube (3-5 days)

1. For the growth control (GC) tube, first, dilute the test culture suspension 1:100 from 1:5 diluted MGIT tube. by adding 0.1 ml of the test culture suspension to 10.0 ml of sterile saline. Mix well by inverting the tube 5-6 times. Use this diluted suspension to add 0.5 ml into the growth control tube.

2. A tube that has been positive for more than 5 days should not be subjected to DST. Instead, it should be subcultured in a fresh MGIT tube supplemented with MGIT 960 Growth Supplement and should be tested in an MGIT 960 instrument until it is positive.

3 Use this tube from one to five days of instrument positivity as described above.

Resource

Mycobacteriology Laboratory Manual   

Assessment

It is extremely important to perform quality control of drug susceptibility testing periodically. The minimum requirement is to test each new batch of reagents, such as SIRE drugs or MGIT medium. If the batch QC fails, all the results obtained within that batch, as well as the new batch of a reagent, should be thoroughly reviewed, and the testing should be repeated. Use M. tuberculosis H37Rv (ATCC [American Type Culture Collection] number 27294) as a QC strain susceptible to all anti-tuberculosis drugs. 

It is not necessary to include a resistant strain, as most of the resistant strains against a drug which are available from ATCC and other culture collections are highly resistant and do not give any added benefit in quality control. The test procedure for QC organisms is the same as described above for clinical isolates. The inoculum should be from a freshly grown culture in the MGIT medium or on an LJ slant. In case the suspension of QC bacteria is made from growth on a solid medium, follow the procedure for suspension preparation as described above. The suspension may be stored in aliquots frozen for up to 6 months at    -70 ºC + 10 ºC.

Resource

Mycobacteriology Laboratory Manual 

Assessment

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

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

Incubation of MGIT DST tubes

1. Tighten the caps and mix the inoculated broth well by gently inverting the tube several times.

2. Susceptibility test "Set Carriers" are provided in different numbers of drug combinations. For a routine SIRE test with critical concentration, a Set Carrier of

   five tubes is used. Place labelled tubes in the correct sequence in the set carrier (GC, STR, INH, RIF, EMB).

3. Enter the susceptibility set carrier into the BACTEC MGIT 960 instrument using the susceptibility test set entry feature. Ensure that the order of the tubes in the AST Set Carrier conforms to Set Carrier definitions. For example, GC, STR, INH, RIF, and EMB for the SIRE standard testing.

4. To check the inoculum's purity, streak the test culture suspension onto a blood agar plate. If blood agar is not available, use chocolate agar or BHI agar. Incubate at 35 ºC + 1ºC for 48 hours and check for any growth. If growth appears, do not set up the susceptibility test. It may be important to establish the purity of the culture before setting up a susceptibility test, particularly if contamination is suspected.

Resource

Mycobacteriology Laboratory Manual 

  Assessment