CDST_LT: Liquid culture troubleshooting

If there is an overall decrease in the culture positivity rate, the following parameters need to be investigated:

A. Incubation:

The majority of mycobacterial species grow well at 37°C ± 1ºC. They may grow slowly or may not grow if the temperature drops below 35°C. If an incubator is used, confirm that the incubator's temperature is 37°C ± 1°C by placing a calibrated thermometer in various locations throughout the incubator or instrument drawers. Monitor the readings several times each day until heating stability is determined. Check the temperature of the MGIT 960 by retrieving the information from the instrument. 

 B. Decontamination Procedure:

1. Confirm that the purity and concentration of all the reagents used in the digestion/ decontamination procedure are satisfactory.

2. Use distilled/ deionized water only for the preparation of reagents.

3. It is better to start with freshly prepared reagents.

4. A high pH of the specimen inoculated into the MGlT medium may influence the performance of MGlT adversely.

5. Do not expose the specimen to the decontamination reagent for longer than the recommended time.

6. Check if MGlT tubes are positive by visual growth but negative by fluorescence.

C. Centrifugation:

1.  Relative Centrifugal Force (RCF) should be 3,000-5,000 x g. Make sure the centrifuge is giving the required RCF.

2. The generation of heat during centrifugation also lowers the recovery due to higher temperature. Avoid the generation of excessive heat by using a refrigerated centrifuge.

D. Use of PANTA:

Check that PANTA is reconstituted with the proper volume.

Resource

Mycobacteriology Laboratory Manual 

Assessment

Detection time can range from 24 hours to six weeks for MGIT and 8 weeks for LJ medium. If the average detection time is significantly longer, these instructions need to be followed:

Digestion/decontamination procedure:

1. Decrease the NaOH concentration and/or time of exposure to NaOH. Higher concentrations of NaOH or longer exposure time will prolong the detection time of mycobacteria.

2. A high pH of the final inoculum will prolong the detection time.

3. Check if the incubation temperature is within specifications. Lower temperatures would delay detection.

4.  In a few instances, too high a concentration of PANTA may delay the detection of certain strains of NTM, especially if the starting number is low.

Procedures Check:

1. Water used to prepare reagents should be pure (distilled/deionized). 

2. All the reagents used should be sterile. 

3. All pipettes and tubes should be sterile.

4. All inoculations should be made in the biological safety hood.

5. Growth Supplement/PANTA mixture should be added to MGIT tubes just before inoculation.

6. The addition of MGlT OADC or MGIT Growth Supplement/PANTA must be done inside a biological cabinet. Leave the tube open for as little time as possible. Leaving the tube open, especially on an open bench top, would increase the contamination rate.

   Resource

Mycobacteriology Laboratory Manual 

Assessment

A high contamination rate indicates improper decontamination procedure. At the same time, too low contamination indicates over-treatment of the specimen that could also lower the culture positivity rate or increase the detection time. If in the MGIT it is more than 7-8%, then the decontamination procedure is not satisfactory and corrective measures should be taken:

 A. Specimen collection and transport:

1. Collect specimens in clean and sterile containers to avoid outside contamination. 

2. Keep the specimen in cool conditions during transport, preferably in an insulated ice box.

3. Transport to the lab as quickly as possible.

4. Upon receipt, keep it in a cool place, preferably in a refrigerator. 

5. Process the specimen as soon as possible.

B. Specimen quality and quantity:

1. The sample should not be too watery or too mucoid. If a mucoid specimen is not completely liquefied, add a small quantity of NALC powder.

2. The volume of the digested and decontaminated specimen should be 2.0–10.0 ml.

C. Specimen processing:

1. NALC-NaOH is the method of choice. 

2. Recommended NaOH concentration of 4% is ideal (the final concentration in the specimen is 1%). 

3. An increase in NaOH usually lowers the contamination rate. 

4. Higher NaOH concentration (up to 1.5% in the specimen) is acceptable when contamination is a serious problem. Once the contamination problem is under control, try to lower the NaOH concentration gradually and bring it to the recommended concentration.

D. Addition of PANTA:

1. Check storage conditions and expiry date of lyophilized PANTA (refrigerated at 2- 8ºC). Improper preparation or storage of PANTA can affect the performance or optimal concentrations.

2. Once reconstituted can be stored at 2-8ºC within 5 days and may not be frozen.

E. Specimen inoculation:

1. The specimen should be inoculated inside a safety cabinet.

2. Tubes should be inoculated with the correct amount (0.5 ml) of the specimen.

3. The inoculated MGIT tubes should be mixed after adding the PANTA and specimen.

Quality control:

1. Process 5 ml sterile buffer (negative control) along with a regular batch of specimens processed in a day. Process the negative control in the same way as clinical specimens and inoculate them into MGIT tubes. This would indicate if there is a source of contamination during the processing.

2. Periodic sterility testing of the reagents, especially a freshly made batch, is required to keep a check on the contamination sources from the reagents. Use a blood agar plate or any other suitable bacteria medium for checking contamination and Middlebrook agar or LJ medium for mycobacterial contamination check.

3. Environmental contamination may be reduced by thoroughly disinfecting the lab, working inside a biosafety hood for all the additions and other processes, and fixing the source of contamination, if established.

   Resource

Mycobacteriology Laboratory Manual 

Assessment:

Usually, more than one specimen is collected from a patient. Therefore, it is not necessary to salvage a contaminated specimen if other specimens from the same patient are positive and not contaminated. However, if it is critical to have the results of a particular specimen that was contaminated, the broth may be reprocessed.

The steps involved are as follows:

1.  The entire contaminated MGIT broth should be transferred into a 50 ml centrifuge tube.

2.   An equal quantity of sterile 4% sterile NaOH solution. 

3. This should be mixed well and allowed to stand for 15-20 minutes while mixing the tube by inverting it periodically.

4.  Phosphate buffer of pH 6.8 should be added after 15-20 mins up to the 40 ml mark on the centrifuge tube and mixed well by inverting the tube. 

5. Centrifugation should be done at 3000g for 15-20 minutes.

6.  The supernatant fluid is poured off.

7.   The sediment is resuspended in 0.5ml phosphate buffer (6.8 pH) and mixed well. 

8.  0.5 ml of this is inoculated into a fresh MGIT tube supplemented with the MGIT growth supplement/PANTA. 

9. The inoculated tubes are left at room temperature for 30 minutes and then placed in the instrument. This is followed up for observation of growth.

10. If no growth is observed, Autoclave all inoculated MGIT tubes before disposal.

    Resource

Mycobacteriology Laboratory Manual 

Assessment