Troubleshooting High Liquid Culture contamination

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: