CDST_LT: Liquid culture contamination

Liquid Culture Contamination and Sources of Contamination

Liquid media are more prone to contamination than solid media. Therefore, it is essential to process specimens with extreme care, adhering very closely to procedures and recommendations.

Sources of Contamination:

1.       Improper or under-decontamination of the specimen
2.       Thick mucoid specimens that are hard to liquefy 
3.       Prolonged storage and transportation time of the specimen after collection. In such situations, especially in hot weather, bacteria tend to overgrow and are hard to kill by routine decontamination procedures.

4.       Use of non-sterile materials such as pipettes, tubes, etc.

         The incidence of contamination with bacteria (other than mycobacteria) varies from laboratory to laboratory, depending upon several factors. According to the NTEP guidelines, up to a 5% contamination rate is acceptable in cultures of clinical specimens on solid media. However, for liquid media, slightly higher contamination may be accepted (up to 7-8%). A very low contamination rate (less than 3%) may indicate too harsh a decontamination process, which would also affect the growth of mycobacteria and may reduce the positivity rate and increase the time-to-detection of positive mycobacterial culture.

   Resource

   Mycobacteriology Laboratory Manual 

5. Assessment

The growth of contaminant bacteria results in positive fluorescence. Therefore, it is important to observe all fluorescent positive MGIT tubes visually for turbidity and to make an AFB smear. If an MGIT tube broth is heavily turbid, contamination is suspected even if the AFB smear is positive. 

Usually, contaminating bacteria causes heavy turbidity, although M. tuberculosis growth appears as particles without significant turbidity, while some of the NTM may produce light turbidity. 

Contamination may be confirmed by the following method: 

1. Make a smear and stain with Ziehl-Neelsen stain. The presence of non-acid-fast contaminant bacteria on the smear confirms contamination.

2. Sub-culture a loopful on blood agar. If blood agar is not available, use chocolate agar or a brain heart infusion (BHI) agar plate. Several specimens (up to 4) may be carefully inoculated on a plate (small streak for each specimen, properly labelled). Divide the plate and identify the specimen number with a marker. Incubate these subcultures at 35ºC ±1ºC and observe after 24-48 hours. If contaminating growth appears, confirm again by gram and ZN stain.

3. If contamination is confirmed with a negative AFB smear from the broth, discard the specimen and report it as contaminated. The isolation procedure can be repeated if contamination is confirmed with a positive AFB smear from the broth.

Resource

Mycobacteriology Laboratory Manual

Assessment