|
B. pseudomallei extraction solution |
Distilled water, normal saline, detergents or enrichment media |
Threonine-basal salt plus colistin 50 mg/L (TBSS-C50 broth) |
|
|
Ashdown broth containing colistin and crystal violet is an alternative |
| Ratio of soil and extraction solution (wt/wt) |
Ranged from 2∶1 to 1∶10 |
1∶1 (10 gram of soil to 10 ml of TBSS-C50 or Ashdown broth) |
| Extraction method |
Manual shaking, vortexing or orbital shaker |
Vortexing for 30 seconds |
|
|
Manual mixing of soil is an alternative option, and may be required if sample is compacted |
| Techniques for detection of B. pseudomallei
|
Culture, PCR or animal inoculation |
Culture (PCR could be added as an additional technique if available) |
| Protocol for culture |
Variable, including direct culture on solid media and quantitation, and qualitative methods relying on broth enrichment |
Incubate the specimen (universal tube with 10 gram of soil plus 10 ml TBSS-C50 or Ashdown broth) for 48 hours |
| Temperature of incubator |
Variable, ranged from 37 to 42°C |
40°C is recommended, and 37–42°C is an alternative option |
| Protocol for sub-culture |
Variable |
Subculture 10 µL of supernatant onto an Ashdown agar plate, and streak to achieve single colonies |
|
|
Incubate plate and examine every 24 hours for 7 days |
| Identification of B. pseudomallei
|
Variable, including basic microbiological tests (which include typical colony morphology, Gram stain, positive oxidase test, inability to assimilate arabinose, resistance to gentamicin and colistin with susceptibility to co-amoxiclav) and biochemical kits (including API20NE [105] and Vitek) with or without additional confirmatory tests (specific latex agglutination test [89], or a specific PCR assay [62], [75], [91], [93]) |
Basic microbiological tests (which include typical colony morphology, Gram stain, positive oxidase test, inability to assimilate arabinose, resistance to gentamicin and colistin with susceptibility to co-amoxiclav) is mandatory plus at least one confirmatory test (API20NE, Vitek system, specific latex agglutination test [89] or a specific PCR assay [62], [75], [91], [93], unless latex test or PCR assay was used during screening) |
|
|
Specific latex agglutination test [89], or a specific PCR assay [62], [75], [91], [93] can be used a screening test |
