Extraction of DNA by Thermal Cell Lysis of Suspended Bacteria from Enrichment Broth
. DNA was extracted from the aliquot of enriched samples in BPW.
Comparison of direct cultivation on a selective solid medium, polymerase chain reaction from an enrichment broth
, and the BD GeneOhm(tm) VanR Assay for identification of vancomycin-resistant enterococci in screening specimens.
Thiosulphate citrate bile salts sucrose agar plates were streaked with a loopful of enrichment broth
after 8 hours and another after 24 hours.
PCR assay from specimen and after 2 and 4 h enrichment: Assays were performed on the 16 MRSA (mecA+ve) positive samples and an equal number (17) of randomly chosen MRSA negative samples (by direct culture) as well as their 2 and 4 h enrichment broths
. Eleven original samples, 10 two hour enrichment broths
and 8 four hour enrichment broths
were positive for mecA PCR (Table II).
To address the selectivity biases associated with enrichment broth
cultures compared with total DNA extractions, comparisons between culture-independent and culture-dependent profiles were made using Sorensen's index of similarity (Nakatsu et al.
There was no bacterial growth detected on any of the directly inoculated blood agar plates or from the enrichment broth
cultures for all of the 30 study samples.
A 1 ml portion from this pre-enrichment culture was transferred to 9 ml of enrichment broth
(Complete Fraser broth, BIO-RAD), and incubated at 35[degrees]C for 24 hours.
In the laboratory, the majority (67%) were detectable in enrichment broth
only, indicating that colonization was occurring at very low density levels, he noted.
Under such conditions, the likelihood of isolating resistant strains using nonselective conditions is dependent upon variables such as relative starting concentration and doubling time of individual strains in the enrichment broth
. Using the selective method minimized potential masking of the effects of such variables.
A portion of the enrichment broth
was used to conduct the BAN test.
The next step is selective plating, which involves streaking the enrichment broth
onto a set of plates and incubating for 24 to 48 hours.
To maximize the recovery of GBS, all specimens should undergo an 18-to-24-hour incubation at 35[degrees] to 37[degrees]C in an appropriate enrichment broth
medium, such as carrot or Lim broth.