Publication details

Indirect imprint sampling technique using a filter paper pad for microbial analysis



Year of publication 2014
Type Conference abstract
Description Introduction There exists a variety of methods how to take samples from surgical wounds for cultivation; nevertheless, an unambiguous and most suitable procedure has so far been not defined with satisfactory precision. The aim of this presentation is to introduce an indirect imprint technique using filter paper pad for microbial analysis of wounds (shows principles of collecting, storaging, transporting, evaluation of samples and interpreting obtained information with all its advantages and disadvantages) and compare it with routinely used swab Method Imprints were carried out using the filter paper Whatman No. 4, cut to a size of 5 x 5 cm and previously sterilized in an autoclaved at 120 oC for 20 minutes and aseptically attached to 24 hour-old blood agar in a Petri dish, for the swabs, we used sterile cotton tips made by the Italian company COPAN; After careful debridement, a sample was taken from the wound surface by the swab and subsequently the same area of the investigation surface was imprinted onto a filter paper. Sheet of filter paper was taken from Petri dish with blood agar by sterile tweezers and put on to surface of wound. After 10 seconds paper became wet and was moved back to the Petri dish and then transported to a microbial laboratory as soon as possible. In the microbial laboratory a filter paper was removed from Petri dish and put on to new blood agar for 10 seconds and then to UriSelect 4 medium and after then to NaCl (salt) blood agar. The cultivation was done in the atmosphere with 10 % CO2 and temperature 37 oC. The final control of Petri dishes happened 48 hours later. The cultivation results of the two techniques were statistically processed using contingency tables and McNemar test. Those samples that were simultaneously cultivation positive in the imprint and negative in the swab were processed in greater detail. Results Result of an indirect imprint was semi-quantitative, one part of the result was type of microbe cultivated from wound and other was density of them accounted by colony forming unit (CFU), an estimate of viable bacterial numbers in wound, the results were given as CFU/cm2. 177 samples from 70 patients were analyzed. Sampling was carried out in 42 men and 28 women. The identical results of both culture methods were in 114 samples and different in 63 samples. The most frequent result of swab was sterile result, in 62 cases; imprint was sterile in 50 cases. Imprint and swab were both negative in 46 cases, and positive in 68 cases. In 15 cases there was a negative imprint and positive swab and in 48 cases there was positive imprint and negative swab. Coincidently, the most frequent cultivation finding with both techniques was a sterile one. The most frequently cultivated microorganism after using swab was Pseudomonas aeruginosa (in 22 cases), compared with Escherichia coli when the filter paper (imprint) was used (in 31 cases). The imprint technique was evaluated as more sensitive compared to swab, at the level of statistical significance P = 0.0001. Of the 177 samples there were 53 samples sterile in the swab and positive in the imprint simultaneously. In three samples CFU was not counted, 22 samples were within the limit of 0 - 25 x 101 CFU/cm2, 20 samples within the limit of 25 x 101 – 25 x 102 CFU/cm2, five within the limit of 25 x 102 – 25 x103 CFU/cm2, and three of more than 25 x 104 CFU/cm2.   Conclusion In our study the imprint technique appears as a highly more sensitive than swabbing. We obtain information not only on the type of the microorganism cultured, but also on the number of viable colonies per square unit, expressed in CFU/cm2.

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