Publikationsdatenbank

Comparison of two established methods for cell count determination (2019)

Art

Poster

Autoren

Schug, Angela R. (WE 7)

Feßler, Andrea T. (WE 7)

Bartel, Alexander (WE 16)

Scholtzek, Anissa D. (WE 7)

Meurer, Marita

Brombach, Julian (WE 7)

Hensel, Vivian

Schwarz, Stefan (WE 7)

Kongress

8th Symposium on Antimicrobial Resistance in Animals and the Environment

Tours Val de Loire - France, 01. – 03.07.2019

Quelle

8th Symposium on Antimicrobial Resistance in Animals and the Environment : Abstracts book : 1-3 July 2019 — UMR 1282 Infectiologie et Santé Publique Institut National de la Recherche Agronomique (Hrsg.)

France: INRA Science & Impact, 2019 — S. 66

Sprache

Englisch

Verweise

URL (Volltext): https://symposium.inrae.fr/arae2019/Abstract-Book

Kontakt

Institut für Veterinär-Epidemiologie und Biometrie

Königsweg 67
14163 Berlin
+49 30 838 56034
epi@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Background and objectives:
Bacterial cell counts are important for many bacteriological tests including inoculum preparation for antimicrobial susceptibility testing (AST) and biocide susceptibility testing. The Clinical and Laboratory Standard Institute (CLSI) provides a cell count method for AST in which 100 μL volumes are plated forenumeration of bacteria [1], whereas the German Veterinary Medical Association (DVG) provides a method for biocide efficacy testing in which total volumes of 1000 μL of two different dilutions are plated for enumeration of bacteria [2]. The aim of the present study was to compare these two methods.

Materials and methods:
The reference strains Staphylococcus aureusATCC®6538, Enterococcus hiraeATCC®10541, and Pseudomonas aeruginosaATCC®15442were tested 14 times for two different cell densities (in the wells), as recommended by CLSI (2.5-5 x 105 cfu/mL = “narrow” range) [1] and DVG (1 x 105 –1 x 106 cfu/mL = “broad” range) [2]. For the CLSI method, 10μL of the inoculum suspension, achieved via one and two dilution steps for the “narrow” and the “broad” range, respectively, were diluted in 10 mL tryptone-saline-diluent (TSD). A total of 100μL of these dilutions was plated in duplicate on tryptic soy agar. For the DVG method, 1:10 dilution series in TSD were prepared and 1 mL (2 x 500 μL) from the dilutions 10-5and 10-6for the “narrow” range and 10-6and 10-7for the “broad” range, was plated. Colonies were counted after 24 h incubation at 37°C. The results were evaluated using the Bland-Altman method for assessing agreement [3].

Results:
The overallcell counts of the CLSI method (100 μL) were 1.3-9.8 x 105cfu/mL for the “narrow” range and 1.3-8.9 x 105cfu/mL for the “broad” range. For the DVG method (1000 μL) 1.4-6.1 x 105cfu/mL for the “narrow” range and 1.5-9.1 x 105cfu/mL for the “broad” range were detected. The Bland-Altman limits of agreement showed a deviation of [-0.5, 0.3] (log10) for the “broad” range compared to [-0.3, 0.3] (log10) for the “narrow” range. No matter which method (CLSI or DVG) was used, these results indicated that the number of dilution steps might have an impact on the cell counts. The tests using less dilution steps showed less variations.

Conclusion:
Since the variations observed were within acceptable ranges, both methods can be used. Our recommendation is to follow the CLSI method, since it is less complex and less time-consuming.