Comparison of two methods for cell count determination in the course of biocide susceptibility testing

Abstract

The inoculum density is an important parameter for numerous experimental approaches in bacteriology, including antimicrobial susceptibility testing (AST), biocide susceptibility testing (BST) and biocide efficacy testing (BET). Methods to determine the inoculum density commonly refer to cell counts and have been described for BET according to the German Medical Veterinary Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) and for AST according to the Clinical and Laboratory Standards Institute (CLSI). In this study, the DVG method using 1000 μL volumes of two different dilution steps and the AST method according to CLSI using a 100 μL volume of a single dilution step from the inoculum suspension were compared. For this, each of the four reference strains, Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442, was comparatively tested 28 times using the inoculum preparation according to DVG. The results were statistically analysed using Bland-Altman plots and 95 % limits of agreement (AL). Moreover, cell counts were correlated with the optical density of the bacterial suspensions used. In comparison, the CLSI method measured lower values for colony-forming units (CFU) of -0.12 log₁₀ compared to the DVG method. Overall, both methods returned an AL of -0.52 to 0.27 log₁₀. Since the variations observed between the two methods were within one log₁₀ step and the measured CFUs did not differ systematically, both methods proved to be suitable for cell count determination. Therefore, the CLSI method, which is less complex and less time-consuming, is recommended.

Keywords: Clinical and Laboratory Standards Institute (CLSI); Enterococcus hirae; Escherichia coli; German Veterinary Medical Society (DVG); Pseudomonas aeruginosa; Staphylococcus aureus.