Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods

Abstract

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.

Phase Variation of LPS and Capsule Is Responsible for Stochastic Biofilm Formation in Francisella tularensis

“…All experiments implementing statistical analysis described in this manuscript were performed independently at least 3 times. For experiments involving stochastic biofilm formation (Figures 1, 3, 4, 6) the log-transformed OD 600 was analyzed by linear mixed effects model, as described previously (Allkja et al, 2021). Analysis was implemented in the Mixed procedure of SAS version 9.4 (SAS Institute, Inc., Raleigh NC).…”

Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidence

“…The level of variability of the untreated controls from our experiments is acceptably small (a repeatability SD of untreated controls <½ log is acceptable) compared to other methods in the peer reviewed literature that have been standardized with AOAC International (Tilt and Hamilton, 1999) or ASTM International (Parker et al, 2014;Goeres et al, 2019Goeres et al, , 2020Allkja et al, 2021).…”

Seaweed Extracts: A Promising Source of Antibiofilm Agents with Distinct Mechanisms of Action against Pseudomonas aeruginosa

“…This makes it necessary to combine the CV assay with the more accurate CFU counting method [33]. Furthermore, Allkja et al, 2021 proved that the CFU counting assay is more responsive in treatment experiments than CV staining, due to potential interaction between the treatment and the dye [27]. Results obtained by adding extracts at t 0 revealed that those derived from the green alga U. lactuca, particularly CH and EA extracts, are the most promising in reducing bacterial adherent biomass, in comparison to the two other algae tested here (Table 2).…”

“…Since the CV staining method showed EA extract originating from the green alga U. lactuca to be the most effective in reducing the formation of the biofilm, as well as in eradicating previously formed biofilms, the potential synergy of the extract with two conventional antibiotics was evaluated following the CFU counting method. The choice of this evaluation method was based on the demonstrated responsiveness of CFU counting method in treatment efficacy testing in comparison with the CV staining method [27]. For comparison, the antibiofilm activity of tobramycin and colistin alone was evaluated on 24 h-old untreated biofilms, while the effect of the antibiotic/EA extract combination was determined on 24 h-old biofilms, previously exposed to the EA extract for 24 h (Figure 5).…”