Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids

Abstract

Biofilm formation is a well-known problem in management of metalworking fluid systems. Due to persistence of microorganisms within biofilms, the reappearance of various species of bacteria, including nontuberculous mycobacteria is often observed after the use of biocides and/or cleaning of delivery systems and replacement of cooling fluid. The aim of this study was to determine the usefulness of the tetrazolium salt assay (MTT assay) for assessing the viability of bacteria in biofilms formed in vitro in fresh and used cutting oils, as well as their susceptibility to antimicrobial biocides. Biofilms were established in the microtiter plate format. The results showed that quantification of formazan, a product of the tetrazolium salt reduction by electron transport system could be used for determination of the propensity of bacteria to form biofilms in these complex media. The use of the assay allows also determination of antimicrobial activity of biocides against biofilms in fresh and used metalworking fluids. Biofilms produced by Gram-negative isolates recovered from field metalworking fluids as well as the wild bacterial communities differed in metabolic activity depending on the type of fresh coolants. The MTT assay has high-throughput potential and can be efficiently used for determination of biofilm-forming capacity of microorganisms from individual machines in metalworking industry. The use of the assay may also guide the selection of the most appropriate biocide to fight these microorganisms.

Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids

“…etalworking fluids (MWF) is a generic term for any chemical used to reduce heat and friction and to safely remove small metal parts in automated mechanical processes such as grinding, cutting, drilling, and polishing (1)(2)(3)(4)(5)(6)(7). In general, MWF can be classified into four groups based on their chemical composition, straight oils, emulsifiable oils, semisynthetic MWF, and synthetic MWF (8).…”

“…First, they hamper the MWF functionality by adhering to surfaces, forming biofilms. These biofilms are complex-structured microbial aggregates that often comprise multiple genera of microbes embedded in a polysaccharide type of matrix (1). Second, as these microbial communities often consist of pathogenic species, they can pose a serious health risk for operators who come into contact with these MWF either directly or through aerosols (8).…”

Microbial contamination and biofilms on machines of metal industry using metalworking fluids with or without biocides

“…According to the previously reported results (Zhao and Liu, 2010;Cady et al 2012;Trafny et al 2013), the amount of the colorimetric product in the MTT assay is proportionally related to the size of microbial population growing in biofilms. The crystal violet assay usually employed for determination of the biofilm mass could not be used throughout the experiments in this study since gave a false-positive result in control samples (i.e.…”

Antibacterial effect of N‐acetylcysteine and taurolidine on planktonic and biofilm forms of Enterococcus faecalis

“…Biofilms of E. faecalis were quantified using a MTT colorimetric method based on the reduction of a tetrazolium salt according to Trafny et al (19). Accordingly, the dentin disks with the adhering bacteria were placed in 24-well plates, and 150 ll of phosphatebuffered saline (PBS) and 50 ll of MTT at a concentration of 0.3% were added to each well.…”