Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm.

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.

A fluorescent method for assessing the antimicrobial efficacy of disinfectant against Escherichia coli ATCC 35218 biofilm.

In this study, a versatile method was developed to assess biocide efficacy against Escherichia coli biofilm growth on carriers made of five different materials. The glucuronidase activity of live E. coli on a fluorogenic substrate (4-methylumbellyferyl-beta-D-glucuronide, MUG) was used as a viability test. Fluorescence emissions from cellular suspensions of E. coli in the test range displayed a linear response with a MUG concentration of 10 microg ml(-1). A glucuronidase activity curve with cellular suspensions of E. coli calculated as colony-forming units per milliliter showed a good correlation (0.9487 and 0.917 for 1 and 18 h of incubation, respectively), with counts obtained from biofilm containing this organism; E. coli cultures in suspension were used as standard. Three agents commonly used as disinfectants, sodium hypochlorite, hydrogen peroxide, and ethanol, were tested at use concentrations and at one-half and decimal dilutions. At decimal dilutions, ethanol at 70% proved to be the least active disinfectant on E. coli biofilm. Unlike other methods, our method permits the testing of disinfectant efficacy against biofilm growth on different materials. In preliminary assays, glass, polyvinyl chloride, polypropylene, polycarbonate, and silicon were tested. Because they gave the lowest E. coli counts after 24 and 48 h, glass and polypropylene were the two materials to which biofilm adhered least strongly.

A fluorescence bioassay to detect residual formaldehyde from clinical materials sterilized with low-temperature steam and formaldehyde.

A microtiter plate toxicity test based on fluorescence was developed to determine the residual concentration of formaldehyde on medical items after LTSF sterilization. The residual formaldehyde on eight common materials, some of which are used in different clinical instruments and devices were analysed after sterilization with LTSF. Formaldehyde residues were detected on cotton, filter paper, natural rubber, PVC, and silicone-coated latex, but not on polyurethane, silicone or glass. Formaldehyde never exceeded the recommended maximum concentration on clinical devices of about 5 microg/cm2. The results were compared with those obtained by means of a chemical method, the correlation being good (R2=0.9396). The biological method proposed here is fast and can be automated, which means that it could be used as a screening method when there are doubts as to the accumulation of residues on clinical materials or instruments that are going to be sterilized with LTSF.

Influence of organic solvents on the sensitivity of a bioluminescence toxicity test with Vibrio harveyi.

The possibility of using organic solvents (OSs) to increase the susceptibility of bioluminescent microorganisms in a bioassay for assessing the toxicity of chemicals dissolved in water was investigated. To conduct the tests acetonitrile, dimethyl sulfoxide, ethanol, methanol, and isopropanol were used as OSs and Cd, Hg, and Zn as reference toxicants. The addition of OSs modified the toxicity of the three metals to Vibrio harveyi, according on the bioluminescence assay used. The sensitivity of the luminescence bioassay for Hg increased in the presence of the five OSs, thus indicating a greater toxic effect. However, the sensitivity of the assay for the other two metals, Cd and Zn, increased or decreased (lesser toxic effect) depending on the concentration at which the OSs were used. No correlation was observed between the concentration of the five OSs and the toxicity of the three reference toxicants. From this it can be deduced that none of these OSs could be recommended for increasing generically the sensitivity of toxicity biotests using V. harveyi.

Correlation of two bioluminescence and one fluorogenic bioassay for the detection of toxic chemicals.

The linear correlation between the EC50 values of 50 substances obtained in luminescence bioassays using Vibrio fischeri and Vibrio harveyi and in a fluorogenic bioassay using Escherichia coli was investigated. As a result, a significant correlation was found between the said values in all three toxicity tests. The bioassay using V. harveyi had a sensitivity similar to that of the fluorogenic bioassay, and the bioassay using V. fischeri was the least sensitive of all. The sensitivity of the three bioassays for each of the tested substances, chiefly heavy metals, organic solvents, orgnochlorated compounds, and pesticides, differed in the majority of the cases. The three bioassays were quantified using the same laboratory apparatus and the data were processed in the same way. The possibility of designing a battery of toxicity tests that can be performed using the same apparatus but different organisms and parameters is discussed.