Dark Field Microscopy-Based Biosensors for the Detection of E. coli in Environmental Water Samples.

Development of sensitive methods for the determination of E. coli bacteria contamination in water distribution systems is of paramount importance to ensure the microbial safety of drinking water. This work presents a new sensing platform enabling the fast detection of bacteria in field samples by using specific antibodies as the biorecognition element and dark field microscopy as the detection technique. The development of the sensing platform was performed using non-pathogenic bacteria, with the E. coli DH5α strain as the target, and Bacillus sp. 9727 as the negative control. The identification of the captured bacteria was made by analyzing the dark field microscopy images and screening the detected objects by using object circularity and size parameters. Specificity tests revealed the low unspecific attachment of either E. coli over human serum albumin antibodies (negative control for antibody specificity) and of Bacillus sp. over E. coli antibodies. The system performance was tested using field samples, collected from a wastewater treatment plant, and compared with two quantification techniques (i.e., Colilert-18 test and quantitative polymerase chain reaction (qPCR)). The results showed comparable quantification capability. Nevertheless, the present method has the advantage of being faster, is easily adaptable to in-field analysis, and can potentially be extended to the detection of other bacterial strains.

A tutorial for analysing the cost-effectiveness of alternative methods for assessing chemical toxicity: the case of acute oral toxicity prediction.

Compared with traditional animal methods for toxicity testing, in vitro and in silico methods are widely considered to permit a more cost-effective assessment of chemicals. However, how to assess the cost-effectiveness of alternative methods has remained unclear. This paper offers a user-oriented tutorial for applying cost-effectiveness analysis (CEA) to alternative (non-animal) methods. The purpose is to illustrate how CEA facilitates the identification of the alternative method, or the combination of methods, that offers the highest information gain per unit of cost. We illustrate how information gains and costs of single methods and method combinations can be assessed. By using acute oral toxicity as an example, we apply CEA to a set of four in silico methods (ToxSuite, TOPKAT, TEST, ADMET Predictor), one in vitro method (the 3T3 Neutral Red Uptake cytotoxicity assay), and various combinations of these methods. Our results underline that in silico tools are more cost-effective than the in vitro test. Battery combinations of alternative methods, however, do not necessarily outperform single methods, because additional information gains from the battery are easily outweighed by additional costs.

Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests.

Application of High Throughput Screening (HTS) to the regulatory safety assessment of chemicals is still in its infancy but shows great promise in terms of facilitating better understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS approaches, we describe in a stepwise manner how a well known cytotoxicity assay can be automated to increase throughput while maintaining reliability. Results generated with selected reference chemicals compared very favourably with data obtained from a previous international validation study concerning the prediction of acute systemic toxicity in rodents. The automated assay was then included in a formal ECVAM validation study to determine if the assay could be used for binary classification of chemicals with respect to their acute oral toxicity, using a threshold equivalent to a dose of 2000 mg/kg b.w. in a rodent bioassay (LD50). This involved the blind-testing of 56 reference chemicals on the HTS platform to produce concentration-response and IC50 data. Finally, the assay was adapted to a format more suited to higher throughput testing without compromising the quality of the data obtained.