Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water. HIGHLIGHTS The high cost of current protozoa detection methods limits their widespread use in limited settings.; Immunomagnetic separation improves detection by cleaning the sample.; Recovery efficiency is maintained for Giardia duodenalis with 50% less beads.; Organisms adhering to beads after dissociation may impact recovery levels.

Electroanalytical properties of chlorophenol red at disposable carbon electrodes: Implications for Escherichia coli detection

The use of coliforms and Escherichia coli as indicator species for assessing the quality of water is well established and a large variety of methods based on β-galactosidase (B-GAL) activity, inherent to the microbes within this classification, have arisen to enable their detection and enumeration. Chlorophenol red (CPR) is widely used as a chromogenic label, but its capacity for translation to electroanalytical devices has yet to be fully explored. The CPR moiety is capable of undergoing oxidation at carbon substrates (+0.7 V) giving rise to a variety of phenolic intermediates. Electrochemical, XPS and enzymatic techniques were employed to characterise the underpinning chemistry and the intermediate identified as a 1,2-quinone derivative in which the chlorine substituent is retained. The latter was found to accumulate at the electrode and, in contrast to the parent CPR, was found to be detected at a significantly less positive potential (+0.3 V). Bacterial hydrolysis of a CPR labelled substrate was demonstrated with the 1,2-quinone oxidation product found to accumulate at the electrode and detected using square wave voltammetry. Proof of concept for the efficacy of the alternative electrode pathway was established through the detection of E.coli after an incubation time of 2.5 h with no interference from the labelled substrates