chlorophyll-a and diacetylfluorescein

In ecotoxicological studies involving environmental contaminants, rapid and multi-parametric optical detection based methods have definite advantages over traditional growth inhibition assays. In this context, a confocal laser scanning microscopy (CLSM) based method to assess ecotoxicity arising out of biocide insult to marine microalgae is reported. Using this technique, the effect of in-use concentrations of chlorine (an oxidizing biocide) on a marine diatom (Cocconeis scutellum Ehrenb) was determined based on inhibition of chlorophyll autofluorescence and esterase activity (probed by fluorescein diacetate (FDA) staining). Determination of mean fluorescence intensity (MFI) per cell by collecting auto-fluorescence from single cells in x, y and z dimensions permitted reproducible toxicity evaluation at single-cell level. Chlorine-induced inhibition of autofluorescence in laboratory cultures was dose-dependent. Additional data on metabolic activity of the diatom cells following chlorine exposure was collected by FDA staining. Our results demonstrate that chlorine, an antifouling biocide commonly used in cooling water systems, causes significant reduction in chlorophyll autofluorescence and esterase activity in diatoms in short-term exposure experiments. Tests employing multiple organisms and multiple toxicity endpoints are superior to standard algal growth inhibition assays for they provide a better understanding of algal-algal interactions and real impact in the environment. The combined autofluorescence-FDAtechnique described here is rapid and has clear advantages in terms of using environmentally relevant toxicant and cell concentrations. Additional microalgal species and toxicity end points can be employed in order to develop multi-species and multiparameter bioassay using confocal microscopy.

Topics: Chlorine; Chlorophyll; Diatoms; Disinfectants; Dose-Response Relationship, Drug; Esterases; Fluoresceins; Fluorescence; Image Processing, Computer-Assisted; Microscopy, Confocal; Toxicity Tests

We used flow cytometry to examine the process of cell death in the bloom-forming alga Heterosigma akashiwo during infection by a double-stranded DNA virus (OIs1) and a single-stranded RNA virus (H. akashiwo RNA virus [HaRNAV]). These viruses were isolated from the same geographic area and infect the same strain of H. akashiwo. By use of the live/dead stains fluorescein diacetate and SYTOX green as indicators of cellular physiology, cells infected with OIs1 showed signs of infection earlier than HaRNAV-infected cultures (6 to 17 h versus 23 to 29 h). Intracellular esterase activity was lost prior to increased membrane permeability during infection with OIs1, while the opposite was seen with HaRNAV-infected cultures. In addition, OIs1-infected cells accumulated in the cultures while HaRNAV-infected cells rapidly disintegrated. Progeny OIs1 viruses consisted of large and small morphotypes with estimated latent periods of 11 and 17 h, respectively, and about 1,100 and 16,000 viruses produced per cell, respectively. In contrast, HaRNAV produced about 21,000 viruses per cell and had a latent period of 29 h. This study reveals that the characteristics of viral infection in algae are virus dependent and therefore are variable among viruses infecting the same species. This is an important consideration for ecosystem modeling exercises; calculations based on in situ measurements of algal physiology must be sensitive to the diverse responses of algae to viral infection.

Topics: Chlorophyll; Chlorophyll A; Culture Media; DNA Viruses; Esterases; Eukaryota; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Organic Chemicals; RNA Viruses; Species Specificity