Studies of eDNA often attempt to image eDNA

Studies of eDNA often attempt to image eDNA of the biofilm matrix , using fluorescence microscopy. However, no comprehensive comparative studies have been done to determine which fluorescent stains are most sensitive and accurate for the visualization of eDNA. Due to the lack of standardization, eDNA images reported in the scientific literature are difficult to compare, and it is therefore equally difficult to evaluate the accuracy of the staining techniques used. To address this problem, we have systematically compared the most commonly used eDNA stains as well as several less common stains, and combined these with different counterstains for cell visualization. This has led us to recommend the combination of TOTO-1 with the cell permeable SYTO 60 as an optimal staining method for the visualization of DNA in dead PRT-060318 and eDNA surrounding living and dead bacteria. The commonly used eDNA stains we evaluated were the intercalating dye propidium iodide, the highly sensitive stain PicoGreen commonly used in DNA quantification, the cyanine monomer SYTOX® Green, and the dye 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) not only sold as a standard for ether-, ester-, and phosphate substrates, but also commonly used as an eDNA stain in biofilms since 2006 (). Additionally, we evaluated the lesser used cyanine monomer TO-PRO-3 and cyanine dimer TOTO-1, all at a final working concentration of 2μM. As counterstains we evaluated the DNA-binding cyanine dyes SYTO 9, SYTO 12, and SYTO 60, as well as the non-fluorescent cell-permanent acetomethoxy derivates of calcein (CAM-red and CAM-green) which become cleaved by esterases upon entering living cells, resulting in the entrapment of fluorescent and membrane-impermeant calcein within the cell. All counterstains were used at a working concentration of 1μM with the exception of SYTO 60, which was determined to function optimally at a concentration of 10μM. All stains were obtained from Life technologies™. Biofilms were all grown for 24h in Lysogeny broth at 37°C, with the exception of which was incubated at 30°C, and which was grown at 37°C in tryptic soy broth. Biofilms were grown in IBIDI μ-plates for optical microscopy (Cat no. 89621), and visualized in PBS using a Zeiss 700 confocal laser scanning microscope. Of the eDNA stains, TOTO-1, PicoGreen and SYTOX® Green allowed excellent visualization of eDNA biofilm structures (a, b and c). However, PicoGreen penetrates the cell membrane over time, eventually producing an erroneous green signal from all cells (data not shown). The excellent visualization by these stains was not surprising, as they are among the most sensitive DNA-binding stains available. The DNA binding affinity of the cyanine dimers is several orders of magnitude higher than their parent monomer dyes, and they furthermore have low intrinsic fluorescence and high quantum yields. The DNA–TOTO-1 complex in particular is very stable (), and it has a high DNA binding affinity due to its four positive charges. Because TOTO-1 has no time constraints and displays excellent sensitivity, it is the most appropriate choice for visualization of eDNA.