A fluorescence-based viability assay for Phytophthora agathidicida oospores

Viability staining is an essential tool in many fields of microbiology. In this study, we aimed to establish a dual fluorescence method for detecting the viability of Phytophthora oospores. Phytophthora is a genus of plant pathogens in the class Oomycete that cause disease in a wide range of agriculturally and ecologically important plants. During the Phytophthora disease cycle, thick-walled oospores can be produced via sexual reproduction. These oospores are essential for long-range dispersal and long-term survival of the pathogen, and therefore methods for the study of oospores are of great interest. In this study, we tested five fluorescent dyes for their ability to stain Phytophthora agathidicida oospores: SYTO 9, FUN-1, fluorescein diacetate, propidium iodide, and TOTO-3 iodide. These dyes represented a selection of total, viable and non-viable cell stains Each dye was assessed individually, and then the best dyes were combined for dual viability staining. In this study, we have identified three dyes — SYTO 9, fluorescein diacetate and TOTO-3 — that can be used to report total, viable and non-viable oospores respectively. We further demonstrate that fluorescein diacetate and TOTO-3 can be used together for dual viability staining. This new method is quantitative and compatible with automated image analysis, allowing oospores to be rapidly and accurately assessed for viability.

Dose Dependent Antimicrobial Cellular Cytotoxicity—Implications for ex vivo Diagnostics

Introduction:Ex vivo and in vitro diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations ex vivo, data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity.Method: After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration (Cmax), and concentrations 10- and 100-fold above.Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax drug concentrations decreased spot forming units/million cells by 32–100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested.Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

Spatial Cleaning Action of Ultrasonic Irrigation on Enterococcus faecalis Biofilm

This study aimed to examine the spatial cleaning effect of ultrasonic irrigation in simulated root canal with oblong cross section in the absence of antimicrobial agent. A 7-day E. faecalis biofilm was cultivated in a rectangular, simulated canal model and subjected to passive ultrasonic irrigation (PUI) with sterile saline for 5 minutes. After that, the biofilm was examined by confocal microscopy after bacterial viability staining at 58 sites around and beyond the endosonic file. Results showed that, at the vicinity of the file, the amount of viable bacteria ranged from 13.1% (2.75 mm from the tip) to 40.5% (4.5 mm level). Lesser amounts of live bacteria were observed within 1 mm from the vibrating file, which amount increased for sites farther away. At 3 mm distance, the amount of bacteria (35.5 to 64.4%) was significantly greater than areas situated close to the vibrating file (P < 0.01). Sites next to the anti-nodes of file had less bacteria remaining than sites near the nodes (P = 0.050). Apically (0.5 mm or more), the amount of bacteria was significantly greater than that at the file tip (16.4%) (P < 0.05). It was concluded that PUI is able to dislodge a single-species biofilm, provided that they are situated in close vicinity to the vibrating file.

Propidium iodide staining underestimates viability of adherent bacterial cells

Combining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids. We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at extracellular DNA (eDNA) being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

Detection of Two Peronospora spp., Responsible for Downy Mildew, in Opium Poppy Seed

Downy mildew is a serious threat to opium poppy production globally. In recent years, two pathogen species, Peronospora somniferi and Peronospora meconopsidis, which induce distinct symptoms, have been confirmed in Australia. In order to manage the spread of these pathogens, identifying the sources of inoculum is essential. In this study, we assessed pathogen presence associated with poppy seed. We developed PCR and qPCR assays targeting the coxI and coxII gene regions, for the detection, differentiation, and quantification of P. somniferi and P. meconopsidis in poppy seed. These results were complemented and compared with direct seed histological examination and a seed washing combined with viability staining for oospore detection. The majority of seed lots from all harvest years contained detectable P. meconopsidis, the earliest (1987) predating the first official record of the disease in Tasmania (1996). In contrast, only seed lots harvested in 2012 or later contained P. somniferi, evidence of its more recent introduction. P. meconopsidis contamination was estimated to be as high as 33.04 pg DNA/g of seed and P. somniferi as high as 35.17 pg DNA/g of seed. Incidence of pathogen contamination of seeds, estimated via a group testing protocol, ranged from 0 to 9% (P. meconopsidis) or 0 to 11% (P. somniferi). Mycelia were predominately found external to the seed coat. Seed washing and viability staining demonstrated that putatively viable oospores were present in the majority of seed lots. Transmission testing confirmed both pathogens can be successfully transmitted from infested seed to infected seedling. PCR and qPCR pathogen assays were found to be reliable and offer a routine test for determining pathogen inoculum in poppy seeds.

Novel Method for Viability Assessment ofBifidobacterium longumATCC 15707 on Non-dairy Foods during drying

ABSTRACTThis study demonstrates a new technique for separating and purifying viable microbes from samples that interfere with viability staining. The viability ofBifidobacterium longumATCC 15707 was assessed using PBDC to separate bacteria from complex non-dairy food matrices and Quantitative Fluorescence Microscopy (QFM) to determine individual cells using LIVE/DEADBacLight bacterial viability staining. Water agar (3%) was used to retain cells ofB. longumand offered a lower fluorescence background withBacLight viability staining, compared with fixation on polycarbonate (PC) black membrane. The effect of drying temperatures and non-dairy foods on viability ofB. longumwas assessed.B. longumcoated on oat, peanut or raisin was separated by filtration, low- and high-speed centrifugation, flotation and sedimentation buoyant density centrifugation. Purified cells were subsequently deposited on water agar for rehydration followed by LIVE/DEADBacLight viability staining and enumeration. Conventional plate counting was also conducted to compare viability results. Finally, the applicability of this novel method for viability assessment was demonstrated and informative information of cell membrane damages ofB. longumincorporated onto non-dairy foods during 24 h drying was observed. Viability assessment ofB. longumcoated onto oat, peanut, or raisin was much lower by plate counting compared to viability staining. Drying appeared to have a greater impact when viability was assessed by plate counting compared to viability staining.IMPORTANCEEnumeration of viable beneficial bacteria from function foods presents a significant bottle neck for product development and quality control. Interference with microscopic and/or fluorescent techniques by ingredients, time required to incubate plated microbes, and the transient nature of the colony forming unit make rapid assessment of viable bacteria difficult. Viability assessment ofBifidobacterium longumATCC 15707 by Percoll Buoyant Density Gradient Centrifugation with LIVE/DEADBacLight viability staining on water agar (3%) was in agreement with serial dilution enumeration. Without the need for incubation viability assessment by staining provided a more rapid means to assess the impact of drying on the viability ofB. longumcoated onto oat, peanut or raisin.

Cell Deposition Microchip with Micropipette Control over Liquid Interface Motion

Positioning single cells on a solid surface is a crucial technique for understanding the cellular functions and cell–cell interactions in cell culture assays. We developed a microfluidic chip for depositing single cells in microwells using a simple micropipette operation. Cells were delivered to microwells by the meniscus motion of liquid interface. The residue deposits of cells were redistributed with air injection, and the isolated single cells were stored in microwells. Different microwell sizes and depths were studied to evaluate the trapping possibility of cells. Medium replacement and cell viability staining with the isolated single cells were achieved in microwells. The chip will serve as a tool for single-cell patterning in an easy-to-use manner.