Fluorescein Diacetate Assay Research Articles

Effects of darkness on multi‐excitation in vivo fluorescence and survival in a marine diatom

Surveys of the California Current System in 1993 revealed high concentrations of photosynthetic pigment biomass at ∼200‐m depth, well below the euphotic zone. The deep fluorescence feature contained an estimated 2.2 × 104 metric tons of carbon and contained ∼2.5 times the amount of chlorophyll observed in surface waters directly above it. Deep phytoplankton assemblages may be a signature of water mass subduction, suggesting the possibility of using phytoplankton as water mass tracers. These field observations led to a laboratory study of the fluorescence characteristics of autotrophic cells as possible indices of acclimation to extended periods of darkness. In vivo multiexcitation Chl a fluorescence of the diatom Thalassiosira weissflogii was monitored for 2 months of total darkness. Numbers of living and dead cells were determined using the vital stain fluorescein diacetate (FDA). By the end of the dark incubation period, in vivo Chl a fluorescence and fluorescence per cell had leveled off to ∼45% and 65% of initial values, respectively. The contribution of accessory pigments to Chl a fluorescence, expressed as multiexcitation fluorescence ratios, was higher in the dark than prior to transfer to darkness but showed no significant changes during 2 months of darkness. The FDA assay indicated that ∼85% of the cells were alive for at least the first 3 weeks during the first dark experiment and for the entire 2 months of second dark incubation. Cell numbers decreased to 65% of initial values and then grew exponentially upon reexposure to a light: dark photoperiod. Our results for T. weissflogii suggest that extended light limitation of photosynthesis does not preclude the survival of subducted phytoplankton assemblages and the consequent accumulation of Chl a at depths below the euphotic zone. If these results extend to natural assemblages, it is not possible to estimate advective time scales based on a maximum persistence time of pigment fluorescence below the euphotic zone. Nevertheless, the deep phytoplankton assemblage we observed provides evidence for water mass subduction and suggests that large, intermittent pulses of phytoplankton carbon are a part of cross‐shelf exchange and vertical flux from surface waters to depth in this region.

Cell-type-specific response to shock waves of suspended or pelleted cells as analysed by flow cytometry or electrical cell volume determination

Shock-wave-induced cell damage of suspended or pelleted bladder cancer cells was analysed with the flow cytometric propidium iodide (PI)/fluorescein diacetate assay, and electrical volume determination using the CASY 1 analyser system and growth curves. The CASY system revealed a smaller fraction of suspended RT4 cells with impaired membrane integrity than the flow cytometric assay. No differences were found for pelleted RT4 cells and suspended J82 cells. The discrepancies of the two viability assays indicated a different response of the cell membrane to shock waves which was dependent on the exposure system and the cell type. Growth curves indicated delayed cell death for suspended RT4 cells and exclusively immediate cell death for pelleted RT4 cells and suspended J82 cells. PI positive suspended RT4 cells were morphologically intact while pelleted RT4 cells and suspended J82 cells were mainly disrupted. From these data it can be concluded that intracellular or membrane alterations seem to be correlated with the occurrence of cavitational effects while cell disruption can likewise occur by the direct impact of the shock wave.

1313 Unilateral lesion of the hippocampal ca1 subfield due to transient forebrain ischemia plus unilateral hyperthermia, and its dynamics of cerebral blood flow and brain temperature

A brief sub-lethal ischaemic stimulus has been reported to protect against subsequent ischaemic damage in vivo, and in vitro following periods of hypoxia or oxygen–glucose deprivation (OGD). Preconditioning against neurotoxic stimuli has been linked to N-methyl-d-aspartate (NMDA) receptors, since receptor blockade prevents the protection afforded by OGD, and low doses of NMDA treatment are capable of preconditioning. The current study demonstrated that NMDA preconditioning also protects against 3-nitropropionic acid (3-NPA), a generator of both excitotoxic and oxidative damage, in addition to glutamate. Cerebellar granule neuronal (CGN) cultures prepared from 8-day neonatal Sprague–Dawley rats were maintained for 8 days prior to NMDA stimulation for 6 h. At 9 days in vitro (DIV), preconditioned and control cultures were subjected to a toxic insult (1 μM–10 mM glutamate or 1 μM–10 mM 3-NPA). Neuronal viability was assessed by use of a fluorescein diacetate assay. Protection was effective with 100 μM NMDA preconditioning for 6 h against 1–100 μM glutamate, and also against 1–500 μM 3-NPA. The study demonstrates that NMDA preconditioning can be beneficial against excitotoxic treatments, even when these are potentially complicated by associated oxidative damage and metabolic compromise, as is the case for 3-NPA.

1312 Effects of dopaminergic transplants on in vivo activity of tryptophan hydroxylase in the striatum of rats with unilateral 6-hydroxydopamine lesions

A brief sub-lethal ischaemic stimulus has been reported to protect against subsequent ischaemic damage in vivo, and in vitro following periods of hypoxia or oxygen–glucose deprivation (OGD). Preconditioning against neurotoxic stimuli has been linked to N-methyl-d-aspartate (NMDA) receptors, since receptor blockade prevents the protection afforded by OGD, and low doses of NMDA treatment are capable of preconditioning. The current study demonstrated that NMDA preconditioning also protects against 3-nitropropionic acid (3-NPA), a generator of both excitotoxic and oxidative damage, in addition to glutamate. Cerebellar granule neuronal (CGN) cultures prepared from 8-day neonatal Sprague–Dawley rats were maintained for 8 days prior to NMDA stimulation for 6 h. At 9 days in vitro (DIV), preconditioned and control cultures were subjected to a toxic insult (1 μM–10 mM glutamate or 1 μM–10 mM 3-NPA). Neuronal viability was assessed by use of a fluorescein diacetate assay. Protection was effective with 100 μM NMDA preconditioning for 6 h against 1–100 μM glutamate, and also against 1–500 μM 3-NPA. The study demonstrates that NMDA preconditioning can be beneficial against excitotoxic treatments, even when these are potentially complicated by associated oxidative damage and metabolic compromise, as is the case for 3-NPA.

Effect of open‐air fumigation with sulphur dioxide and ozone on phyllosphere and endophytic fungi of conifer needles

ABSTRACTThe phyllosphere microbial populations inhabiting the needles of three conifer species, Scots pine (Pinus sylvestris L.), Sitka spruce (Picea sitchensis L.) and Norway spruce (Picea abies (L.) Karst.), exposed to SO2 and O3, in an open‐air fumigation experiment were analysed over a 3 year period using serial dilution after washing, direct plating and a fluorescein diacetate (FDA) enzyme assay. Total fungal populations ranged from 102 to 105 colonyforming units (CPU) g−1 fresh weight of needles. The dominant fungi isolated from needles varied with tree species and isolation technique; Aureobasidium pullulans (de Bary) Arnaud was most common on Scots pine and Norway spruce and white yeasts on Sitka spruce using the dilution plating method. However, direct plating of needle segments onto culture media indicated that Sclerophoma pythiophila (Corda) Hohnel was dominant on Scots pine and A. pullulans on Sitka and Norway spruce. Green needles of Sitka spruce were found to be endophytically colonized by Rhizosphaera kalkhoffii Bubak, but seldom by Lophodermium piceae (Fuckel) Hohn during extensive sampling in 1990. Statistical analyses revealed significant differences (P<0.05) between plots in the 3 year mean of the total fungal populations or the fungal biomass (FDA assay) on all three tree species. Differences between plots were also observed for a number of dominant component species. Data were also analysed for treatment effects. A significant effect of SO2 treatment was observed on the total fungal populations on Sitka spruce (P<0.05) which were reduced markedly by the low‐SO2 treatment, while the O3 treatment caused a significant increase in total fungal numbers on Scots pine (P<0.05). The FDA activity on needles of both Scots pine and Sitka spruce was noticeably higher in the 03‐only treatment plot, but the overall O3 effect was not significant. Treatment effects were also detected on the occurrence of component species. The serial dilution method revealed an SO2 effect (P<0.05) of a reduction in the occurrence of pink yeasts on Sitka spruce and an O3 effect (P<0.05) of an increase in the occurrence of S. pythiophila on Sitka spruce (P<0.01) but a decrease of Epicoccum nigrum Link and Cladosporium spp. on Scots pine. The direct‐plating method revealed an SO2 effect of an increase in S. pythiophila on Norway spruce (P<0.05). Ozone treatment caused a significant increase in the isolation of a black strain of A. pullulans on Norway spruce (P<0.05). Endophytic colonization of Sitka spruce needles by R. kalkhoffii was found to be increased on two occasions by O3 exposure.

Phospholipid and FDA activity measurements adapted to biological GAC

Abstract Established microbial ecology analytical techniques for measuring the quantity and activity of bacteria were examined for use on biological granular activated carbon (GAC). Activity was determined using the fluorescein diacetate (FDA) assay. The assay was tested and accordingly corrected to account for the adsorption of the fluorescein on the GAC by utilizing a standard curve containing GAC from the reactor. The quantity of FDA solution and incubation times were also examined. Normalization of fluorescein adsorbance to incubation time appears to be feasible provided that the concentration of the FDA solution does not become inhibiting. The amount of biomass present was determined by measuring the phosphorous content of the phospholipids in cellular membranes. The assay can be normalized for sample size. The combination of the two assays successfully measured the microbial characteristics of the biological GAC.

An Attempt to Quantify Relative Mycelial Development of Phanerochaete chrysosporium Using the Fluorescein Diacetate Spectrophotometric Assay

The spectrophotometric fluorescein diacetate assay was used to quantify viable mycelial growth of Phanerochaete chrysosporium on Pinus banksiana wood chips. Tests were conducted on inoculated nonsterile and sterile chips with noninoculated nonsterile and sterile chips as control. Additionally, some chips were compressed to make nutrients more readily accessible by mechanically rupturing the wood parenchyma cells. Comparisons were made of chips heavily colonized with P. chrysosporium against chips with only scattered mold mycelium to test the effectiveness of the fluorescein diacetate assay for this particular species. It was found that P. chrysosporium did not produce significant hydrolysis of fluorescein diacetate as compared to other resident microflora.

Effects of Jet Fuel Spills on the Microbial Community of Soil

Hydrocarbon residues, microbial numbers, and microbial activity were measured and correlated in loam soil contaminated by jet fuel spills resulting in 50 and 135 mg of hydrocarbon g of soil. Contaminated soil was incubated at 27 degrees C either as well-aerated surface soil or as poorly aerated subsurface soil. In the former case, the effects of bioremediation treatment on residues, microbial numbers, and microbial activity were also assessed. Hydrocarbon residues were measured by quantitative gas chromatography. Enumerations included direct counts of metabolically active bacteria, measurement of mycelial length, plate counts of aerobic heterotrophs, and most probable numbers of hydrocarbon degraders. Activity was assessed by fluorescein diacetate (FDA) hydrolysis. Jet fuel disappeared much more rapidly from surface soil than it did from subsurface soil. In surface soil, microbial numbers and mycelial length were increased by 2 to 2.5 orders of magnitude as a result of jet fuel contamination alone and by 3 to 4 orders of magnitude as a result of the combination of jet fuel contamination and bioremediation. FDA hydrolysis was stimulated by jet fuel and bioremediation, but was inhibited by jet fuel alone. The latter was traced to an inhibition of the FDA assay by jet fuel biodegradation products. In subsurface soil, oxygen limitation strongly attenuated microbial responses to jet fuel. An increase in the most probable numbers of hydrocarbon degraders was accompanied by a decline in other aerobic heterotrophs, so that total plate counts changed little. The correlations between hydrocarbon residues, microbial numbers, and microbial activity help in elucidating microbial contributions to jet fuel elimination from soil.

A Biochemical Method for Estimating Viability of Teliospores ofTilletia controversa

(...) Lipase activity was detected consistently in extracts from viable teliospores by a fluorescein diacetate (FDA) assay. No fluorescence activity was observed in extracts from autoclaved spores. By comparison, lipase detection was inconsistent when 4-methyl-umbelliferyl-palmitate was used as substrate. Extracts of teliospores that did not germinate on soil extract agar exhibited no lipase or glucosidase activity. Viability of individual teliospores could not be assessed by lipase assay because spore coats were impermeable to FDA. The FDA assay reduced the time needed to assess the average viability of a teliospore population from 2 mo to 1 hr

Microencapsulation of CHO cells in a hydroxyethyl methacrylate-methyl methacrylate copolymer

Chinese hamster ovary fibroblasts, as model cells, have been microencapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer (HEMA-MMA) by interfacial precipitation. The polymer containing ∼- 75 mol% HEMA, dissolved in polyethylene glycol 200 (PEG 200) was coextruded with the cell suspension (4–6 × 105 cells/ml in the α-MEM with 10% foetal calf serum ± Ficoll 400/PBS) through a concentric needle assembly. Polymer solution droplets, containing cells, were blown off the end of the needle assembly by a coaxial filtered air stream into a nonsolvent bath containing phosphate buffered saline (PBS) with 5 ppm Pluronic L101, overlaid with hexadecane. The nascent capsules hang at the hexadecane/PBS interface while the solvent is extracted into the aqueous nonsolvent, to precipitate the polymer around the cells. The resultant capsules were 500 μm-1 mm in diam. with a microporous sponge-like interior, and also very tough and flexible. The cells survived encapsulation based on subculture ability, retention of some fluorescein diacetate (FDA) activity over 5 d and direct light microscopic evidence of cell growth over 10 d after histological sectioning and staining. However, cell growth was not uniformly observed (especially in the FDA assay) and this was attributed to space limitations for growth within the microporous interior. Continued development of this process and adaptation to cells such as pancreatic islets is expected to lead to hybrid artificial organs which are capable of ameliorating metabolic disorders such as diabetes.

Osmotic tolerance of human granulocytes.

Human granulocytes are injured when returned to isotonic conditions after exposure at 0 degree C to hyperosmotic solutions of NaCl or sucrose with osmolalities above 0.6 osmolal. The damage was expressed as a loss of membrane integrity [fluorescein diacetate (FDA) assay] only after 60-90 min incubation at 37 degrees C. Survival after exposure to a 1.4-osmolal solution at 0 degree C was dependent on the extent of subsequent dilution. Dilution to below 0.6 osmolal was damaging, but cells could be returned to near-osmotic conditions provided that the solute concentration was increased again to 0.64 osmolal before the cells were incubated at 37 degrees C. Granulocyte cell volumes were measured under various osmotic conditions by computer-assisted micrometry. The cells did not display a minimum volume but behaved as osmometers over the observed range of 0.2-1.4 osmolal. Granulocyte volume at a given osmolality was independent of whether the cells had first been exposed to a strongly hyperosmotic medium, indicating that no solute loading occurred in hyperosmotic sucrose solutions. Even though the cells did not survive sequential exposure to greater than 0.6 osmolal solutions, subsequent return to isotonicity, and incubation at 37 degrees C, neither cell lysis nor loss in FDA-positive cells occurred after the first two steps. This finding is not consistent with the critical-surface area-increment theory of freezing injury. The mechanism of cell injury in hyperosmotic solutions is thus not known. However, the results show that osmotic stress is potentially a major damaging factor both in the equilibration of cells with protective additives and during freezing and thawing.

Development of preovulatory follicles and oocytes during the oestrous cycle of mature and aged rats.

Abstract. The percentage of viable granulosa cells and the viability of the oocyte of each antral follicle on each day of the oestrous cycle of mature and aged rats was determined by trypan blue exclusion and fluorescein diacetate assays, respectively. In mature rats, the number of mid-sized follicles (350–550 μm in diameter) increased from oestrus through di-oestrus. The percentage of viable granulosa cells increased in only mid-sized follicles which contained a viable oocyte. On pro-oestrus, two types of preovulatory follicles (> 550 μm in diameter) were observed in the mature pro-oestrous rat: those with a high granulosa cell viability (> 60%) and a viable oocyte and those with a lower percentage of viable granulosa cells (< 50%) and a non-viable oocyte. This suggests that in mature rats only viable oocytes would be ovulated since presumably, only those preovulatory follicles with a high percentage of viable granulosa cells are able to ovulate. In aged rats, preovulatory-sized follicles were present on each day of the cycle. The percentage of viable granulosa cells within those preovulatory-sized follicles with nonviable oocytes increased on met-oestrus, decreased on di-oestrus and increased by pro-oestrus. This pattern resulted in the development of 3 to 4 preovulatory follicles per ovary with all preovulatory-sized follicles possessing a high percentage of viable granulosa cells regardless of the functional state of the oocyte. It is proposed that in aged rats, all the preovulatory follicles would ovulate but many of the ovulated oocytes would be defective. This mechanism would account for the lower fertility of these older animals.