Showing papers by "Douglas B. Kell published in 1992"

Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry

Abstract: A.S. KAPRELYANTS AND D.B. KELL. 1992. The fluorescent dye rhodamine 123 (Rh 123) is concentrated by microbial cells in an uncoupler-sensitive fashion. Steady-state fluorescence measurements with Micrococcus luteus indicated that provided the added dye concentration is below approximately 1 mmol/1, uptake is fully uncoupler-sensitive and is not accompanied by significant self-quenching of the fluorescence of accumulated dye molecules. ‘Viable’ and ‘non-viable’ cells are easily and quantitatively distinguished in a flow cytometer by the extent to which they accumulate the dye. The viability of a very slowly growing chemostat culture of Mic. luteus is apparently only about40–50%, as judged by plate counts, but most of the ‘non-viable’ cells can be resuscitated by incubation of the culture in nutrient medium before plating. The extent to which individual cells accumulate rhodamine 123 can be rapidly assessed by flow cytometry, and reflects the three distinguishable physiological states exhibited by the culture (‘non-viable’, ‘viable’ and ‘non-viable-but-resuscitable’). Gram-negative bacteria do not accumulate rhodamine 123 significantly because their outer membrane is not permeable to it; a simple treatment overcomes this. Flow cytometry using rhodamine 123 should prove of general utility for the rapid assessment of microbial viability and vitality.

Channelling can decrease pool size.

Abstract: It is widely considered that a possible advantage of metabolite channelling, in which a product of an enzyme is transferred to the next enzyme in a metabolic pathway without being released to the 'bulk' solution, is that channelling can decrease the steady-state concentrations of 'pool' intermediates. This then spares the limited solvent capacity of the cell, and reduces the loss of pathway flux due to leakage or instability of the free intermediate. Recently, however, based on simulations of a particular model of a 'dynamic' channel, Cornish-Bowden ["Failure of channelling to maintain low concentrations of metabolic intermediates" (1991) Eur. J. Biochem. 195, 103-108] has argued that this is not in fact the case; his simulations indicated that the channel was rather ineffective at decreasing the concentration of the pool intermediate, and in some cases actually increased it. However, although his simulations were restricted to very specific thermodynamic and kinetic parameters, he generalised his conclusions, arguing that "channelling has no effect on the free concentration of a channelled intermediate in a pathway". By showing that, for a number of kinetic cases, the concentration of the pool intermediate did decrease substantially with increased channelling, we demonstrate here that the conclusion of Cornish-Bowden is not correct. In particular, if the reaction catalysed by the enzymes forming the channel has an equilibrium constant K higher than 1, and if the enzyme removing the product of the channel reaction is kinetically competent, channelling in the model system studied by Cornish-Bowden (1991) can decrease the steady-state concentration of the pool by a factor of 1000, independently of the mechanism of the terminal reaction and under conditions of essentially constant overall flux. If the channel is a 'static' channel, the decrease in the pool can be to arbitrarily low levels. This conclusion also holds for a system in which other reactions may consume the pool intermediate. Thus, channelling can maintain metabolite concentrations at low levels.

Physiological Studies on the Solid-state Quinoa Tempe Fermentation, Using On-line Measurements of Fungal Biomass Production

Abstract: A quantitative approach to the on-line measurement of fungal biomass, based on the biomass-dependent changes in electrical capacitance at 0.30 MHz, was exploited to optimise the solid-substrate tempe fermentation of Chenopodium quinoa Willd by Rhizopus oligosporus Saito. Variables including the mould strain, the initial pH, the inoculum density and the substrate moisture content influenced the mycelial development and quality of quinoa tempe prepared in petri dish fermentation units. It was found that R oligosporus isolate UCW-FF8001 at an inoculation density of 33 x lo4 colony forming units per gram of quinoa substrate at 620 g kg-I moisture content yielded both the highest biomass and the best quality tempe.

The Protonmotive Force as an Intermediate in Electron Transport-Linked Phosphorylation: Problems and Prospects

Abstract: Publisher Summary This chapter describes the protonmotive force (pmf) as an intermediate in electron transport-linked phosphorylation and its problems and prospects. An important and unresolved general problem in cellular biochemistry concerns the question, in a metabolic pathway, of whether the intermediary metabolites for whatever reason, are passed directly among the enzymes that catalyze their interconversion. If redox chains and ATP hydrolases pump protons across membranes, and given that the diffusion of protons is one of the fastest chemical reactions known, then the result of this pumping of protons is the setting up of an electrochemical potential difference for protons among the aqueous phase that the coupling membrane serves to separate. This pmf should be created at a rate, and possess a magnitude, sufficient to account for ATP synthesis. Another important prediction of the chemiosmotic coupling hypothesis is that, if the pmf is the energy-coupling intermediate in ETP, an artificially applied pmf should drive phosphorylation at a rate greater than or equal to the in vivo rate.

Rapid Determination, Using Dielectric Spectroscopy, of the Toxicity of Organic Solvents to Intact Cells

Abstract: Dielectric spectroscopy utilising dual-frequency measurements has been used to study the toxic effects of a number of organic solvents to suspensions of Saccharomyces cerevisie. Solvents of a highly apolar nature, such as hexadecane, were identified as being non-cytotoxic, and thus suitable for use with whole-cell systems. A novel approach to aid biotransformations, using mixed organic solvents, has also been studied. This has revealed that the cytotoxic nature of polar organic solvents, such as octan-l-ol, may be negated by dissolving them first in apolar, non-cytotoxic organic solvents, such as hexadecane, before exposure to the cell suspension. The use of mixed organic solvents with immobilised cell systems was shown to be possible by the growth of Lactobacillus brevis , (immobilised within hollow ceramic microspheres), in the presence of 5% (v/v) octan-l-ol dissolved within 5% (v/v) hexadecane, added to the media. Dispersion of these immiscible solvents was improved by the addition of 5% (v/v) ethanol and 0.05% (v/v) tween 80. Cell growth occurred (as measured by dielectric spectroscopy) over a 70- hour period.