Techniques for biochemical respiration measurements.

A small membrane-covered oxygen electrode is described. This electrode is used either as a stationary electrode in stirred solutions or as a vibrating electrode in unstirred solutions. An amplifier system for registration of the electrode current and its time derivative is also described as are two specialized reaction vessels, a miniature vessel of ca. 7-μl volume and a closed vessel for sampling during respiration measurements. The kinetics of oxygen uptake from the atmosphere of respiring solutions is investigated. The uptake follows first-order kinetics and may be calculated from simple equations. The uptake may be prevented by continuously adjusting the air space oxygen tension to the oxygen tension of the solution. This is done with the controlled gas mixer which is described. It makes possible reliable respiration measurements in open reaction vessels (e.g., photometric cuvettes). The techniques described have been developed for work with mitochondria but they have wide applicability.     

Instructions to authors

A bacterial staining method using fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is described for quantifying actively respiring bacteria that adhere to commercially pure titanium surfaces coated with cross-linked albumin. This has not been possible to date using ordinary DNA stains such as propidium iodide (PI) or Hoechst, both of which produce a very bright background. With this technique, it was demonstrated that the cross-linked albumin inhibited the adherence of Staphylococcus aureus and Staph. epidermidis to the titanium surface.     

Characterization of a thermo-osmotic gas transport mechanism in Alnus glutinosa (L.) Gaertn.

Summary A gas transport system based upon the physico-chemical effect of thermo-osmosis of gases in described for the black alder, Alnus glutinosa (L.) Gaertn. Air is transported through the alder's stem to the roots, thus improving O₂ supply to respiring tissues of the root system. The gas transport system is investigated by means of a tracer gas technique (11% ethane in air, v/v). Gas transport depends on any source of radiant heat generating a temperature difference between the tree's stems and the atmosphere. The amount of gas transported in leafless trees is four times higher than the amount of gas reaching the roots by gas diffusion. Two-thirds of the gas is transported in the wood, only one-third in the bark. Intercellular spaces inside the porous lenticels of the bark are responsible for this kind of gas transport. Their diameters are estimated by the effusion rates of different tracer gases to be in the range of 1 m.     

Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration.

1. Aerobic respiration by cells of Paracoccus dentrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium. Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation. Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described. 2. If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P. dentrificans respiring aerobically in 100 mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid/NaOH or 100mM-NaH2PO4/NaOH. This potential, however, is decreased by more than 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM-K+. 3. Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150mV in described suspension media. The presence of these species under aerobic conditions, moreover, has negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration. These data indicate that none of these molecules exert a significant uncoupling effect on the protonmotive force. 4. No 204Tl+ uptake into respiring cells was detected. This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore not an indicator of membrane potential as has been proposed. The absence of respiration-driven 204Tl+ uptake indicates that P. denitrificans cells grown under the conditions specified in the present work do not possess K+-transport systems of either the Kdp or TrkA types that have been described in Escherichia coli.     

Brain-derived respiring mitochondria exhibit homogeneous, complete and cyclosporin-sensitive permeability transition

The mitochondrial permeability transition (mPT) is increasingly implicated in neuronal cell death. In the present study, isolated respiring brain mitochondria were examined for their ability to undergo calcium-induced mPT and their sensitivity to mPT inhibition by cyclosporin A (CsA). Previous studies have suggested a heterogeneous response to calcium, a limitation of CsA inhibition, and a relative resistance in the ability of respiring brain mitochondria to undergo mPT. Using fluorometric and electron microscopic analyses, we found that virtually the whole population of respiring brain mitochondria readily undergo mPT and swell upon calcium exposure. Further, brain mitochondria were highly sensitive to CsA which potentiated morphological recovery after transient swelling as well as completely blocked mPT induction in the presence of a low concentration of ADP. Using flow cytometry, which allows analysis of individual mitochondria, we demonstrate that both brain and liver mitochondria display homogeneous responses to calcium-induced mPT. We conclude that the mPT is one likely target for the broad in vivo neuroprotective effects displayed by CsA when allowed to penetrate the blood-brain barrier, and that development of compounds inhibiting mPT may prove beneficial for the treatment of severe brain disease.     

Cartesian-diver microrespirometry: a technique for the accurate measurement of respiratory flux in plant cells

Abstract. A Cartesian-diver microrespirometer system is described which can be used to measure respiratory fluxes of oxygen accurately for cells of higher plants in a liquid phase. This microrespirometry technique has been adapted from protozoological and microfaunal studies to plant physiology. The Cartesian-diver has considerable scope for investigation of oxygen flux in plant cells and has several advantages compared to the oxygen electrode in terms of sensitivity to changing oxygen levels in respiring material. Because the volumes of liquid are small in the Cartesian-divers, diffusional distances arc measured in micrometres and there is no need for stirring to overcome diffusional problems, thus minimizing the risk of mechanical damage to the experimental material. In addition, only very small quantities of experimental material are required for the Cartesian-diver which is invaluable where only limited amounts of tissue or numbers of cells can be obtained. Examples of respiratory oxygen consumption by protoplasts from intercalary meristematic regions of light-grown barley ( Hordeum vulgare L.c.v. Patty) seedlings, in response to abscisic and gibberellic acids, are presented. The advantages and disadvantages of Cartesian-diver microrespirometry compared to oxygen electrodes are also discussed.     

Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration.

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Respiring bacteria deposit accumulated INT-formazan intracellularly as dark red spots. Corresponding to electron transport system activity, these deposits attain a size and a degree of optical density which allows them to be examined by light microscopy. If polycarbonate filters and epifluorescence microscopy are applied to analyze an INT-treated water sample, it is possible to differentiate between respiring and apparently nonrespiring bacteria. This differentiation, which permits determinations of the total number of bacteria and the proportion thereof involved in respiration, is realized directly within one and the same microscopic image. Initial applications of the present method for hydrobiological purposes showed that the proportion of respiring aquatic bacteria ranged between 6 to 12% (samples taken from coastal areas of the Baltic Sea) and 5 to 36% (samples taken from freshwater lakes and ponds). Cells of 1.6 to 2.4 micrometer (freshwater) and 0.4 micrometer (Baltic Sea) account for the highest proportion of respiring bacteria.     

Properties of mitotic cells prepared by mechanically shaking monolayer cultures of Chinese hamster cells

A technique has been developed for the selective detachment of mitotic cells from monolayer cultures of Chiness hamster cells with a simple reciprocating shaking machine. Cultures prepared by the shake treatment and placed in spinner flasks are routinely obtained, in which the mitotic fraction drops from 0.95–0.05 in 19 minutes. Some properties of mitotic cells prepared by this technique are described, along with a simple procedure for producing large quantities of mitotic cells. Cells chilled immediately after collection from a series of shake treatments complete mitosis synchronously upon subsequent resuspension in warm medium.     

Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration.

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Respiring bacteria deposit accumulated INT-formazan intracellularly as dark red spots. Corresponding to electron transport system activity, these deposits attain a size and a degree of optical density which allows them to be examined by light microscopy. If polycarbonate filters and epifluorescence microscopy are applied to analyze an INT-treated water sample, it is possible to differentiate between respiring and apparently nonrespiring bacteria. This differentiation, which permits determinations of the total number of bacteria and the proportion thereof involved in respiration, is realized directly within one and the same microscopic image. Initial applications of the present method for hydrobiological purposes showed that the proportion of respiring aquatic bacteria ranged between 6 to 12% (samples taken from coastal areas of the Baltic Sea) and 5 to 36% (samples taken from freshwater lakes and ponds). Cells of 1.6 to 2.4 micrometer (freshwater) and 0.4 micrometer (Baltic Sea) account for the highest proportion of respiring bacteria.     

Improved Method for Determination of Respiring Individual Microorganisms in Natural Waters

A method is reported that combines the microscopic determinations of specific, individual, respiring microorganisms by the detection of electron transport system activity and the total number of organisms of an estuarine population by epifluorescence microscopy. An active cellular electron transport system specifically reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, which is recognized as opaque intracellular deposits in microorganisms stained with acridine orange. In a comparison of previously described sample preparation techniques, a loss of >70% of the counts of INT-reducing microorganisms was shown to be due to the dissolution of INT-formazan deposits by immersion oil (used in microscopy). In addition, significantly fewer fluorescing microorganisms and INT-formazan deposits, both ≤0.2 μm in size, were found for sample preparations that included a Nuclepore filter. Visual clarity was enhanced, and significantly greater direct counts and counts of INT-reducing microorganisms were recognized by transferring microorganisms from a filter to a gelatin film on a cover glass, followed by coating the sample with additional gelatin to produce a transparent matrix. With this method, the number of INT-reducing microorganisms determined for a Chesapeake Bay water sample was 2-to 10-fold greater than the number of respiring organisms reported previously for marine or freshwater samples. INT-reducing microorganisms constituted 61% of the total direct counts determined for a Chesapeake Bay water sample. This is the highest percentage of metabolically active microorganisms of any aquatic population reported using a method which determines both total counts and specific activity.     

Factors Affecting the Determination of Respiratory Activity on the Basis of Cyanoditolyl Tetrazolium Chloride Reduction with Membrane Filtration

Deficiencies in traditional bacterial enumeration techniques which rely on colony formation have led to the use of total direct counting methods, such as the acridine orange direct count technique for the enumeration of planktonic bacteria. As total direct counts provide no information on the viability or activity of the organisms, demonstration of respiratory activity with the fluorochrome cyanoditolyl tetrazolium chloride (CTC) has been employed. We have modified this technique by performing filtration prior to CTC incubation. Cells captured on a polycarbonate membrane were incubated on absorbent pads saturated with medium containing CTC. Following counterstaining with DAPI (4(prm1),6-diamidino-2-phenylindole) total and respiring cells were enumerated by epifluorescence microscopy. Factors affecting CTC reduction by Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli K-12 were investigated. With K. pneumoniae, nutrient additions to the CTC medium did not increase the number of respiring cells detected. CTC reduction by all three organisms decreased in response to an increase of the pH of the CTC medium above pH 6.5. Increasing phosphate concentrations contributed to this inhibitory effect. CTC-membrane filter counts of K. pneumoniae, S. typhimurium, and E. coli K-12 and of bacteria in well water corresponded closely with plate counts (r = 0.987). The results show that careful attention should be given to the composition of CTC-containing media which are used to enumerate respiring bacteria. With an appropriate medium, reliable enumeration of respiring bacteria can be achieved within a few hours.     

Measurement of stem respiration of sycamore (Platanus occidentalis L.) trees involves internal and external fluxes of CO2 and possible transport of CO2 from roots

CO(2) released by respiring cells in tree stems can either diffuse to the atmosphere or dissolve in xylem sap. In this study, the internal and external fluxes of CO(2) released from respiring stems of five sycamore (Platanus occidentalis L.) trees were calculated. Mean rates of stem respiration were highest in mid-afternoon and lowest at night, and were positively correlated with air temperature. Over a 24 h period, on average 34% of the CO(2) released by respiring cells in the measured stem segment remained within the tree. CO(2) efflux to the atmosphere consisted of similar proportions of CO(2) derived from local respiring cells (55%) and CO(2) that had been transported in the xylem (45%), indicating that CO(2) efflux does not accurately estimate respiration. A portion of the efflux of transported CO(2) appeared to have originated in the root system. A modification of the method for calculating stem respiration based on internal and external fluxes of CO(2) was developed to separate efflux due to local respiration from efflux of transported CO(2).     

Activity of yeast multidrug resistance pumps during growth is controlled by carbon source and the composition of growth-depleted medium: DiS-C3(3) fluorescence assay

Like other tested wild-type strains (DTXII and IL-125-2B), exponential glucose- and/or fructose-grown cells of Saccharomyces cerevisiae BY4742 exhibit the previously described high activity of Pdr5p and Snq2p pumps (measured as export of the potentiometric fluorescent probe diS-C₃(3)). Upon saccharide depletion from the medium the pump activity in these cells, which differ from other strains in having a lower membrane potential, sharply drops to a very low level similar to that found in cells grown on ethanol or glycerol. This negligible pump activity in respiring cells thus appears to have a universal character.

Addition of glucose or fructose to respiring BY4742 cells grown to low culture densities restores multidrug resistance pump activity due partly to pump synthesis in pre-existing cells and partly to the high pump activity of newly grown cells; no such pump activity boost occurs when the sugar is added to high-density cultures of ethanol-grown or post-diauxic glucose-grown cells, even if these cultures are diluted to low density by their original growth-depleted medium. A strong sugar-induced increase in pump activity is found solely if respiring cells from high-density cultures are resuspended in fresh YPD or YPE medium before sugar addition. Its absence in respiring cells suspended in growth-depleted medium reflects an as yet unidentified effect of the composition of the growth-exhausted medium (depletion of some components and/or accumulation of extracellular metabolites during yeast growth) on sugar-induced pump activity rise.     

Testing a computer-assisted bending machine for manufacturing orthodontic treatment elements]

The use of suitable orthodontic devices producing desired defined force systems is of importance for successful orthodontic treatment. Bending loops can be difficult and time-consuming. Computerised fabrication would enable very precise reproduction of individual loops. A bending machine has now been developed within the framework of a computer-assisted treatment concept. In this study, a prototype machine was used to fabricate U-, T- and delta loops made of stainless steel, cobalt chromium and titanium molybdenum wire. The various geometric parameters of each loop were measured to determine how precisely they had been produced. Furthermore, the force system of each loop were experimentally investigated during simulated activation in an orthodontic measurement and simulation system. The results indicate that the geometric parameters had an average error of 2.8 degrees for angles and 0.9 mm for lengths. Owing to the fabrication errors, loops of the same type produced different force systems. Overall, the new bending machine can fabricate different types of loop, but the requirements of very precise fabrication are currently not met. This fact, together with further limitations in terms of configuration, means that the machine cannot be used routinely at present. However, the machine can nevertheless be considered a good basis for further development.     

A "slow" temperature jump apparatus built from a stopped-flow machine

A simple modification to a standard thermostated stopped-flow machine is described which allows it to be used as a temperature jump machine. Temperature jumps larger than 10 degrees C can be achieved in less than 150 ms which makes it useful for the range of times where conventional rapid temperature jumps are not applicable. The apparatus has a sample size of 300 microliters and can produce temperature jumps both above and below the initial temperature.     

Method for Coding Data on Protozoan Strains for Computers

SYNOPSIS. Traditionally, observations on the nature of protozoa have been published in periodicals or books, or remain buried in research notebooks. The retrieval and processing of information on a particular species or strain are dependent solely upon individual investigators. Although various modern methods have been applied to the study of protozoa, no attempt has been made to develop a system with which information on protozoan strains can be stored, retrieved easily, and processed for various analyses by computer technology. Based upon an existing system for encoding data on bacterial strains, a complementary system applicable to protozoan strains was developed and is described herein.     

The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states

The intensity of light scattered at 90° to the incident beam and the effective hydrodynamic radii of mitochondria incubated under a variety of conditions have been measured. Addition of high concentrations of uncouplers to respiring mitochondria resulted in a decrease in scatter which was not due to swelling. Addition of valinomycin to mitochondria depleted of substrate in K⁺-free medium produced an increase in scatter that was not due to shrinking. It is concluded that changes in the intensity of scattered light are not reliable indices of changes of volume of mitochondria, and that changes in conformation with changes in metabolic state dominate changes in light scatter. A molecular mechanism for the effect of metabolic state upon the scattered intensity is suggested.     

Development of multicolour digital image analysis system to enumerate actively respiring bacteria in natural river water

AIMS: To develop a rapid and simple method for the assessment of metabolic activity of bacteria in natural environment. METHODS AND RESULTS: A rapid and simple multicolour digital image analysis system for enumerating viable bacteria based on active fluorescent staining has been developed. This system can accurately differentiate actively respiring bacteria and non-respiring bacteria by distinctive colour information in the digital image captured by an epifluorescence microscope equipped with low magnification objective lens. An algorithm to distinguish bacteria from considerable detritus, which produced bright fluorescence in different colours, by colour segmentation has also been developed. This system was applied to river water samples, and the total and respiratory active bacterial counts by digital image analysis were highly related to those by epifluorescence microscopy (r2 = 0.96 and 0.93, respectively). CONCLUSION: This system allowed the rapid and simple differentiation of bacteria from detritus and concurrent assessment of their metabolic activity, with results being available within 1 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The low magnification image analysis allows more rapid and simple quantification of bacteria and could considerably decrease image data amount and acquisition time. This system could be easily applied to the rapid analysis of microbes in various environments.     

Investigations into the number of respiring bacteria in groundwater from sandy and gravelly deposits

Samples were collected from organically polluted and unpolluted groundwater of sandy and gravelly deposits. After filtration onto polycarbonate filters (0.2m pore size) the number of respiring bacteria was recorded by microscopically counting cells containing red INT-formazan spots, which characterize respiring bacteria. The total number of bacteria was simultaneously recorded by epifluorescence microscopy after staining with acridine orange. The number of respiring bacteria in the groundwater samples (55–490×10³/cm³) is within the range of values for other aquatic biotopes, but as the total number of bacteria in groundwater was usually higher, the proportion of respiring groundwater bacteria (0.66–7. 4%) was lower. Mainly larger bacteria, rods, and bacteria on particles could be identified as being active, whereas hardly any respiratory activity could be detected among small cocci and free interstitial bacteria. If the supply of dissolved organic matter (DOM) is adequate, the biomass of respiring bacteria correlates well with oxygen concentration, but there is no direct correlation between DOM concentration in groundwater and active bacterial biomass. Nor could any relationship be observed between the biomass of total and respiring bacteria, or between the quantity of respiring bacteria and heterotrophic bacterial activity.     

A stable, sensitive, and inexpensive amplifier for oxygen electrode studies

A stable, compact, and inexpensive amplifier is described that permits using the Clark oxygen electrode to measure the oxygen consumption of even slowly respiring mammalian cells at the lowest cell densities at which exponential growth will take place. A modification of the usual spinner flask used in mammalian cell culture greatly reduces the noise picked up by the electrode circuit and makes possible measurements at the highest sensitivity range.