A method for the experimental determination of light absorption by aquatic heterotrophic bacteria

A method has been set up for the experimental determinationof the volume coefficient for light absorption in vivo by aquaticheterotrophic bacteria. The application described here is theabsorption measurement of the bacterial fraction that passesthrough the commonly used GF/F ifiter and remains unaccountedfor. The experimental samples were prepared by successive waterfiltra tions through GF/F and 0.22 µm Millipore membranes.Light-transmission and light-reflection measurements of thefilter-retained samples were performed using a dual-beam spectrophotometerequipped with an integrating sphere attachment. Sample absorptionwas derived from the data by a procedure that corrects for thecontamination of the results due to the high degree of lightscattering by the bacteria. The bacterial absorption was discriminatedfrom fine detritus absorption by bleaching the bacterial respiratorypigments using a K₂S₂O₈ solution. The absorption amplificationcaused by multiple scattering in the filter was corrected forby an expression that was obtained experimentally. A test ofthe method, including error analysis, was performed on samplescollected in both marine and inland waters. The relative contributionsto light absorption by heterotrophic bacteria and various typesof particulate matter were also measured for a typical situation.Combining the measured volume absorption coefficients with backscatteringcoefficients computed by Mie theory yields a set of input datato multicomponent optical models that is needed to assess thecontribution of these heterotrophic bacteria to the radiativetransfer process.     

A sensitivity analysis of the 'Transmittance-Reflectance' method for measuring light absorption by aquatic particles

A thorough sensitivity analysis of the ‘Transmittance–Reflectance’(T-R) method for measuringin vivo light absorption by aquaticparticles retained on glass-fiber filters has been carried out.Particular attention has been devoted to the error contributionby the variability of the optical properties of the GF/F filtersused. The overall error of the measurement of the filter-retainedparticle optical density, OD_s, has been evaluated as ~0.002(1), with 0.0015 error contribution by the filter variability.The corresponding error of the optical density of the particlesuspension, OD_sus, has been obtained by differentiation of theequation expressing the experimental correlation (OD_susversusOD_s); the error increases with the optical density value, from= 0.0015 (for OD_sus = 0.05) to = 0.027 at the upper limit ofthe optical density range tested (OD_sus = 0.59). The validityrange of the experimental (OD_sus versus OD_s) correlation hasbeen shown to extend to OD_s = 0.75. The importance of the accuratecorrection for light backscattering performed by the T-R methodhas been investigated by comparison with results given by thestandard ‘Transmittance’ (T) method; it has beenshown that the coarse correction for light scattering includedin the T method may cause serious errors in the measured opticaldensity spectra of mineral detritus. This evidence confirmsthe need to resort to the T-R method for the measurement ofparticle samples of detritus-rich coastal waters. The papersummarizes the latest developments of the T-R method and includesan Appendix with asynopsis of the routine now used by the authors.     

Variability of the amplification factor of light absorption by filter-retained aquatic particles in the coastal environment

The amplification of light absorption by aquatic particles retainedon glass-fiber filters, the so-called ß factor, has beenmeasured for 29 stations located in the varying coastal environmentof the northern basin of the Adriatic Sea. The spectral valuesof the optical density of particles have been determined onglass-fiber filters by the standard transmittance (T) methodas well as by the transmittance-reflectance (T-R) method, whichperforms an accurate correction for light backscattering bythe particles, and on glass slides by the modified filter–transfer–freeze(FTF) method, which eliminates the pathlength amplification.It has been shown that the relationships between the opticaldensities of particles on glass slides (OD_sus) and on glass-fiberfilters (OD_f) have a low dispersion when OD_f is measured bythe T-R method: in this case, the use of a single expressionfor all stations adds an error that does not exceed the typicaluncertainty of the measurement of the filter-retained particleabsorption. On the contrary, the OD_sus versus OD_f plots obtainedby the T method exhibit considerable variability, apparentlydue to the approximate correction for light backscattering performedby this method, and do not permit the use of a single equationfor all stations.     

Measurement of light absorption by aquatic particles retained on filters: determination of the optical pathlength amplification by the 'transmittance-reflectance' method

The optical pathlength amplification (ß factor), inducedby multiple scattering in glass-fibre filters used for lightabsorption measurements of aquatic particles, has been measuredby the transmittance-reflectance (T-R) method for algal specieswith cell diameter in the range from 1 to 5 µm. Theßfactors obtained showed a much lower dispersion than that observedin similar measurements carried out by the standard transmittancemethod, suggesting that this could be due, at least in part,to the incomplete account of the scattering losses taken bythe latter method. An experimental test has verified that theß factor expression derived from measurements carriedout by the T-R method on an algal culture is also applicableto detrital particles.     

In-beam light absorption measurement of anthracyclines in cells with a flow-through system

A flow-through cuvette in which cells attach as a monolayer to a quartz plate was developed for measurement of the light absorbance of anthracyclines in cells. Despite the drawback of a short path-length (of the order of the cell diameter), a dynamic flow-through set-up and baseline storage made it possible to measure intracellular absorbance and obtain spectral data for daunomycin and carminomycin. Stopping the flow and allowing the drug to equilibrate between medium and cells led to a 20% decrease of molar light absorption of cellular anthracycline, which permitted measurement of the total cellular concentration. Furthermore, accumulation and efflux kinetics were determined for H35 rat hepatoma cells. On the basis of the reported formation constant of the iron-complex of carminomycin, which is of the order of 10(34), we expected to find this complex within the cells. However, the spectrum of cellular drug did not show absorbance bands characteristic of the complex. A red shift and hypochromism were found in the daunomycin spectrum after intracellular binding, which corresponds with the spectral change observed after intercalation of daunomycin into DNA.     

Automated chromatographic system for the simultaneous measurement of plasma pregnenolone and 17-hydroxypregnenolone by radioimmunoassay

A new, simple, rapid and highly practicable automated chromatographic system for the separation, and a sensitive radioimmunoassay system for the subsequent measurement of pregnenolone and 17-hydroxypregnenolone has been developed. Pregnenolone and 17-hydroxypregnenolone were extracted with methylene chloride and separated from cross-reacting steroids by mechanised Sephadex-LH20 multi-column chromatography. Anti-pregnenolone and anti-17-hydroxypregnenolone were obtained by immunising rabbits with pregnenolone-20-oxime-BSA and 17-hydroxypregnenolone-20-oxime-BSA. The lower detection limit of the assay is 0.15 and 0.28 nmol/l for pregnenolone and 17-hydroxypregnenolone, respectively. Normal values for this assay in young male adults, in adult females, and in prepubertal boys and girls were established as a basis for the functional diagnosis of androgen excess syndromes/steroidogenesis defects.     

An integrated portable apparatus for the simultaneous field measurement of photosynthetic CO2 and water vapour exchange, light absorption and chlorophyll fluorescence emission of attached leaves

Abstract. A portable apparatus has been constructed to measure simultaneously the quantum yield of CO₂ assimilation, light absorption, chlorophyll fluorescence emission and water vapour exchange of attached intact leaves in the field. The core of the instrument is a light-integrating spherical leaf chamber which includes ports for a light source, photosynthetically active radiation sensor, fluorescence probes and gas inlet and outlet manifolds. Measurement of the quantum flux inside the empty chamber and with a leaf present allows determination of leaf absorptance. An open gas exchange system is employed using an infra-red analyser to measure leaf CO₂ exchange. Using a DC white light source the quantum yield of CO₂ assimilation based on absorbed light (φ_abs) may be determined rapidly in either ambient air or artificial gas mixtures. Inclusion of capacitance humidity probes into the gas inlet and outlet ports allows simultaneous determination of water vapour exchange and subsequent estimation of stomatal conductance to CO₂ and intercellular CO₂ concentration. Measurement of fluorescence emission by the sample leaf exposed to white light is achieved by a modulated fluorescence detection system. In addition to determination of the minimal, maximal and variable fluorescence levels, a further analysis allows the photochemical and non-photochemical components of fluorescence quenching, to be estimated. The theory and design of this apparatus is described in detail. The use of the apparatus in the field is demonstrated through a study of the photosynthetic performance of a maize and bean crop during the growing season and by analysis of the photosynthetic performance of crops subjected to nitrogen-stress and a herbicide treatment.     

Proof of a law for calculating absorption of light by cellular suspensions

A statistical argument is used to prove that, under a wide range of conditions, the effect of a layer of liquid containing randomly distributed cells on the transmission of light can be calculated by multiplying the individual effects due to each cell. This rule is in use, but treated as an approximation. The proof relies on the exponential dependence of transmission on path length to show that, for randomly distributed cells, the rule is almost exact. Extensions to allow for light scattering are discussed.     

A direct method for the simultaneous measurement of ceramide and phospholipase D activity

Both ceramide and phospholipase D (PLD) have important roles in a variety of signal transduction pathways. Recent evidence suggests that ceramide is a novel second messenger with specific biological effects. Publications in this field have increased rapidly in the last few years. However, a method to directly and rapidly measure cermide production has been lacking. Herein, we report on a novel, inexpensive, direct and rapid assay for the measurement of ceramide and the simultaneous measurement of PLD activity. This method uses labeling of cells with [(14)C]myristic acid and a TLC solvent of ethyl acetate/acetic acid/trimethylpentane. This method avoids the loss of radioactivity and variability due to changes in DAG kinase activity that are associated with the commonly-used DAG kinase assay.     

Determination of second virial coefficient of proteins using a dual-detector cell for simultaneous measurement of scattered light intensity and concentration in SEC-HPLC

A method is proposed for the measurement of the B22 value of proteins in aqueous solutions in flow-mode that utilizes a novel fabricated dual-detector cell, which simultaneously measures protein concentration and the corresponding scattered light intensity at 90 degrees , after the protein elutes from a size-exclusion column. Each data point on the chromatograms obtained from the light scattering detector and the concentration (ultraviolet) detector is converted to Rayleigh's ratio, Rtheta, and concentration, c, respectively. The B22 value is calculated from the slope of the Debye plot (Kc/Rtheta versus c) generated from a range of concentrations obtained from these chromatograms for a single protein injection. It is shown that this method provides reliable determination of the B22 values for such proteins as lysozyme, chymotrypsinogen, and chymotrypsin in various solution conditions that agree well with those reported in literature.     

Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions

For particulate suspensions and for solutions that scatter light measurably the total absorbance A generally contains contributions due to specific absorption (Aa) and scattering of light (As). The quantity As is closely related to the turbidity tau. In general, spectrophotometry of such systems requires proper modification of the spectrophotometer used in order to permit accurate determination of the absorbance A and of the derived quantities Aa and As. Apparent deviation from Beer's law in such systems is often due to inappropriate experimental technique. After a discussion of the parameters that determine the intensity of light scattered by solutes, an account is given of the experimental precautions to be taken for determination of the absorbance of light scattering suspensions and solutions and of techniques for correcting absorbance spectra for scattering of light. Measurement of the turbidity is briefly confronted with determination of the scattering ratio i90 degrees/Io and the impact of erroneous turbidity measurements on derived molecular parameters is discussed.     

The consequences of delayed greening during leaf development for light absorption and light use efficiency

Many species of rainforest plants have an unusual form of leaf development such that leaves delay greening until after full leaf expansion. Chlorophyll accumulation was measured during leaf development in five woody rainforest species, three with white young leaves, and two with ‘normal’ greening. In the three species with white leaves, the chlorophyll content of the expanding leaves was about 0.4mg dm^?2, whereas in the two species with green young leaves, chlorophyll content was about 2.1 mg dm^?2. Chlorophyll accumulation in greenhouse and field experiments was independent of light level. During leaf expansion, species with delayed chloroplast development only absorb 18–25% of the maximum possible light, compared with 80% for species with normal greening. Furthermore, species with delayed greening have low chlorophyll contents and reduced absorption for at least 30 d after full expansion. At a PPFD typical of the forest under story, the photosynthetic light use efficiency based upon incident radiation was 0.030–0.036 for species with delayed chloroplast development and 0.068–0.085 for the two species with normal greening. The lower light use efficiency of white species was primarily due to decreased light absorption. However, they also had a slightly lower light use efficiency based upon absorbed radiation, suggesting that development of other components of the photo-synthetic apparatus also may be delayed. Despite the fact that delayed greening decreases light absorption and light use efficiency during leaf development, it is extremely common in shade-tolerant species. We suggest that an advantage of delayed greening is that resources are not invested in the leaf until it is fully expanded and better defended from herbivores.