Experimental problems using electronic particle counters

The following scheme lists the problems which have been and will continue to be encountered in grazing experiments on natural particle assemblages. In some cases there are solutions, but many of the problems listed below remain intractable. To solve them will require innovative approaches, most probably combining the use of particle counters with other techniques to approach the problem to be solved from several angles at once. Variance between samples due to

- statistical causes (especially caused by large particles) and

- differences between experimental bottles and between experimentals and controls at start of experiment. Shifts in particle distribution during experiment due to

- increase in small particles

- growth of bacteria and other organisms due to excretion of grazers and

- breakage during handling by grazers into fragments. Processes, independent of grazing, leading to changes in particle size distribution due to

- primary production

- grazing by additional components and

- bacterial growth on detrital particles and the formation of detrital flocs.

     

Specific problems using electronic particle counters

Some specific problems of electronic particle counters (Coulter Counter, Elzone) are discussed. Conductivity of the medium does not influence the size response of the instrument, but might change the size of the particles through osmotic stress. An important problem of electronic particle counters is count loss by coincidence of passage of particles through the orifice and caused by dead time of the instruments. Several correction equations were tried out. It turned out that the count losses were much higher than the ones predicted by the manufacturers. In instruments equipped with multi-channel size analysers dead time count loss is much more important than the count loss by coincidence. A maximal counting frequency of 200 counts/second is recommended for accurate work. The width of the size distribution of particles depended on the diameter of the aperture tube.     

Precision of size determination of resistive electronic particle counters

The precision of the size determination of resistive particlecounting systems was studied by: (i) measuring the apparentsize of a single particle cycled repeatedly through the sensingelectrodes: (ii) by measuring the apparent size distributionof a population of particles of known and limited size range.(iii) by moving individual particles through a large-scale modelof the electrode assembly, and (iv) by measuring the size ofalgal cells microscopically and (subsequently) with a resistivecounting system. Three systematic errors were indicated (i)An ‘edge error’ results when a particle passes throughthe orifice near the perimeter of the orifice (     

Detection of wine-spoiling yeasts by electronic methods

Electronic techniques for the estimation of micro-organisms have been used widely in the food industry because they entail minimal sample preparation, provide continuous automatic monitoring, produce relatively rapid results and incur low running costs. Electrical measurements have been evaluated for use in the alcoholic beverage industry to differentiate between wine-spoiling and non-wine-spoiling yeast contaminants. Suitable media, supplemented with ethanol to inhibit the growth of non-wine-spoilage yeasts, are described for use with the Bactometers models 32 and M123. A procedure for detecting spoilage yeasts in opaque alcohol-based drinks is also described.     

Functionalization of liposomes: microscopical methods for preformulative screening

Abstract

The development of smart delivery systems able to deliver and target a drug to the site of action is one of the major challenges in the field of pharmaceutical technology. The surface modification of nanocarriers, such as liposomes, is widely investigated either for increasing the blood circulation time (by pegylation) or for interacting with specific tissues or cells (by conjugation of a selective ligand as a monoclonal antibody, mAb). Microscopical analysis thereby is a useful approach to evaluate the morphology and the size owing to resolution and versatility in defining either surface modification or the architecture and the internal structure of liposomes. This contribution aims to connect the outputs obtained by transmission electron (TEM) and atomic force (AFM) microscopical techniques for identifying the modifications on the liposomal surface. To reach this objective, we prepared liposomes applying two different pegylation technologies and further modifying the surface by mAb conjugation. This work demonstrates the feasibility to apply the combined approach (TEM and AFM analysis) in the evaluation of the efficacy of a surface engineering process.     

Interpretation of the electron microscopical appearance of collagen fibrils from corneal stroma

The appearance in the electron microscope of mechanically-dispersed corneal collagen has been observed after positive staining with phosphotungstic acid and/or uranyl acetate and after negative staining with phosphotungstate ions. The distributions of positive stains (both cationic and anionic) were similar to those observed in other type I collagens (e.g. skin, tendon). A high correlation was found between charge density in the fibril and the distribution of charged amino acids predicted from the sequence of calf skin collagen. This correlation could be improved by including type III sequence data, suggesting the presence of 20% type III collagen within each fibril. Negative staining showed the usual collagen D-periodicity but without a clear gap/overlap structure. Detailed analysis revealed at least six sites where stain penetration was inhibited. Specific staining of glycosides using N,N,N′,N′-tetramethylethylenediamine(TEMED)-osmate suggested that these sites identify the covalent attachment of disaccharides to the collagen. Using synchroton X-ray diffraction from TEMED-osmate stained corneas we have determined the locations of the stain ions (and hence the glycosides) in the moist tissue. The results demonstrate that even though the detailed charge distribution and axial molecular packing in corneal collagen are similar to other type I collalgens, carbohydrate material, probably disaccharide, is attached at fairly regular intervals. This does not occur in other type I collagens. In particular, the presence of glycoside in the overlap region may play a role in producing the narrow uniform fibrils which are essential for the transparency of the cornea.     

The electronic spectrum of adenine by electron impact methods

The electron energy loss spectrum of adenine has been recorded over an excitation energy range of approximately 3-22 eV for scattering angles of 3 degrees and 6 degrees. In addition to reporting accurate vapor phase peak positions, the present work supports the long-held contention that the group of peaks in the 6- to 10-eV range belong to transitions that originate from valence pi orbitals. In the vapor phase spectrum, a Rydberg transition corresponding to an n----3s excitation is readily observed with a term value of 3.45 eV relative to the first long-pair ionization potential.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

Image analysis of transparent particle systems by mathematical morphology methods

The particle images are often distorted by apparent holes due to undeflected transmitted light. These apparent holes must be disregarded for a consequent image analysis. The reconstruction of the real particles by methods of mathematical morphology was used. The problems associated with touching particles were also dealt with. The method is illustrated on an ion-exchanger system.     

The electronic spectra of porphyrin-like cobalt-tetracarboxy-phthalocyanines as modified by polylysine and polyglycols

The effect of poly-L-lysine and polyglycols on the electronic spectral properties of cobalt-tetracarboxy-phthalocyanine, compound (I), was studied in order to determine the effect of the polymers on the molecular stacking properties of compound (I). In the present study we have coupled, both covalently and electrostatically, the poly-L-lysine to compound (I). The electrostatic interaction with the polylysine resulted in a bathochromic shift of absorbance by compound (I) from 680 to 695 nm, and the generation of shoulders in the 590 and 580 nm region. The bathochromic shift indicates that the polylysine either misaligns the units of compound (I) in the molecular stack or alters the angular relationship of the planar compound (I) molecules. The covalent linkage with the polylysine resulted in stabilization of the monomeric form of compound (I) with no hypochromism in the 680 nm region. The reaction of the polyglycols, dextran and DEAE dextran with compound (I) prior to the addition of the polylysine, stabilized the polymeric stacked form of compound (I) in the presence of the polylysine. Ammonium ion and ethanolamine resulted in the appearance of peaks in the 730-760 nm region; a trihydroxy containing primary amine and monomeric lysine generated no such peak at wavelengths above 700 nm. The polyglycol binding capacity of compound (I) facilitated the separation of the unbound compound (I) from the polymeric complexes.     

Interpretation of thermal perturbation spectra of proteins.

The thermal perturbation difference spectrum of reduced lysozyme has a long wave length extremum at 304 nm at pH 6.15 and a very small extremum at 306 nm at pH 1.5. These results differ from those of Leach & Smith (1972), which showed an extremum at 293 nm, the same as for model tryptophyl compounds. Our result may arise from a conformational difference between the two sample temperatures. The interpretation of thermal perturbation spectra of proteins is discussed. Contributions from thermally induced concentration differences, buried chromophores, and chromophores in crevices are considered in the interpretation of the thermal perturbation spectrum of bovine serum albumin. It is suggested that chromophores in pauci-aqueous crevices may appear buried toward thermal perturbation spectroscopy but accessible toward solvent perturbation and chemical reagents.     

Interpretation of the resonance Raman spectra of hemoproteins

The interpretation of the resonance Raman spectra of hemoproteins is given based on the normal coordinate analysis of model compounds (Cu octamethylporphin and Cu octaethylporphin). The correlation between the form of normal vibrations and the sensitivity of vibrational frequencies to the valence and spin state of the Fe atom is discussed.     

Interpretation of fluctuation spectra in lipid bilayer simulations

Atomic resolution and coarse-grained simulations of dimyristoylphosphatidylcholine lipid bilayers were analyzed for fluctuations perpendicular to the bilayer using a completely Fourier-based method. We find that the fluctuation spectrum of motions perpendicular to the bilayer can be decomposed into just two parts: 1), a pure undulation spectrum proportional to q⁻⁴ that dominates in the small-q regime; and 2), a molecular density structure factor contribution that dominates in the large-q regime. There is no need for a term proportional to q⁻² that has been postulated for protrusion fluctuations and that appeared to have been necessary to fit the spectrum for intermediate q. We suggest that earlier reports of such a term were due to the artifact of binning and smoothing in real space before obtaining the Fourier spectrum. The observability of an intermediate protrusion regime from the fluctuation spectrum is discussed based on measured and calculated material constants.     

Comparison between direct methods for determination of microbial cell volume: electron microscopy and electronic particle sizing.

Size frequency distributions of different phototrophic and heterotrophic microorganisms were determined by means of scanning and transmission electron microscopy and electronic particle sizing. Statistically significant differences existed among the three techniques used in this study. Cells processed for electron microscopy showed lower mean cellular volumes than those processed for electronic particle sizing, reflecting a shrinkage by factors ranging from 1.1 to 6.2 (mean, 2.3). Processing of cells for scanning electron microscopy caused higher shrinkage than processing for transmission electron microscopy. Shrinkage was dependent neither on the size nor on the cell wall type of the microorganism. When processed for scanning electron microscopy, phototrophic bacteria were strongly shrunken, whereas heterotrophic microorganisms were less affected. A direct relationship existed among phototrophic bacteria between percentage of shrinkage and specific pigment content. This was probably a consequence of the pigment extraction by organic solvents during the dehydration process, previous to the critical point drying, necessary to examine the specimens under the scanning electron microscope.     

Estimation of the disruption in freeze-pressed Saccharomyces cerevisiae by an electronic particle counter

An electronic particle counter has been used to estimate the disintegration by freeze-pressing baker's yeast. A counter threshold level which just yielded the maximum count for intact cells was selected. The conductivity of the suspending medium was chosen such that maximum counts were obtained. Under these conditions, the electronic counts agreed well with the visual counts. At a certain threshold level the maximum count was obtained at a lower resistivity (higher conductivity) in the suspending solution with the freeze-pressed suspension than with untreated cells, indicating that damage to the permeability barrier may occur without disruntion of the cell envelope. Fresh baker's yeast cells do not behave as nonconducting particles. This has to be taken into account when volume determinations with electronic particle counters are performed.     

Isotopic effects in the electronic spectra of tryptophan

Summary. No influence of isotopic substitution in deuterium-substituted tryptophan on the florescence excitation spectrum has previously been found out. Here, the isotopic effects of electronic excitation of deuterium-substituted tryptophan were experimentally and theoretically analyzed for first time. It was shown a short-wave shift of the UV-absorption maximum at 220 nm corresponding to the 360 cal/mol and short-wave shift for fluorescence spectrum corresponding to the 210 cal/mol. To account for this effect, the quantum chemical calculations of the geometric and electron structure, frequencies of normal vibrations and transition energies have been performed. The isotopic effects originate from the zero-point energies of ground and excited states. It was found that isotopic shifts depend on the position of isotope in the molecule and kind of transition. So, it can be utilized in the analysis of proteins structure and complexation.