Statistical evaluation of electronic and plate counts for estimating bacterial populations

Bacteria from cultures of Staphylococcus aureus, Pseudomonas geniculata, and Rhodotorula glutenis were counted by use of an electronic counter and by plate counts from broth cultures at selected periods from 0 to 24 hr. Variations in the two methods were noted, and the results were compared after calculating correlations, coefficients of variation, and nested analyses of variance. It was not possible to determine the absolute accuracy of the two methods; however, the precision of the results obtained with an electronic counter was better than that obtained with plate counting. It appears that electronic counting, because of ease and convenience, provides better comparative counts at a particular stage of growth, especially during the early stages of the growth period, whereas plate counting yields the best results in determining growth curves.     

Glass micromodel study of bacterial dispersion in spatially periodic porous networks

Successful implementation of bioremediation clean-up strategies depends on accurate predictions of the transport of bacteria within the subsurface. In this study, etched flat-plate glass micromodels were used to examine bacterial transport in a homogenous network. These networks were created by acid-etching interconnected channels into a glass plate and then fusing it to an unetched plate forming semi-cylindrical pores. The transparent nature of the micromodel allows for both qualitative observations of the bacteria within the pores and quantitative measurements of their concentration. The micromodels are designed to allow establishment of a well-characterized step change in bacterial concentration (Escherichia coli NR50) within the network. During the experiments, bacteria are dispersed through the network by flow. Light scattering is used to detect the change in turbidity within the pores as the bacteria travel through the network. The change in turbidity is used to construct breakthrough curves and spatial concentration profiles of bacteria within the network. The breakthrough curves are fit to the one-dimensional advection/dispersion equation to determine dispersion coefficients at different interstitial fluid velocities. From the breakthrough curves, dispersion coefficients were reproducible for replicate experiments over a range of velocities in the advection-dominated regime. The dispersivity values for two network designs resembling an interconnecting capillary network and a spatially periodic network of cylinders were 0.28 and 0.33 cm respectively, which are slightly greater than the literature values found for other pore networks. Experiments were also conducted within the diffusion-dominated regime to examine the effects of bacterial motility on dispersion. The accumulation of bacteria on the pore walls became significant at the low flow rates and extended experimental times thereby rendering the use of light scattering to determine concentrations ineffective. Bacterial chemotaxis, created by a self-imposed oxygen gradient, was also observed in the micromodel under stagnant fluid conditions.     

Quantitative evaluation of the effectiveness of the formation of sister chromatid exchanges and chromosome aberrations as affected by chemical mutagens

Dose curves of five chemicals were studied to compare the efficiency of SCE and chromosomal aberration induction by different chemical mutagens. SCEs were found to increase linearly with the dose, whereas chromosomal aberrations--nonlinearly. Using regression coefficients obtained from the dose curves it was found that the efficiency of the studied chemical mutagens in induction of SCEs is 100-300 times as high as that in the induction of chromosomal aberrations.     

An improved approximate solution of the Lamm equation for the simultaneous estimation of sedimentation and diffusion coefficients from sedimentation velocity experiments

Sedimentation and diffusion coefficients are important parameters to describe size and shape of macromolecules in solution. The data can be obtained from sedimentation velocity experiments by a nonlinear fitting procedure using approximate solutions for the Lamm equation. Here, we present a modification of such a model function that was originally proposed by Fujita [H. Fujita, Mathematical Theory of Sedimentation Analysis, Wiley, New York, 1962]. The extended model function is well suitable to study low molecular mass compounds. The improvement of this solution given here is based on using an adjustable value for the explicit integration variable, z, the reduced radius. This modification leads to more accurate sedimentation and diffusion coefficients compared to using a constant value of 0.5 as used by Fujita. The advantage of our modification was demonstrated by the analysis of noise-free curves calculated using the finite element method, as well as experimental curves obtained for the peptides angiotensin I and II. The relatively low sedimentation and diffusion coefficients found for both substances indicate that the peptides exist as extended chains of about 3.65 nm (angiotensin I) or 3.04 nm length (angiotensin II) in solution. The lack of higher-order structure of the peptides that was derived also from CD spectra might facilitate receptor binding, and could be one reason for the fast proteolytic digestion of the free peptides.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Hydrophobic interaction chromatography of proteins. II. Binding capacity,recovery and mass transfer properties

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.     

Spectral sensitivity of photoreceptors in the compound eye of the locust

Three types of receptor with different _max of 360, 430, and 530 nm were found in the locust retina by extracellular recording. Their spectral sensitivity curves were considerably broader than the absorption curves of the corresponding pigments. Possible coefficients of electrical coupling between different receptor types in ommatidia were calculated on the basis of the spectral sensitivity curves obtained for photoreceptors, assuming that each receptor contains only one light-sensitive pigment. The resulting values resembled coefficients measured in the locust by Shaw and Lillywhite. The way in which spectral sensitivity curves spread in comparison with pigment absorption curves may thus be caused by electrical coupling between cells.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 69–76, January–February, 1986.     

Species–area curves indicate the importance of habitats’ contributions to regional biodiversity

We examined species–area curves, species composition and similarity (Jaccard's coefficients), and species richness in 17 vegetation types to develop a composite index of a vegetation type's contribution to regional species richness. We collected data from 1 to 1000 m² scales in 147 nested plots in Rocky Mountain National Park, Colorado, USA to compare three species–area curve models’ abilities to estimate the number of species observed in each vegetation type. The log(species)–log(area) curve had the largest adjusted coefficients of determination (r² values) in 12 of the 17 types, followed by the species–log(area) curve with five of the highest values. When the slopes of the curves were corrected for species overlap among plots with Jaccard's coefficients, the species–log(area) curves estimated values closest to those observed. We combined information from species–area curves and measures of heterogeneity with information on the area covered by each vegetation type and found that the types making the greatest contributions to regional biodiversity covered the smallest areas. This approach may provide an accurate and relatively rapid way to rank hotspots of plant diversity within regions of interest.     

Hill coefficients,dose–response curves and allosteric mechanisms

Hill coefficients (n _H) derived from four parameter logistic fits to dose–response curves were compared to calculated realistic reaction schemes and related to experimental data: (1) Hill coefficients may give information on the number of interacting sites but cannot distinguish between competitive, non-competitive or ortho-, iso-, or allosteric mechanisms. (2) For enzymatic dose–inhibition curves, Hill coefficients smaller than one do not indicate anticooperative binding but show that at least one ternary complex has enzymatic activity. (3) Hill coefficients different from one are proof for multiple ligand binding. The large variations of reported Hill coefficients corresponds to multiple allosteric binding, where induced conformational changes cause loss of the active conformation. Such a denaturation mechanism is in stark contrast to the desired specificity of drugs. The discussion is open.     

Hill coefficients of dietary polyphenolic enzyme inhibitiors: can beneficial health effects of dietary polyphenols be explained by allosteric enzyme denaturing?

Inspired by a recent article by Prinz, suggesting that Hill coefficients, obtained from four parameter logistic fits to dose–response curves, represent a parameter allowing distinction between a general allosteric denaturing process and real single site enzyme inhibition, Hill coefficients of a number of selected dietary polyphenol enzyme inhibitions were compiled from the available literature. From available literature data, it is apparent that the majority of polyphenol enzyme interactions reported lead to enzyme inhibition via allosteric denaturing rather than single site inhibition as judged by their reported Hill coefficients. The results of these searches are presented and their implications discussed leading to the suggestion of a novel hypothesis for polyphenol biological activity termed the insect swarm hypothesis.     

The validation of a portable force plate for measuring force-time data during jumping and landing tasks

The purpose of this study was to determine the reliability and validity of a portable force plate when analyzing jumping and landing tasks. Subjects performed 3 drop vertical jumps and 3 drop landings on both a standard strain gauge laboratory force plate and a portable force plate. In contrast to typical laboratory installed force plates, the portable 6-component force plate can be easily transported and used onsite at various training or data collection sites and incorporates Hall effect technology. The measured parameters included maximum force and time to maximum force for initial stance of the both tests, maximum takeoff force, and time to maximum takeoff force for the drop vertical jump. The Pearson correlation coefficients for the drop landing and the drop vertical jump for maximum force (r = 0.942, r = 0.940), time to maximum force (r = 0.891, r = 0.920) and for drop jump maximum jumping force (r = 0.971), and time to maximum takeoff force (r = 0.917) were all high and indicate that the force data collected by a resistor-type portable force plate provide similar measures to a standard strain-gauge laboratory force plate. Additionally, the within session reliability of the drop landing and the drop vertical jump measured by the portable force plate showed high interclass correlation coefficients for examined variables of 0.979 and 9.67 for maximum landing force and 0.917 and 0.920 for time to maximum landing force, respectively. The interclass correlation coefficients for the maximum takeoff force and time to maximum takeoff force during the drop vertical jump were 0.991 and 0.86. The results indicate the force and timing measurements from the portable force plate were both valid and reliable. Use of the portable force plate may facilitate methods of force measurement that can be applied out into the field and therefore a valuable tool for on site landing and jump force measurements in a variety of settings for large numbers of subjects.     

A Kinetic Model for the Action of Xylocaine on Receptors for Acetylcholine

A kinetic scheme postulating the rapid formation of a partially active acetylcholine-receptor-drug complex from Xylocaine (or a derivative) and the active acetylcholine-receptor complex can account for the effects of Xylocaine and its derivatives at the neuromuscular junction. Transmembrane currents generated by an analogue computer programmed according to the scheme can exactly match end plate currents produced by nerve stimulation in the presence of the drugs. The scheme also accounts for the qualitatively different effects of the drugs on the end plate potential and on responses to iontophoretically applied acetylcholine. The analysis presented is consistent with very rapid reactions between acetylcholine and receptors, characterized by rate coefficients in the range 10⁴ to 10⁶ sec^-1. It is based on the hypothesis that the activation of receptors by acetylcholine changes the structure of the receptors and thus their affinity for Xylocaine. The analysis does not require pharmacological separability of sodium and potassium conductances during the end plate current.     

The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[alpha]pyrene in Salmonella typhimurium strains

The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-1-oxide (4NQO) and benzo[alpha]pyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system (S9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5 micrograms/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 micrograms/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 micrograms/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60% of the sum of separate application at doses ranging from 25 to 100 micrograms/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of S9 mix, quercetin may affect the metabolic pathway of these mutagens.     

Growth of the African elephant (Loxodonta africana)

The three-stage desk calculation of the von Bertalanffy equation to describe growth in height and weight with age in the elephant is compared with a new approach to calculating the three coefficients in the function by a computer. The two methods give different results with respect to the weight/age calculations. Theoretical von Bertalanffy equations calculated by both methods to describe growth in height and weight with age in the African elephant in Zambia are compared with previously published equations for the elephant in East Africa. Details are given of growth in height in two known-age African elephants, a female ‘Diksie’ and a male ‘Kartoum’. Theoretical growth in height curves for the female African and Asiatic elephant are compared. The coefficients K and / for growth in height are not transferable to the growth in weight equations. Inherent inaccuracies in the calculation of the coefficients in the von Bertalanffy equation are discussed, and it is concluded that in animals which have a long life-span such as the elephant, the equation serves as a purely empirical representation of weight-at-age data and that there is little biological significance in the parameters it contains. The computer-calculated curves give the best fit to the data. The regression of log age on log shoulder height from 2–20 years of age provides a more realistic approach to comparative growth studies. The increase in adrenal weight with age is linear. Tusk growth in relation to age and sex in Zambia is compared with East Africa. It is concluded that the tusks in Zambia are smaller and are more difficult to sex correctly than their East African counterparts, possibly a consequence of the Zambian elephant having a greater degree of tusk wear. Allometric growth is described with emphasis on the estimation of body weight from shoulder height. The most reliable estimates are obtained from a purely empirical representation of the data, a semilog plot of log body weight on shoulder height.     

Nonparametric mixed effects models for unequally sampled noisy curves

We propose a method of analyzing collections of related curves in which the individual curves are modeled as spline functions with random coefficients. The method is applicable when the individual curves are sampled at variable and irregularly spaced points. This produces a low-rank, low-frequency approximation to the covariance structure, which can be estimated naturally by the EM algorithm. Smooth curves for individual trajectories are constructed as best linear unbiased predictor (BLUP) estimates, combining data from that individual and the entire collection. This framework leads naturally to methods for examining the effects of covariates on the shapes of the curves. We use model selection techniques--Akaike information criterion (AIC), Bayesian information criterion (BIC), and cross-validation--to select the number of breakpoints for the spline approximation. We believe that the methodology we propose provides a simple, flexible, and computationally efficient means of functional data analysis.     

Evaluation of the Spiral Plate Maker for the Enumeration of Micro-organisms in Foods

A statistically based comparison of five methods of enumerating bacteria in foods has been undertaken to assess the potential application of the Spiral Plate Maker (Gilchrist et al. 1973) which is claimed to reduce very significantly the costs involved in the quantitative estimation of viable micro-organisms in foods. The performance of the Spiral Plate Maker was compared with that of three conventional methods (pour plate, surface spread plate and drop count) for the examination of four types of food by four different operators. Analysis of variance showed that there were no differences between the methods at the 5% level although some isolated interactions occurred. Regression and correlation coefficients between the various methods were all highly significant and the results obtained by the spiral plate method were within the limits of error for traditional quantitative methods. The spiral plate method, in many cases, can replace advantageously any of the other methods for the quantitative estimation of viable microorganisms in foods. Labour requirements for the enumeration of micro-organisms by the spiral plate method were only 31% of that required for one conventional method. In addition considerable savings in materials were achieved.     

Use of implicit methods from general sensitivity theory to develop a systematic approach to metabolic control. I. Unbranched pathways

It is shown that metabolic control theory (MCT), is its present form, is a particular case of general sensitivity theory, which studies the effects of parameter variations on the behavior of dynamic systems. It has been shown that metabolic control theory is obtained from this more general theory for the particular case of steady-state and linear relationships between velocities and enzyme concentrations. In such conditions the relationships between elasticities and flux control coefficients are easily obtained. These relationships are in the form of a matrix product constructed in a priori form. Relationships between combined response coefficients and concentration control coefficients are presented. The use of implicit methodology from general sensitivity theory provides a generalization of MCT, which is applied to unbranched pathways. For this particular case, provided the matrices have been properly constructed, the matrix of global properties (flux and concentration control coefficients) can be obtained by inversion of the matrix of local properties (elasticities). The theorems of MCT (concentration summation, flux summation, flux connectivity, and concentration connectivity) applicable for unbranched pathways are directly obtained by inspection of the matrix product. With these results, the present theoretical basis of MCT is extended with a more structured framework that allows a wider range of application. The results make clearer the relatedness of MCT to the more general approach provided by biochemical systems theory (BST).     

A neural network model to predict knee adduction moment during walking based on ground reaction force and anthropometric measurements

The external knee adduction moment (KAM) is a major variable for the evaluation of knee loading during walking, specifically in patients with knee osteoarthritis. However, assessment of the KAM is limited to locations where full motion laboratories are available. The purpose of this study was to develop and test a simple method to predict the KAM using only force plate and anthropometric measurements. Three groups of 28 knees (asymptomatic, mild osteoarthritis, and severe osteoarthritis) were studied. Walking trials were collected at different speeds using a motion capture system and a force plate. The reference KAM was calculated by inverse dynamics. For the prediction, inter-subject artificial neural networks were designed using 11 inputs coming from the ground reaction force and the mechanical axis alignment. The predicted KAM curves were similar to the reference curves with median mean absolute deviation (MAD) of 0.36%BW*Ht and median correlation coefficient of 0.966 over 756 individual trials. When comparing mean group curves, the median MAD was 0.09%BW*Ht and the median correlation coefficient 0.998. The peak values and the angular impulses extracted from the predicted and reference curves were significantly correlated, and the same significant differences were obtained among the three groups when the predicted or when the reference curves were used for 95% of the comparisons. In conclusion, this study demonstrated that a simple method using a generic artificial neural network can predict the KAM curve during walking with a high level of significance and provides a practical option for a broader evaluation of the KAM.     

Time-dependent total extinction analysis for simple reactions involving intermediates

When the individual species in a simple series reaction A → B → C have different light absorption or scattering extinction coefficients, the progress curves obtained from extinction measurements are generally biphasic. If the rate constants are not too dissimilar, the curves can exhibit maxima or minima, or be s-shaped or monotonic. The shape of the curves depends mainly on the relative values of the extinction coefficients. A few simple rules are developed, which usually suffice to give important qualitative information concerning the nature of the intermediate.     

Growth Curves of Anaerobic Bacteria in Solid Media

Simple pour plate and spectrophotometric techniques for the evaluation of growth curves of several anaerobic bacteria on solid media are described. Three basic patterns of anaerobic growth were observed. The curves obtained were very reproducible when studied on separate occasions. The curves obtained by spectrophotometric measurement were comparable to those obtained by the pour plate method, especially when a large bacterial inoculum was used. Limitations in the interpretation of the results are discussed. The methods and principles reported could provide the basis for the determination of bacterial growth on solid media using other organisms and different experimental conditions.