Evaluation of Methods for Sampling, Recovery, and Enumeration of Bacteria Applied to the Phylloplane

Determining the fate and survival of genetically engineered microorganisms released into the environment requires the development and application of accurate and practical methods of detection and enumeration. Several experiments were performed to examine quantitative recovery methods that are commonly used or that have potential applications. In these experiments, Erwinia herbicola and Enterobacter cloacae were applied in greenhouses to Blue Lake bush beans (Phaseolus vulgaris) and Cayuse oats (Avena sativa). Sampling indicated that the variance in bacterial counts among leaves increased over time and that this increase caused an overestimation of the mean population size by bulk leaf samples relative to single leaf samples. An increase in the number of leaves in a bulk sample, above a minimum number, did not significantly reduce the variance between samples. Experiments evaluating recovery methods demonstrated that recovery of bacteria from leaves was significantly better with stomacher blending, than with blending, sonication, or washing and that the recovery efficiency was constant over a range of sample inoculum densities. Delayed processing of leaf samples, by storage in a freezer, did not significantly lower survival and recovery of microorganisms when storage was short term and leaves were not stored in buffer. The drop plate technique for enumeration of bacteria did not significantly differ from the spread plate method. Results of these sampling, recovery, and enumeration experiments indicate a need for increased development and standardization of methods used by researchers as there are significant differences among, and also important limitations to, some of the methods used.     

Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples

AIMS: To compare the sensitivities of two nested PCR assays for the detection of Legionella pneumophila to each other and to the plate counting technique (ISO 11731) in a wide range of aquatic samples. METHODS AND RESULTS: The nested PCR assay with the primer set LEG 225-LEG 858 revealed 56% of the 46 analysed aquatic samples as being positive for Legionella spp., while the primer set JFP-JRP yielded 98% positive samples. The detection was confirmed by sequencing the PCR products. These results are considerably higher than the result obtained with the plate counting technique (41%), indicating the higher sensitivity of PCR-based diagnostic methods. As the PCR assay with the LEG 225-LEG 858 primer set resulted in a lower number of positive samples, it is considered not sensitive enough for aquatic samples. Similar results for the respective primer sets were obtained for the detection of the species L. pneumophila, responsible for 90% of all human Legionella infections, in the aquatic samples analysed. Both microbial community analysis by PCR-denaturing gradient gel electrophoresis and the analysis of biotic and abiotic water quality parameters revealed no relation between L. pneumophila-positive and -negative samples and the physico-chemical and bacteriological characteristics of the aquatic samples. CONCLUSIONS: The results show the additional value of the PCR assay with the JFP-JRP primer set compared with the plate counting technique, as well as its applicability in a wide range of aquatic samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the importance of comparing different primer sets for nested PCR assays for the detection of L. pneumophila in aquatic samples, as well as the lower sensitivity of the widely accepted plate counting technique (ISO 11731).     

A procedure for the statistical evaluation of Ames Salmonella assay results. Comparison of results among 4 laboratories

Ames Salmonella test data collected in our laboratory and 3 National Cancer Institute contract laboratories were analyzed to study the distribution of experimental errors associated with the test. It is shown that the Poisson distribution is not appropriate, and that the power transformation model Y = (revertants/plate)lambda, with lambda = 0.2 as estimated by the methods of Box and Cox, produced a measurement scale on which the experimental errors could be adequately described by a normal (Gaussian) distribution with a constant variance. The modeling procedure enables one to properly use analysis of variance, regression analysis, and Student's t test to analyze Ames Salmonella test results, and well-known statistical quality control procedures to monitor laboratory performance. The method detects weak mutagenic activity and measures the amount and uncertainty of the increase in revertants/plate. The development of the power transformation model is discussed and examples of its use in the interpretation of Ames Salmonella assay results are included.     

Stress: a Factor to be Considered in Heterotrophic Microorganism Enumeration from Aquatic Environments

Heterotrophic microorganisms in water samples are susceptible to the transient stress of warmed agar used in the standard methods pour plate procedure, causing significantly decreased recoveries in comparison with a spread plate technique. Microbial starvation can increase susceptibility to a transient warming stress. The standard plate count procedure, as presently described, should not be considered for quantitation of microorganisms from aquatic environments.     

How to optimize the drop plate method for enumerating bacteria

The drop plate (DP) method can be used to determine the number of viable suspended bacteria in a known beaker volume. The drop plate method has some advantages over the spread plate (SP) method. Less time and effort are required to dispense the drops onto an agar plate than to spread an equivalent total sample volume into the agar. By distributing the sample in drops, colony counting can be done faster and perhaps more accurately. Even though it has been present in the laboratory for many years, the drop plate method has not been standardized. Some technicians use 10-fold dilutions, others use twofold. Some technicians plate a total volume of 0.1 ml, others plate 0.2 ml. The optimal combination of such factors would be useful to know when performing the drop plate method.This investigation was conducted to determine (i) the standard deviation of the bacterial density estimate, (ii) the cost of performing the drop plate procedure, (iii) the optimal drop plate design, and (iv) the advantages of the drop plate method in comparison to the standard spread plate method. The optimal design is the combination of factor settings that achieves the smallest standard deviation for a fixed cost. Computer simulation techniques and regression analysis were used to express the standard deviation as a function of the beaker volume, dilution factor, and volume plated. The standard deviation expression is also applicable to the spread plate method.     

水生动物进口风险评估综述

随着全球水产养殖业的发展,水产动物活体或水产品贸易日益频繁,不可避免地给进口国带来病原风险,造成疾病引入和传播,甚至生物入侵,严重危害水产养殖业的发展。水生动物进口风险评估(IRA),是指从别国或地区进口水生动物活体包括其受精卵、稚鱼、幼鱼、苗种、成体以及商品等的风险分析。风险分析是指对风险事件进行科学、透明、系统分析的一个过程,它由危害识别、风险评估、风险交流和风险管理4个部分构成。目前,常用的风险评估方法有定性风险评估、半定量风险评估和定量风险评估3种。定性风险评估具有灵活性强、适用范围广、易掌握的特点,能够综合各种资料、数据和信息,尤其适合初次风险评估,但容易受评估人员主观因素的影响。定量风险评估可避免主观因素的影响,评估结果准确、可靠,但需要收集大量数据,工作量巨大,评估成本也很高。通常,定性评估结果若能够提供很好的防范措施,则不必进行定量评估。将外来水生病原阻止在引进之初远比引入后根除更加容易。因此,开展水生动物进口风险评估对于阻止水生动物疾病传播和水生态环境破坏具有重要意义,同时也可为各国进行水产贸易提供参考。     

The Practical Significance in the Microbiological Examination of Cold Stored Foods of the Allegedly Low Heat Resistance among Psychrotrophic Organisms

Summary: The significance in food microbiology of the observation of Stapert, Sokolski & Northam (1962), that some bacteria occurring in water have such low heat resistances that they would be affected by the warm agar used for pouring plates, was tested. Forty-two samples of 6 different foods stored for 7 days at 3° to enrich their psychrotrophic microflora, were examined using the same medium, but in thin poured plates and on spread plates, respectively, incubated at 14°. The logarithmic average count of the foods was c. 10⁸/g and included about 60% Gram-negative rods and 30% cocci. In 90% of the samples no differences were observed between the results from the two methods of counting. The slightly higher spread plate counts in the remaining instances could be accounted for by the increased disruption of bacterial agglomerations always observed in this procedure. Hence there is no reason to query the results of poured plate counts in general. Where it is yet probably safer to use spread plates, rigorous asepsis in pouring and storing should be observed to prevent the development of colonies of contaminants too small to be detected when the plates are inoculated, but leading to erroneous counts after incubation.     

Novel technique for measuring the size distribution of granules from anaerobic reactors for wastewater treatment

A new technique for measuring the size distribution of anaerobic granular sludge, which involves the use of digital image analysis, is presented. Sludge samples are embedded in gelatin, spread over glass dishes, which are then placed over a flat-bed scanner where an image is captured. The images are processed with an image analysis software. The technique is simple, reliable and does not need any special equipment. © Rapid Science Ltd. 1998     

An automated method of sample preparation of biofluids using pierceable caps to eliminate the uncapping of the sample tubes during sample transfer

Biological samples are normally collected and stored frozen in capped tubes until analysis. To obtain aliquots of biological samples for analysis, the sample tubes have to be thawed, uncapped, samples removed and then recapped for further storage. In this paper, we report an automated method of sample transfer devised to eliminate the uncapping and recapping process. This sampling method was incorporated into an automated liquid-liquid extraction procedure of plasma samples. Using a robotic system, the plasma samples were transferred directly from pierceable capped tubes into microtubes contained in a 96-position block. The aliquoted samples were extracted with methyl-tert-butyl ether in the same microtubes. The supernatant organic layers were transferred to a 96-well collection plate and evaporated to dryness. The dried extracts were reconstituted and injected from the same plate for analysis by liquid chromatography with tandem mass spectrometry.     

Comparison of different drying and storage methods on quantifiable concentrations of fecal steroids in the cheetah

Fecal steroid analysis is a powerful tool that can provide important information on the health, physiology, and reproductive status of nondomestic species. However, studying free‐ranging animals requires that feces be stored and transported from the collection site to the laboratory in a manner that prevents degradation or alteration of steroid metabolites. To determine the effects of different handling and storage methods on fecal steroids, 30 fresh fecal samples from five captive cheetahs were collected, thoroughly mixed, separated into aliquots, and processed (stored or dried) under different conditions. Concentrations of gonadal and adrenal steroid hormones were analyzed in feces stored frozen at –20°C or at room temperature in 95% ethanol. Both frozen and ethanol‐stored aliquots were desiccated using a lyophilizer, solar oven, or conventional oven. The steroid values from aliquots stored and desiccated using the different methods were compared to those obtained using the optimal storage method of freezing at –20°C and desiccating in a lyophilizer (control). Concentrations of corticoid, estrogen, progestagen, and androgen metabolites in fecal extracts were quantified by radioimmunoassay. Androgen metabolite concentrations were not significantly affected (P > 0.05) by storage or drying methods. Fecal samples stored at room temperature in ethanol and lyophilized also had steroid concentrations that did not differ (P > 0.05) from controls. However, the concentrations of corticoid and estrogen metabolites were significantly lower (P < 0.05), and progestagen metabolites were significantly higher (P < 0.05) in samples desiccated in solar and conventional ovens without regard to storage method. These results suggest that storage of fecal samples at room temperature in ethanol is the best alternative to freezing for subsequent analysis of steroid hormone concentrations. Differences in measured concentrations of hormones in oven‐desiccated samples could be due to hormone degradation or shifts in the immunodominant metabolite. Therefore, validation of storage and processing techniques should be included in the development of any new fecal steroid analysis methodology. Zoo Biol 21:215–222, 2002. © 2002 Wiley‐Liss, Inc.     

Rapid and quantitative detection of the microbial spoilage in chicken meat by diffuse reflectance spectroscopy (600-1100 nm)

AIMS: To evaluate the feasibility of visible and short-wavelength near-infrared (SW-NIR) diffuse reflectance spectroscopy (600-1100 nm) to quantify the microbial loads in chicken meat and to develop a rapid methodology for monitoring the onset of spoilage. METHODS AND RESULTS: Twenty-four prepackaged fresh chicken breast muscle samples were prepared and stored at 21 degrees C for 24 h. Visible and SW-NIR was used to detect and quantify the microbial loads in chicken breast muscle at time intervals of 0, 2, 4, 6, 8, 10, 12 and 24 h. Spectra were collected in the diffuse reflectance mode (600-1100 nm). Total aerobic plate count (APC) of each sample was determined by the spread plate method at 32 degrees C for 48 h. Principal component analysis (PCA) and partial least squares (PLS) based prediction models were developed. PCA analysis showed clear segregation of samples held 8 h or longer compared with 0-h control. An optimum PLS model required eight latent variables for chicken muscle (R = 0.91, SEP = 0.48 log CFU g(-1)). CONCLUSIONS: Visible and SW-NIR combined with PCA is capable of perceiving the change of the microbial loads in chicken muscle once the APC increases slightly above 1 log cycle. Accurate quantification of the bacterial loads in chicken muscle can be calculated from the PLS-based prediction method. SIGNIFICANCE AND THE IMPACT OF THE STUDY: Visible and SW-NIR spectroscopy is a technique with a considerable potential for monitoring food safety and food spoilage. Visible and SW-NIR can acquire a metabolic snapshot and quantify the microbial loads of food samples rapidly, accurately, and noninvasively. This method would allow for more expeditious applications of quality control in food industries.     

流域环境要素空间尺度特征及其与水生态分区尺度的关系——以辽河流域为例

指标体系构建是流域水生态分区技术框架中的一项重要内容。目前提出的指标体系尚缺乏一定的科学理论支持,如所选指标的生态尺度与各级分区大小之间的对应关系。生态学研究认为,不同尺度上的生态过程和格局不同。以辽河流域为例,对影响水生态系统的几个大尺度环境要素(降水、地形和植被)的空间尺度特征进行分析,得出了各要素空间变异最为显著的尺度。其中,降水的空间尺度约为75 km,地形要素和指标均大致存在16 km、32 km、64 km和128 km多个景观特征尺度。在界定水生态一、二级分区范围基础上,讨论了各环境要素作为水生态一、二级分区指标的适用性,以期为辽河流域水生态分区指标选取提供一定科学依据,同时希望能为其他流域提供一定参考。     

Rates of Transformation of Methyl Parathion and Diethyl Phthalate by Aufwuchs Microorganisms

Using batch cultures, we determined transformation rates for low concentrations of two toxicants—an insecticide, methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate), and a plasticizer, diethyl phthalate—by aufwuchs, aquatic microbial growth attached to submerged surfaces or suspended in streamers or mats. Aufwuchs samples were collected from field sites, an indoor channel, and a continuous-flow fermentor. Aufwuchs fungi, protozoa, and algae did not transform methyl parathion or diethyl phthalate, but bacteria rapidly transformed both chemicals. Second-order transformation rate coefficients, K_b, based on total plate counts of bacteria in aufwuchs, were determined for potential use in a mathematical model capable of predicting the transport and fate of chemicals in aquatic systems. K_b for both methyl parathion and diethyl phthalate decreased as the concentration of total bacteria, [B], increased in aufwuchs. This effect resulted from the proportion of nontransformer to transformer bacteria increasing as [B] increased and from the rate of transformation per transformer cell decreasing as [B] increased. First-order transformation rate coefficients, K₁, were relatively stable per unit of surface area colonized by aufwuchs, because K_b decreased as [B] increased (K₁ = K_b × [B]).     

Survival of Salmonella enterica in freshwater and sediments and transmission by the aquatic midge Chironomus tentans (Chironomidae: Diptera)

Survival of a nalidixic acid-resistant strain of Salmonella enterica serovar Typhimurium mr-DT-104 in water and sediments was tested using artificially contaminated aquaria. Water samples remained culture positive for salmonella for up to 54 days. Sediment samples were culture positive up to 119 days. In addition, potential mechanisms for spreading salmonella in the environments by chironomid larvae and adults were tested. We evaluated the acquisition of mr-DT-104 by chironomids from contaminated aquatic sediments and subsequent spread to uncontaminated sediments. Larval chironomids raised in contaminated sediments became culture positive, and the bacteria were carried over to adults after emergence. Contamination of clean sediments by chironomid larvae was not demonstrated. These findings clearly suggest that mr-DT-104 serovar organisms can survive in aquatic sediments for at least several months. Uptake of salmonellae by chironomid larvae and adults suggests that they are possible vectors of mr-DT-104 in both aquatic and terrestrial environments, although the role of larval defecation in movement of bacteria to new sediments was not demonstrated.     

BioDry: An Inexpensive,Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.), freezing, or bulky storage/sampling equipment. Gel electrophoresis analysis of nucleic acid extracts from samples treated in the lab with the BioDry method indicated that molecular integrity was protected in samples stored at room temperature for up to 30 days. Analysis of 16S/18S rRNA genes for presence/absence and relative abundance of microorganisms using both 454-pyrosequencing and TRFLP profiling revealed statistically indistinguishable communities from control samples that were frozen in liquid nitrogen immediately after collection. Seawater and river water biomass samples collected with a portable BioDry “field unit", constructed from off-the-shelf materials and a battery-operated pumping system, also displayed high levels of community rRNA preservation, despite a slight decrease in nucleic acid recovery over the course of storage for 30 days. Functional mRNA and protein pools from the field samples were also effectively conserved with BioDry, as assessed by respective RT-PCR amplification and western blot of ribulose-1-5-bisphosphate carboxylase/oxygenase. Collectively, these results demonstrate that BioDry can adequately preserve a suite of biomolecules from aquatic biomass at ambient temperatures for up to a month, giving it great potential for high resolution sampling in remote locations or on autonomous platforms where space and power are limited.     

Go with the flow: water velocity regulates herbivore foraging decisions in river catchments

Foragers typically attempt to consume food resources that offer the greatest energy gain for the least cost, switching between habitats as the most profitable food resource changes over time. Optimal foraging models require accurate data on the gains and costs associated with each food resource to successfully predict temporal shifts. Whilst previous studies have shown that seasonal changes in food quantity and quality can drive habitat shifts, few studies have shown the effects on habitat choice of seasonal changes in metabolic foraging costs. In this study we combined field and literature data to construct an optimal foraging model to examine the effect of seasonal changes in food quantity, food quality and foraging costs on the timing of a switch from terrestrial to aquatic habitat by non‐breeding mute swans Cygnus olor in a shallow river catchment. Feeding experiments were used to quantify the functional response of swans to changes in aquatic plant biomasses. By sequentially testing alternative models with fixed or variable values for food quantity, food quality and foraging cost, we found that we needed to include seasonal variance in foraging costs in the model to accurately predict the observed habitat switch date. However, we did not need to include seasonal variance in food quantity and food quality, as accurate predictions could be obtained with fixed values for these two parameters. Therefore, the seasonal changes in foraging costs were the key factor influencing the behavioural decision to switch feeding habitats. These seasonal changes in foraging costs were driven by changes in water velocity; the profitability of aquatic foraging was negatively related to water velocity, as faster water required more energy to be expended in swimming. Our results demonstrate the importance of incorporating seasonal variation in foraging costs into our understanding of the foraging decisions of animals.     

NMR-based metabonomics of bovine blood: an investigation into the effects of long term storage on plasma samples

Freezers in research institutions often contain a plethora of samples left over from studies performed years or even decades ago. Along with samples stored in biobanks, these could prove to be treasure troves for metabonomic research. Although the influence of sample handling and short to medium term storage on conventionally determined blood parameters has been reported, little is known about the effects of long term storage (years to decades) on plasma samples. The aim of this study was to investigate the influence of long term storage on the metabolite profile and to assess the value of archived samples for metabonomic studies. Heparinised plasma of 22 heifers that had been stored at ?20 °C for between 2 and 15 years was analysed using NMR spectroscopy and statistical analysis techniques. Lactate (principal component 1) explained 79.6 % of variance between all spectra, but was not correlated with storage time. The highest correlation with storage time (R ² = 0.474) was found for betaine, with other metabolites (acetoacetate, histidines, glycerol, lipids and glucose) also showing moderate correlation (R ² values between 0.217 and 0.437). Our results indicate that samples stored for extended periods of time can potentially be used in metabonomics studies, if precautions are taken during data analysis.     

Survival of Salmonella enterica in Freshwater and Sediments and Transmission by the Aquatic Midge Chironomus tentans (Chironomidae: Diptera)

Survival of a nalidixic acid-resistant strain of Salmonella enterica serovar Typhimurium mr-DT-104 in water and sediments was tested using artificially contaminated aquaria. Water samples remained culture positive for salmonella for up to 54 days. Sediment samples were culture positive up to 119 days. In addition, potential mechanisms for spreading salmonella in the environments by chironomid larvae and adults were tested. We evaluated the acquisition of mr-DT-104 by chironomids from contaminated aquatic sediments and subsequent spread to uncontaminated sediments. Larval chironomids raised in contaminated sediments became culture positive, and the bacteria were carried over to adults after emergence. Contamination of clean sediments by chironomid larvae was not demonstrated. These findings clearly suggest that mr-DT-104 serovar organisms can survive in aquatic sediments for at least several months. Uptake of salmonellae by chironomid larvae and adults suggests that they are possible vectors of mr-DT-104 in both aquatic and terrestrial environments, although the role of larval defecation in movement of bacteria to new sediments was not demonstrated.     

Asymmetric response of early warning indicators of phytoplankton transition to and from cycles

Phytoplankton populations often exhibit cycles associated with nuisance blooms of cyanobacteria and other algae that cause toxicity, odor problems, oxygen depletion, and fish kills. Models of phytoplankton blooms used for management and basic research often contain critical transitions from stable points to cycles, or vice-versa. It would be useful to know whether aquatic systems, especially water supplies, are close to a critical threshold for cycling blooms. Recent studies of resilience indicators have focused on alternate stable points, although theory suggests that indicators such as variance and autocorrelation should also rise prior to a transition from stable point to stable cycle. We investigated changes in variance and autocorrelation associated with transitions involving cycles using two models. Variance rose prior to the transition from a small-radius cycle (or point) to a larger radius cycle in all cases. In many but not all cases, autocorrelation increased prior to the transition. However, the transition from large-radius to small-radius cycles was not associated with discernible increases in variance or autocorrelation. Thus, indicators of changing resilience can be measured prior to the transition from stable to cyclic plankton dynamics. Such indicators are potentially useful in management. However, these same indicators do not provide useful signals of the reverse transition, which is often a goal of aquatic ecosystem restoration. Thus, the availability of resilience indicators for phytoplankton cycles is asymmetric: the indicators are seen for the transition to bloom–bust cycles but not for the reverse transition to a phytoplankton stable point.     

Environment and Spatial Influences on Aquatic Insect Communities in Cerrado Streams: the Relative Importance of Conductivity,Altitude, and Conservation Areas

The aquatic insect community is an important element for stream functionality and diversity, but the effects of altitude and conservation areas on the aquatic insect community have been poorly explored in neotropical ecozone. The lack of studies about the relative importance of space and environment on community structure is another obstacle within aquatic insect ecology, which precludes the inclusion of these studies in more current frameworks, like the metacommunity dynamics. We evaluated the relationship between the aquatic insect community structure at 19 streams in the Brazilian Cerrado and spatial and environmental variables, namely geographical distance among sites, stream altitude, chemical variables, and environmental protection areas. We partitioned the variance explained by spatial and environmental components using a partial redundancy analysis. The environment exhibited a strong spatial structure for abundance and number of genera, increasing these community parameters with elevated water conductivity. Only community composition had a large unexplained portion of variance, with a small portion constrained by environmental (altitude and conductivity) and spatial factors. A relevant point in the result was the streams with high conductivity were located outside of the conservation areas. These results suggest that the relationship between number of genera and abundance with environmental conditions is always associated with spatial configuration of streams. Our study shows that altitude is an important determinant of community structure, as it exerts indirect influences, and electrical conductivity directly determines community composition, and that some national parks may be inefficient in maintaining the diversity of aquatic insects in the Cerrado region.