Parameters affecting spore recovery from wipes used in biological surface sampling

The need for the precise and reliable collection of potential biothreat contaminants has motivated research in developing a better understanding of the variability in biological surface sampling methods. In this context, the objective of this work was to determine parameters affecting the efficiency of extracting Bacillus anthracis Sterne spores from commonly used wipe sampling materials and to describe performance using the interfacial energy concept. In addition, surface thermodynamics was applied to understand and predict surface sampling performance. Wipe materials were directly inoculated with known concentrations of B. anthracis spores and placed into extraction solutions, followed by sonication or vortexing. Experimental factors investigated included wipe material (polyester, cotton, and polyester-rayon), extraction solution (sterile deionized water [H(2)O], deionized water with 0.04% Tween 80 [H(2)O-T], phosphate-buffered saline [PBS], and PBS with 0.04% Tween 80 [PBST]), and physical dissociation method (vortexing or sonication). The most efficient extraction from wipes was observed for solutions containing the nonionic surfactant Tween 80. The increase in extraction efficiency due to surfactant addition was attributed to an attractive interfacial energy between Tween 80 and the centrifuge tube wall, which prevented spore adhesion. Extraction solution significantly impacted the extraction efficiency, as determined by statistical analysis (P < 0.05). Moreover, the extraction solution was the most important factor in extraction performance, followed by the wipe material. Polyester-rayon was the most efficient wipe material for releasing spores into solution by rank; however, no statistically significant difference between polyester-rayon and cotton was observed (P > 0.05). Vortexing provided higher spore recovery in H(2)O and H(2)O-T than sonication, when all three wipe materials and the reference control were considered (P < 0.05).     

Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces

Aims: Having and executing a well-defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and nonporous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes. Methods and Results: Eight collection tools, five swabs and three wipes, were used. Three collection/preservation solutions were evaluated: an ink jet aerosol generator was used to apply Bacillus subtilis endospores to five porous and nonporous surfaces. The collection efficiencies of the swabs and wipes were compared using a statistical multiple comparison analysis. Conclusions: The ScottPure^® wipe had the highest collection efficiency and phosphate-buffered saline (PBST) with 0·3% Tween was the best collection solution of those tested. Significance and Impact of the Study: Validated sampling for potential biological warfare is of significant importance and this study answered some relevant questions.     

Evaluation of a wipe surface sample method for collection of Bacillus spores from nonporous surfaces

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of +/-0.12, and for painted wallboard it was 0.29 with a standard deviation of +/-0.15. Evaluation of a sonication extraction method for the polyester-rayon blend wipes produced a mean extraction efficiency of 0.93 with a standard deviation of +/-0.09. Wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel sample area and 105 CFU per unit of painted wallboard sample area. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling following the release of a biological agent such as Bacillus anthracis.     

Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores from Nonporous Surfaces

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of ±0.12, and for painted wallboard it was 0.29 with a standard deviation of ±0.15. Evaluation of a sonication extraction method for the polyester-rayon blend wipes produced a mean extraction efficiency of 0.93 with a standard deviation of ±0.09. Wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel sample area and 105 CFU per unit of painted wallboard sample area. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling following the release of a biological agent such as Bacillus anthracis.     

Slow Recovery from Inbreeding Depression Generated by the Complex Genetic Architecture of Segregating Deleterious Mutations

The deleterious effects of inbreeding have been of extreme importance to evolutionary biology, but it has been difficult to characterize the complex interactions between genetic constraints and selection that lead to fitness loss and recovery after inbreeding. Haploid organisms and selfing organisms like the nematode Caenorhabditis elegans are capable of rapid recovery from the fixation of novel deleterious mutation; however, the potential for recovery and genomic consequences of inbreeding in diploid, outcrossing organisms are not well understood. We sought to answer two questions: 1) Can a diploid, outcrossing population recover from inbreeding via standing genetic variation and new mutation? and 2) How does allelic diversity change during recovery? We inbred C. remanei, an outcrossing relative of C. elegans, through brother-sister mating for 30 generations followed by recovery at large population size. Inbreeding reduced fitness but, surprisingly, recovery from inbreeding at large populations sizes generated only very moderate fitness recovery after 300 generations. We found that 65% of ancestral single nucleotide polymorphisms (SNPs) were fixed in the inbred population, far fewer than the theoretical expectation of ∼99%. Under recovery, 36 SNPs across 30 genes involved in alimentary, muscular, nervous, and reproductive systems changed reproducibly across replicates, indicating that strong selection for fitness recovery does exist. Our results indicate that recovery from inbreeding depression via standing genetic variation and mutation is likely to be constrained by the large number of segregating deleterious variants present in natural populations, limiting the capacity for recovery of small populations.     

Cryptosporidium and giardia recoveries in natural waters by using environmental protection agency method 1623

Relatively few studies have examined recoveries from source waters by using Environmental Protection Agency method 1623 with organism spike doses that are environmentally realistic and at turbidity levels commonly found in surface waters. In this study, we evaluated the filtration capacities and recovery efficiencies of the Gelman Envirochek (standard filter) and the Gelman Envirochek high-volume (HV) sampling capsules under environmental conditions. We also examined the performance of method 1623 under ambient conditions with matrix spike experiments using 10 organisms/liter. Under turbid conditions, the HV capsule filtered approximately twice the volume filtered by the standard filter, but neither could filter 10 liters without clogging. In low-turbidity waters, oocyst, but not cyst, recoveries were significantly higher when the HV capsule was used. In turbid waters, organism recoveries were lower than those in nonturbid waters and were not significantly different for the different filters. When the HV capsule was used, Cryptosporidium recoveries ranged from 36 to 75%, and Giardia recoveries ranged from 0.5 to 53%. For both organisms, recoveries varied significantly by site. Turbidity could explain variation in Giardia recoveries (r(2) = 0.80) but not variation in Cryptosporidium recoveries (r(2) = 0.16). The inconsistent recoveries across sites suggested that the background matrix of the ambient water affected recovery by method 1623. A control sample collected at the height of the winter rainy season detected one organism, highlighting the difficulty of using this method to accurately measure pathogen abundance under natural conditions. Our findings support the use of the HV filter under field conditions but suggest that designing a cost-effective and statistically valid monitoring program to evaluate sources and loads of protozoan pathogens may be difficult.     

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.     

Searching for the Optimal Sampling Solution: Variation in Invertebrate Communities,Sample Condition and DNA Quality

There is a great demand for standardising biodiversity assessments in order to allow optimal comparison across research groups. For invertebrates, pitfall or flight-interception traps are commonly used, but sampling solution differs widely between studies, which could influence the communities collected and affect sample processing (morphological or genetic). We assessed arthropod communities with flight-interception traps using three commonly used sampling solutions across two forest types and two vertical strata. We first considered the effect of sampling solution and its interaction with forest type, vertical stratum, and position of sampling jar at the trap on sample condition and community composition. We found that samples collected in copper sulphate were more mouldy and fragmented relative to other solutions which might impair morphological identification, but condition depended on forest type, trap type and the position of the jar. Community composition, based on order-level identification, did not differ across sampling solutions and only varied with forest type and vertical stratum. Species richness and species-level community composition, however, differed greatly among sampling solutions. Renner solution was highly attractant for beetles and repellent for true bugs. Secondly, we tested whether sampling solution affects subsequent molecular analyses and found that DNA barcoding success was species-specific. Samples from copper sulphate produced the fewest successful DNA sequences for genetic identification, and since DNA yield or quality was not particularly reduced in these samples additional interactions between the solution and DNA must also be occurring. Our results show that the choice of sampling solution should be an important consideration in biodiversity studies. Due to the potential bias towards or against certain species by Ethanol-containing sampling solution we suggest ethylene glycol as a suitable sampling solution when genetic analysis tools are to be used and copper sulphate when focusing on morphological species identification and facing financial restrictions in biodiversity studies.     

Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces

To comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and species-specific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.     

Preliminary evaluation of several disinfection/sterilization techniques for use with microdialysis probes

This study evaluated the suitability of some disinfection and sterilization methods for use with microdialysis probes. Disinfection or sterilization should minimize the tissue inflammatory reaction and improve the long-term health of rats on study and ensure the quality of data obtained by microdialysis sampling. Furthermore, the treatment should not negatively impact probe integrity or sampling performance. The techniques chosen for evaluation included two disinfection methods (70% ethanol and a commercial contact lens solution) and two sterilization methods (hydrogen peroxide plasma, and e-beam radiation). Linear microdialysis probes treated by these processes were compared to untreated probes removed from the manufacturer's packaging as if sterile (the control group). The probes were aseptically implanted in the livers of rats and monitored for 72 hours. The parameters chosen to evaluate probe performance were relative sample mass recovery and the relative in vivo extraction efficiency of the probe for caffeine. Post mortem bacterial counts and histopathology examination of liver tissue were also conducted. The probes remained intact and functional for the entire study period. The methods tested did not acutely alter the probes although hydrogen peroxide plasma and contact lens solution groups showed reduced extraction efficiencies. Minimal tissue damage was observed surrounding the probes and acute inflammatory reaction was mild to moderate. Low numbers of bacterial colonies from the implantation sites indicates that the health of animals in this study was not impaired. This was also true for the control group (untreated probe).     

Efficient methods for large-area surface sampling of sites contaminated with pathogenic microorganisms and other hazardous agents: current state,needs, and perspectives

The recovery operations following the 2001 attacks with Bacillus anthracis spores were complicated due to the unprecedented need for large-area surface sampling and decontamination protocols. Since this event, multiple reports have been published describing recovery efficiencies of several surface sampling materials. These materials include fibrous swabs of various compositions, cloth wipes, vacuum socks, and adhesive tapes. These materials have reported recovery efficiencies ranging from approximately 20% to 90% due to the many variations in their respective studies including sampling material, composition of surface sampled, concentration of contaminant, and even the method of deposition and sample processing. Additionally, the term recovery efficiency is crudely defined and could be better constructed to incorporate variations in contaminated surface composition and end user needs. While significant efforts in devising protocols for large-area surface sampling have been undertaken in the years since the anthrax attacks, there is still a general lack of consensus in optimal sampling materials and the methodology in which they are evaluated. Fortunately, sampling efforts are continuing to be supported, and the knowledge gaps in our procedures, methodology, and general understanding of sampling mechanisms are being investigated which will leave us better prepared for the future.     

Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores

In this study, four commonly-used sampling devices (vacuum socks, 37 mm 0.8 μm mixed cellulose ester (MCE) filter cassettes, 37 mm 0.3 μm polytetrafluoroethylene (PTFE) filter cassettes, and 3M™ forensic filters) were comparatively evaluated for their ability to recover surface-associated spores. Aerosolized spores (~ 10⁵ CFU cm^− 2) of a Bacillus anthracis surrogate were allowed to settle onto three material types (concrete, carpet, and upholstery). Ten replicate samples were collected using each vacuum method, from each material type. Stainless steel surfaces, inoculated simultaneously with test materials, were sampled with pre-moistened wipes. Wipe recoveries were utilized to normalize vacuum-based recoveries across trials. Recovery (CFU cm^− 2) and relative recovery (vacuum recovery/wipe recovery) were determined for each method and material type. Recoveries and relative recoveries ranged from 3.8 × 10³ to 7.4 × 10⁴ CFU cm^− 2 and 0.035 to 1.242, respectively. ANOVA results indicated that the 37 mm MCE method exhibited higher relative recoveries than the other methods when used for sampling concrete or upholstery. While the vacuum sock resulted in the highest relative recoveries on carpet, no statistically significant difference was detected. The results of this study may be used to guide selection of sampling approaches following biological contamination incidents.     

从蛇足石杉中超声提取石杉碱甲和石杉碱乙

用正交试验确立了超声提取蛇足石杉生物碱的最佳条件。以石杉碱甲和石杉碱乙回收率为指标,考察了溶剂倍数、溶剂浓度、超声时间、超声功率等因素的影响。结果表明,在室温下超声提取的最优条件为：酸浓度O．8％(v／v),液固比例20：1,超声功率600W,超声15min。三次重复实验所得石杉碱甲和石杉碱乙回收率分别是9r7．3％和93．5％,相对标准偏差分别为1．31％和1．40％(n=3)。与传统的回流提取工艺相比,过程时间从2h缩短为15min,回收率提高了10％以上。     

Intragenomic variation in non-adaptive nucleotide biases causes underestimation of selection on synonymous codon usage

Patterns of non-uniform usage of synonymous codons vary across genes in an organism and between species across all domains of life. This codon usage bias (CUB) is due to a combination of non-adaptive (e.g. mutation biases) and adaptive (e.g. natural selection for translation efficiency/accuracy) evolutionary forces. Most models quantify the effects of mutation bias and selection on CUB assuming uniform mutational and other non-adaptive forces across the genome. However, non-adaptive nucleotide biases can vary within a genome due to processes such as biased gene conversion (BGC), potentially obfuscating signals of selection on codon usage. Moreover, genome-wide estimates of non-adaptive nucleotide biases are lacking for non-model organisms. We combine an unsupervised learning method with a population genetics model of synonymous coding sequence evolution to assess the impact of intragenomic variation in non-adaptive nucleotide bias on quantification of natural selection on synonymous codon usage across 49 Saccharomycotina yeasts. We find that in the absence of a priori information, unsupervised learning can be used to identify genes evolving under different non-adaptive nucleotide biases. We find that the impact of intragenomic variation in non-adaptive nucleotide bias varies widely, even among closely-related species. We show that the overall strength and direction of translational selection can be underestimated by failing to account for intragenomic variation in non-adaptive nucleotide biases. Interestingly, genes falling into clusters identified by machine learning are also physically clustered across chromosomes. Our results indicate the need for more nuanced models of sequence evolution that systematically incorporate the effects of variable non-adaptive nucleotide biases on codon frequencies.     

DNA extraction protocol for rapid PCR detection of pathogenic bacteria

Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT + Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays.     

Dispersal Ability Determines the Role of Environmental,Spatial and Temporal Drivers of Metacommunity Structure

Recently, community ecologists are focusing on the relative importance of local environmental factors and proxies to dispersal limitation to explain spatial variation in community structure. Albeit less explored, temporal processes may also be important in explaining species composition variation in metacommunities occupying dynamic systems. We aimed to evaluate the relative role of environmental, spatial and temporal variables on the metacommunity structure of different organism groups in the Upper Paraná River floodplain (Brazil). We used data on macrophytes, fish, benthic macroinvertebrates, zooplankton, periphyton, and phytoplankton collected in up to 36 habitats during a total of eight sampling campaigns over two years. According to variation partitioning results, the importance of predictors varied among biological groups. Spatial predictors were particularly important for organisms with comparatively lower dispersal ability, such as aquatic macrophytes and fish. On the other hand, environmental predictors were particularly important for organisms with high dispersal ability, such as microalgae, indicating the importance of species sorting processes in shaping the community structure of these organisms. The importance of watercourse distances increased when spatial variables were the main predictors of metacommunity structure. The contribution of temporal predictors was low. Our results emphasize the strength of a trait-based analysis and of better defining spatial variables. More importantly, they supported the view that “all-or- nothing” interpretations on the mechanisms structuring metacommunities are rather the exception than the rule.     

Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies

DNA metabarcoding refers to the DNA-based identification of multiple species from a single complex and degraded environmental sample. We developed new sampling and extraction protocols suitable for DNA metabarcoding analyses targeting soil extracellular DNA. The proposed sampling protocol has been designed to reduce, as much as possible, the influence of local heterogeneity by processing a large amount of soil resulting from the mixing of many different cores. The DNA extraction is based on the use of saturated phosphate buffer. The sampling and extraction protocols were validated first by analysing plant DNA from a set of 12 plots corresponding to four plant communities in alpine meadows, and, second, by conducting pilot experiments on fungi and earthworms. The results of the validation experiments clearly demonstrated that sound biological information can be retrieved when following these sampling and extraction procedures. Such a protocol can be implemented at any time of the year without any preliminary knowledge of specific types of organisms during the sampling. It offers the opportunity to analyse all groups of organisms using a single sampling/extraction procedure and opens the possibility to fully standardize biodiversity surveys.     

Automated Processing of Imaging Data through Multi-tiered Classification of Biological Structures Illustrated Using Caenorhabditis elegans

Quantitative imaging has become a vital technique in biological discovery and clinical diagnostics; a plethora of tools have recently been developed to enable new and accelerated forms of biological investigation. Increasingly, the capacity for high-throughput experimentation provided by new imaging modalities, contrast techniques, microscopy tools, microfluidics and computer controlled systems shifts the experimental bottleneck from the level of physical manipulation and raw data collection to automated recognition and data processing. Yet, despite their broad importance, image analysis solutions to address these needs have been narrowly tailored. Here, we present a generalizable formulation for autonomous identification of specific biological structures that is applicable for many problems. The process flow architecture we present here utilizes standard image processing techniques and the multi-tiered application of classification models such as support vector machines (SVM). These low-level functions are readily available in a large array of image processing software packages and programming languages. Our framework is thus both easy to implement at the modular level and provides specific high-level architecture to guide the solution of more complicated image-processing problems. We demonstrate the utility of the classification routine by developing two specific classifiers as a toolset for automation and cell identification in the model organism Caenorhabditis elegans. To serve a common need for automated high-resolution imaging and behavior applications in the C. elegans research community, we contribute a ready-to-use classifier for the identification of the head of the animal under bright field imaging. Furthermore, we extend our framework to address the pervasive problem of cell-specific identification under fluorescent imaging, which is critical for biological investigation in multicellular organisms or tissues. Using these examples as a guide, we envision the broad utility of the framework for diverse problems across different length scales and imaging methods.     

Evaluation of alcohol wipes used during aseptic manufacturing

Aim: During aseptic manufacturing and specifically during the transfer of items into an isolator, disinfection of surfaces is essential for reducing the risk of final product contamination. Surface disinfection can be carried out by a variety of methods, however the most accepted current practice is a combination of spraying with 70% alcohol and wiping. The aim of this study was to evaluate the effectiveness of two wipe systems by determining their ability to remove, kill and transfer bacterial contaminants from standardized surfaces. Methods and Results: The protocol used to achieve these objectives was based on a newly published method specifically designed to test wipes. Alcohol impregnated wipes performed better at reducing microbial bioburden than the alcohol spray/dry wipe applications. Impregnated wipes drastically reduced (1–2 log₁₀ reduction) a small bioburden (approx. 2 log₁₀) of spores of Bacillus subtilis and methicillin‐resistant Staphylococcus aureus from the surface, but failed to remove (<0·2 log₁₀ reduction) Staphylococcus epidermidis. The alcohol spray/dry wipes did not manage to remove (<0·2 log₁₀ reduction) spore or bacterial bioburden from surfaces and was able to transfer some viable micro‐organisms to other surfaces. Both wipe types showed poor antimicrobial efficacy (<1 log₁₀ reduction) against the test bacteria and spores. Conclusions: As far as the authors are aware this is the first time that such a practical study has been reported and our results suggest that the best wipes for surface disinfection in aseptic units are the alcohol (IPA) impregnated wipes when compared with the dry wipes sprayed with alcohol. Significance and Impact of the Study: The impregnated wipes performed better than the dry wipes sprayed with alcohol and should be used for surface disinfection in aseptic units.     

Growth requirement for N as a criterion to assess the effects of physical manipulation on nitrate uptake fluxes in spinach

The effects of physical manipulation of hydroponically grown plants of spinach (Spinacia oleracea L., cvs Subito and Glares) on nitrate uptake fluxes were studied in a long-term experiment (3 days), and in short-term label experiments (2 h) with ¹³N-nitrate and ¹⁵N-nitrate. In the long-term experiment, net nitrate uptake rate (NNUR) was measured by following the nitrate depletion in the uptake solution, which was replaced at regular intervals. In the short-term experiments, NNUR and nitrate influx were measured by simultaneous application of ¹³N-nitrate and ¹⁵N-nitrate. Plants were gently transferred into the labelled uptake solution, as is usually done in nutrient uptake studies. In addition, a more severe physical manipulation was carried out, including blotting of the roots, to mimic pretreatments which involve more handling of the plants prior to uptake measurements. Nitrate influx was measured immediately after physical manipulation and after 2 h of recovery. To assess the impact of the physical manipulation the experimentally determined nitrate uptake fluxes were compared with the N demand for growth, defined as relative growth rate (RGR) times plant nitrogen concentration (PNC) of parallel plants, which were left undisturbed. Nitrate influx and efflux were both subject to changes after physical manipulation of the plants. Physical handling, however, did not always result in an alteration of NNUR, which complicates the determination of the length of the recovery period. The impact of the handling and the time course of the recovery depended on the severity of the disturbance and were independent of the light conditions during the experiments. Even after a gentle transfer of the plants, recovery, in most cases, was not complete within 2 h. The data emphasise the need for minimal disturbance of plants during the last hours prior to nutrient uptake measurements.