Cherry-picking in an orchard: unattended LC/MS analysis from an autosampler with >32,000 samples online

The 1,536-well microplate format has widely supplanted the 384-well microplate format for high-throughput screening and for IC(50) assays. Previously, liquid chromatography/mass spectrometry (LC/MS) analyses of such samples required manual transfers of the wells of interest from a 1,536-well plate into a 384-well plate. Because this manual transfer introduced a source of potential error, it became clear that a more appropriate solution would be to sample directly from the 1,536-well plates. Currently, commercially available 1,536-well plate auto samplers are not compatible with Waters LC/MS systems. The authors have modified their CTC PAL autosampler to support injection from up to twenty-four 1,536-well plates. This allows them to cherry-pick any sample from up to 36,864 wells on the autosampler. Because of its success at this Institute, sampling from 1,536-well plates has not only become the preferred method for LC/MS analysis from IC(50) plates but also become the standard format used for the handling of and the sampling from large combinatorial libraries.     

Automated online liquid chromatographic/mass spectrometric metabolic study for prodrug stability

In vitro metabolic stability studies are performed routinely in drug discovery to determine the rate of metabolism as well as the metabolic fate of compounds. These studies are labor intensive, involving incubation of the compound with a biological matrix, sampling at various time points, stopping the reaction, and sample preparation for analysis. All of these steps involve manual pipetting in the conventional method. An automated method for in vitro metabolism studies is reported here. The method reduces the time and manual labor required and has other advantages, such as better reproducibility and unattended operation. This method utilizes an autosampler custom configured with cooling and incubation capabilities. The autosampler is programmed to directly inject incubation samples at set time points onto an online extraction column. The extracted sample then enters an analytical column for separation and ultimately the mass spectrometer for detection. The injection has the dual function of stopping the reaction and starting the analysis on the LC-MS. This method was used for the metabolic stability study of a prodrug in plasma and liver S9 fractions of five different species. The stability data from the automated method were similar to those obtained using the conventional method. The potential for this method to increase throughput of metabolic stability studies in drug discovery is demonstrated.     

Quantifying antimicrobial resistance at veal calf farms

This study was performed to determine a sampling strategy to quantify the prevalence of antimicrobial resistance on veal calf farms, based on the variation in antimicrobial resistance within and between calves on five farms. Faecal samples from 50 healthy calves (10 calves/farm) were collected. From each individual sample and one pooled faecal sample per farm, 90 selected Escherichia coli isolates were tested for their resistance against 25 mg/L amoxicillin, 25 mg/L tetracycline, 0.5 mg/L cefotaxime, 0.125 mg/L ciprofloxacin and 8/152 mg/L trimethoprim/sulfamethoxazole (tmp/s) by replica plating. From each faecal sample another 10 selected E. coli isolates were tested for their resistance by broth microdilution as a reference. Logistic regression analysis was performed to compare the odds of testing an isolate resistant between both test methods (replica plating vs. broth microdilution) and to evaluate the effect of pooling faecal samples. Bootstrap analysis was used to investigate the precision of the estimated prevalence of resistance to each antimicrobial obtained by several simulated sampling strategies. Replica plating showed similar odds of E. coli isolates tested resistant compared to broth microdilution, except for ciprofloxacin (OR 0.29, p≤0.05). Pooled samples showed in general lower odds of an isolate being resistant compared to individual samples, although these differences were not significant. Bootstrap analysis showed that within each antimicrobial the various compositions of a pooled sample provided consistent estimates for the mean proportion of resistant isolates. Sampling strategies should be based on the variation in resistance among isolates within faecal samples and between faecal samples, which may vary by antimicrobial. In our study, the optimal sampling strategy from the perspective of precision of the estimated levels of resistance and practicality consists of a pooled faecal sample from 20 individual animals, of which 90 isolates are tested for their susceptibility by replica plating.     

An automated technique for sampling the contents of stoppered gas-collection vials

Summary We have built an autosampler system that delivers the contents of pressurized gas collection vials to the injection port of a gas chromatograph. The three-part system consists of a shuttle base upon which vials move sequentially past a static sampling point, a sampling needle that is driven through vial septa by an air-driven piston, and an air-actuated sample valve that alternately places a sample loop in line with either a sample delivery line from the sample needle or a carrier stream leading to the gas chromatograph. We have used the system to analyze several thousand gas samples taken from soil cores assayed for denitrification activites, and have found the system reliable and capable of producing highly repeatable results.Journal Article No. 11491 of the Michigan State Agricultural Experiment Station, East Lansing, Michigan 48824, USA     

Ethanol analysis from biological samples by dual rail robotic autosampler

Detection, identification, and quantitation of ethanol and other low molecular weight volatile compounds in liquid matrices by headspace gas chromatography-flame ionization detection (HS-GC-FID) and headspace gas chromatography-mass spectrometry (HS-GC-MS) are becoming commonly used practices in forensic laboratories. Although it is one of the most frequently utilized procedures, sample preparation is usually done manually. Implementing the use of a dual-rail, programmable autosampler can minimize many of the manual steps in sample preparation. The autosampler is configured so that one rail is used for sample preparation and the other rail is used as a traditional autosampler for sample introduction into the gas chromatograph inlet. The sample preparation rail draws up and sequentially adds a saturated sodium chloride solution and internal standard (0.08%, w/v acetonitrile) to a headspace vial containing a biological sample, a calibrator, or a control. Then, the analytical rail moves the sample to the agitator for incubation, followed by sampling of the headspace for analysis. Using DB-624 capillary columns, the method was validated on a GC-FID and confirmed with a GC-MS. The analytes (ethanol, acetonitrile) and possible interferences (acetaldehyde, methanol, pentane, diethyl ether, acetone, isopropanol, methylene chloride, n-propanol, and isovaleraldehyde) were baseline resolved for both the GC-FID and GC-MS methods. This method demonstrated acceptable linearity from 0 to 1500 mg/dL. The lower limit of quantitation (LOQ) was determined to be 17 mg/dL and the limit of detection was 5 mg/dL.     

The estimation of pooled-sample sensitivity for detection of Salmonella in turkey flocks

Aims:  To investigate the effectiveness of pooled sampling methods for detection of Salmonella in turkey flocks.
Methods and Results:  Individual turkey droppings were taken from 43 flocks, with half the dropping tested for S almonella as an individual sample and the other half included in a pool of five. A pair of boot swabs and a dust sample were also taken from each flock. The results were analysed using Bayesian methods in the absence of a gold standard. This showed a dilution effect of mixing true-positive with negative samples, but even with this the pooled faecal samples were found to be a highly efficient method of testing compared with individual faecal samples. The more samples included in the pool, the more sensitive the pooled sampling method was predicted to be. The sensitivity of dust sampling was much more sensitive than faecal sampling at low prevalence.
Conclusions:  Pooled faecal sampling is an efficient method of Salmonella detection in turkey flocks. The additional testing of a dust sample greatly increased the effectiveness of sampling, especially at low prevalence.
Significance and Impact of the Study:  This is the first study to relate the sensitivity of the sampling methods to the within-flock prevalence.     

Comparison of sampling methods to detect Salmonella infection of turkey flocks

Aims:  To compare the efficiency of various sampling methods for detection of Salmonella in turkey flocks.
Methods and Results:  In a field study that compared various sampling methods one pair of boot swabs taken from the whole turkey house provided suitably sensitive results for fattening and rearing flocks and was no less sensitive than two pairs, each from half the house, tested as a pooled sample. The sensitivity was further enhanced by adding a dust sample. The dust sample appeared to be particularly useful in flocks with a low prevalence, especially in breeding flocks, and was more sensitive than a method which used five pairs of boot swabs per flock. Combined incubation of a boot swab and a dust sample showed no interference between the two sample types and a maximum sensitivity of detection. Litter samples and commercial sponge drag swabs provided a lower level of detection.
Conclusions:  A single pair of boot swabs taken from the whole house is recommended for routine sampling of commercial rearing or fattening flocks. An additional dust sample could be added to increase detection in flocks with a low prevalence or in breeding flocks, but adding an additional pair of boot swabs would not increase detection compared with a single pair.
Significance and Impact of the Study:  This study demonstrates that significant efficiencies can be made in sampling programmes for detection of Salmonella in turkey flocks without detracting from the sensitivity. Similar studies are recommended for other poultry sectors, particularly in chicken breeding flocks.     

Standardized sampling plan for Aphis gossypii based on the cotton cultivar,plant phenology and crop size

A standardized sampling plan is the starting point for developing a decision‐making system for pest control. Aphis gossypii (Hemiptera: Aphididae) is a destructive sap‐feeding pest on cotton worldwide. However, research addressing cotton cultivar, plant phenology and field size with the aim of developing a sampling plan for A. gossypii has not been done. Therefore, in this study, we developed a standardized sampling for A. gossypii as a function of these factors. To accomplish this, A. gossypii densities in four experimental cotton cultivars were sampled weekly during year one to determine the ideal aphid characteristic to sample (by individual or colony). During year one and two, A. gossypii densities were sampled weekly in the same cultivars to determine sampling unit, sampling technique and the number of samples for an A. gossypii sampling plan. Using the sample number determined, the sampling time was recorded for cotton field size of 1, 5, 10, 50, 100 and 150 ha in order to estimate the sampling cost. In cotton, the count of individuals was the best characteristic for the assessment of A. gossypii. Leaves of the most apical branches for the vegetative and reproductive cotton plant stage were the best sampling units. The best sampling technique was direct counting. The cotton cultivar did not affect the development of the sampling plan. The A. gossypii sampling plan involved the evaluation of 58 samples per zone and required 20 min (<0.35 min/sample) for the evaluation of these samples. However, the walking time between samples was the main factor responsible for the total sampling time and cost in cotton fields, and this factor strongly depends on the size of the cotton field.     

DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species

There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z-linked locus and a sex-typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/μL). The number of microsatellite loci amplified per sample (0-5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA and is applicable across a wide range of bird species. Nonetheless, genotyping errors should be properly estimated for each species by using a suite of highly polymorphic loci. These errors may be resolved by sampling only recently laid eggs (to avoid non-maternal DNA contamination) or by performing several PCR replicates per sample (to avoid allelic dropout and false alleles) and/or by increasing the amount of DNA used in the PCR through increasing the volume of the PCR or increasing the concentration of template DNA.     

Sequential sampling: a novel method in farm animal welfare assessment

Lameness in dairy cows is an important welfare issue. As part of a welfare assessment, herd level lameness prevalence can be estimated from scoring a sample of animals, where higher levels of accuracy are associated with larger sample sizes. As the financial cost is related to the number of cows sampled, smaller samples are preferred. Sequential sampling schemes have been used for informing decision making in clinical trials. Sequential sampling involves taking samples in stages, where sampling can stop early depending on the estimated lameness prevalence. When welfare assessment is used for a pass/fail decision, a similar approach could be applied to reduce the overall sample size. The sampling schemes proposed here apply the principles of sequential sampling within a diagnostic testing framework. This study develops three sequential sampling schemes of increasing complexity to classify 80 fully assessed UK dairy farms, each with known lameness prevalence. Using the Welfare Quality herd-size-based sampling scheme, the first ‘basic’ scheme involves two sampling events. At the first sampling event half the Welfare Quality sample size is drawn, and then depending on the outcome, sampling either stops or is continued and the same number of animals is sampled again. In the second ‘cautious’ scheme, an adaptation is made to ensure that correctly classifying a farm as ‘bad’ is done with greater certainty. The third scheme is the only scheme to go beyond lameness as a binary measure and investigates the potential for increasing accuracy by incorporating the number of severely lame cows into the decision. The three schemes are evaluated with respect to accuracy and average sample size by running 100 000 simulations for each scheme, and a comparison is made with the fixed size Welfare Quality herd-size-based sampling scheme. All three schemes performed almost as well as the fixed size scheme but with much smaller average sample sizes. For the third scheme, an overall association between lameness prevalence and the proportion of lame cows that were severely lame on a farm was found. However, as this association was found to not be consistent across all farms, the sampling scheme did not prove to be as useful as expected. The preferred scheme was therefore the ‘cautious’ scheme for which a sampling protocol has also been developed.     

Optimizing noninvasive sampling of a zoonotic bat virus

Outbreaks of infectious viruses resulting from spillover events from bats have brought much attention to bat‐borne zoonoses, which has motivated increased ecological and epidemiological studies on bat populations. Field sampling methods often collect pooled samples of bat excreta from plastic sheets placed under‐roosts. However, positive bias is introduced because multiple individuals may contribute to pooled samples, making studies of viral dynamics difficult. Here, we explore the general issue of bias in spatial sample pooling using Hendra virus in Australian bats as a case study. We assessed the accuracy of different under‐roost sampling designs using generalized additive models and field data from individually captured bats and pooled urine samples. We then used theoretical simulation models of bat density and under‐roost sampling to understand the mechanistic drivers of bias. The most commonly used sampling design estimated viral prevalence 3.2 times higher than individual‐level data, with positive bias 5–7 times higher than other designs due to spatial autocorrelation among sampling sheets and clustering of bats in roosts. Simulation results indicate using a stratified random design to collect 30–40 pooled urine samples from 80 to 100 sheets, each with an area of 0.75–1 m², and would allow estimation of true prevalence with minimum sampling bias and false negatives. These results show that widely used under‐roost sampling techniques are highly sensitive to viral presence, but lack specificity, providing limited information regarding viral dynamics. Improved estimation of true prevalence can be attained with minor changes to existing designs such as reducing sheet size, increasing sheet number, and spreading sheets out within the roost area. Our findings provide insight into how spatial sample pooling is vulnerable to bias for a wide range of systems in disease ecology, where optimal sampling design is influenced by pathogen prevalence, host population density, and patterns of aggregation.     

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.     

Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories

Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.     

Simulated Sampling Strategies for Nematodes Distributed According to a Negative Binomial Model

A FORTRAN computer program was developed to simulate nematode soil sampling strategies consisting of various numbers of samples per field, with each sample consisting of various numbers of soil cores. The program assumes that the nematode species involved fit a negative binomial distribution. Required input data are estimates of the mean and k values, the number of samples per field and cores per sample in the strategy to be investigated, and the number of times the simulation is to be replicated. Output consists of simulated values of the relative deviation from the mean and standard error to mean ratio, both averaged over all replications. The program was used to compare 150 simulated sampling strategies for Meloidogyne incognita, involving all combinations of two mean values (2.0 and 10.0 la.rvae/10 cm³ soil), three k values (1.35, 0.544, and 0.294), five different numbers of samples per field (1, 2, 4. 10, 20), and five different numbers of cores per sample (1, 2, 4, 10, 20). Simulations resulting from different mean values were similar, but best results were obtained with higher k values and 20 cores per sample. Relatively few 20-core samples were needed to obtain average deviations from the mean of 20-25%.     

A Feasibility Study to Monitor the Macroinvertebrate Diversity of the River Nile Using Three Sampling Methods

The River Nile is one of the world’s major rivers. Its’ catchment in Egypt has a population of 75,000,000. River flow is highly regulated and there are known discharges of pollutants. 1035 km of the river downstream of the Aswan high dam was studied to test the hypothesis that representative qualitative samples can be used to estimate macroinvertebrate biodiversity. Benthic macroinvertebrates are difficult to sample in large rivers and a reliable sampling strategy is required to evaluate their ecological status. Three methods for sampling have been investigated. Ekman Grab, macrophyte sweep netting and Artificial Substrate Samplers (ASS) were used to sample 15 sites from Aswan to Cairo between September 2001 and June 2002. Organisms were identified to species level where possible. Taxon accretion curves indicated that the all taxa present at a site should be collected using either 15 grab samples, 10 macrophyte samples or 5 ASS. The best time to sample was May–June. The biodiversity of macroinvertebrates in the Nile was recorded as 50 taxa with values of 7–31 at individual bank-side sites. Mid-stream biodiversity was much lower (0–19). Lowest biodiversity occurred at sites with known pollution inputs whilst highest occurred at sites with high levels of sedimentation. A regular programme for biomonitoring is recommended which will allow current status to be confirmed and future changes detected.     

Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae.

An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters.     

Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces

AIM: To evaluate US Centers for Disease Control and Prevention recommended swab surface sample collection method for recovery efficiency and limit of detection for powdered Bacillus spores from nonporous surfaces. METHODS AND RESULTS: Stainless steel and painted wallboard surface coupons were seeded with dry aerosolized Bacillus atrophaeus spores and surface concentrations determined. The observed mean rayon swab recovery efficiency from stainless steel was 0.41 with a standard deviation (SD) of +/-0.17 and for painted wallboard was 0.41 with an SD of +/-0.23. Evaluation of a sonication extraction method for the rayon swabs produced a mean extraction efficiency of 0.76 with an SD of +/-0.12. Swab recovery quantitative limits of detection were estimated at 25 colony forming units (CFU) per sample area for both stainless steel and painted wallboard. CONCLUSIONS: The swab sample collection method may be appropriate for small area sampling (10 -25 cm2) with a high agent concentration, but has limited value for large surface areas with a low agent concentration. The results of this study provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequence of an inappropriate public health response founded on information gathered using an ineffective or unreliable sample collection method has the potential for undesired social and economic impact.     

Efficiency of variable-intensity and sequential sampling for insect control decisions in cole crops in the Netherlands

A total of 24 commercial fields of cabbages and Brussels sprouts were sampled in a grid fashion with 20–25 equally spaced cells with four plants per cell. Using this data base of 80–100 plants, we conducted computer stimulations to compare the treatment decisions that would be made for the major insect pests using published sequential sampling programs and a newly developed variable-intensity sampling program. Additionally, we compared the number of samples required to make the decision. At low thresholds (10–20%) for both Lepidoptera and cabbage aphids, variable intensity-sampling required a smaller sample size and provided more reliable decisions, while at high thresholds (40–50%) sequential sampling provided more reliable decisions. In both procedures, the occurrence of incorrect decisions was minimal. The number of cases in which a decision would not be reached after a 40-plant sample was lower for variable-intensity sampling. Considering the number of samples required to make a correct decision and the greater need for reliable decisions at lower thresholds, variable-intensity sampling was superior to sequential sampling. Additionally, variable-intensity sampling has the advantage of requiring samples to be taken in a greater area of the field and thus increases the probability of detecting localized infestations. Although variable-intensity sampling was not designed to classify pest populations for treatment decisions but rather to achieve sampling precision around the population mean, our present studies indicate that it can also be an effective method to aid in treatment decisions.     

A comparison of sampling methods for bluegreen lucerne aphid (Acyrthosiphon kondoi) in Canterbury,New Zealand

Abstract

Sampling of bluegreen lucerne aphid showed that the most efficient method differs according to the time of year and the height of the lucerne. In late June 1976, when the lucerne was short (mean height 3.6 cm), an area removal sample of 700 cm² was more efficient to achieve a 10% degree of precision (half-width of a confidence limit) than area samples of 80 and 300 cm², suction sampling of the same areas, and stem sampling. In early spring (lucerne height 11.7 cm) the 80 cm² area removal sample was the most efficient. Just before the second hay cut (late January), the length of lucerne stems (62.5 cm) precluded use of any area removal technique; stem samples were most efficient. The cost in time was fuliy considered in sampling method comparisons. Possible differences with population density were not considered.     

Comparison of environmental sampling methods for detecting Salmonella in commercial laying flocks in the UK

Aims: To investigate the performance of the Salmonella National Control Programme (NCP) sampling/testing methods in laying flocks of domestic fowl. Methods and Results: Eighty‐five visits were made to 69 flocks representative of the main production systems (cage, barn and free‐range) infected with Salmonella. In each visit, three methodologies were compared: (i) the European Union (EU) baseline survey method (five faeces and two dust samples); (ii) an in‐house (Veterinary Laboratories Agency, VLA) ‘wet’ method that involved collecting 10 dust and 10 faeces samples into jars with buffered peptone water; and (iii) a method involving two samples of pooled faeces and one of dust (cultured as one sample of each type), which has been adopted for the NCP for laying flocks across the EU. Conclusions: The ‘wet’ method was the most sensitive, and the NCP the least, although individual NCP samples were the most sensitive ones. Significance and Impact of the Study: The apparent lower sensitivity of the NCP method may be compensated by repeated sampling of flocks (twice during rear and several times during lay). Sampling using VLA methodology should be advocated for farms aiming to disclose low‐level Salmonella before restrictions on the sale of eggs from Salmonella Enteritidis or Salmonella Typhimurium‐infected flocks are in place.