Risk Of Cross-contamination Research Articles

Occurrence of Listeria monocytogenes in Camel Milk Cheese from two township markets of Borno and Kano States, Nigeria

Introduction: Camel milk cheese is one of the major camel milk products consumed in Northern Nige-ria. The conditions in which the cheese is sold in markets pose a risk of cross-contamination with foodborne pathogens such as Listeria monocytogenes. The study was carried out to evaluate the microbial quality of raw camel milk cheese sold in two different markets. Methods: A total of 200 cheese samples, (100 samples from each market) of Kano and Borno States, of Northern Nigeria were analysed for total aerobic plate count (TAPC) and total coliform count (TCC). Listeria species were isolated from the samples and the isolates were subjected for antimicrobial susceptibility test with commonly used antibiotics. Results: The results of the study show that mean TAPC of the sampled cheese was 5.90 log10 cfu/g and 5.70 log10 cfu/g for the Kano and Borno markets, respectively. While the mean for TCC per gram sampled was 3.75 log10 cfu/g and 3.81 log10 cfu/g for Kano and Borno, respectively. The occurrence rate of Listeria monocytogenes was 1(0.5%) in Kano and none in Borno market. However other Listeria species were isolated in both markets with overall occurrence rate of L. ivanovii 6(3%), L. welshimeri 1(0.5%) and L. grayi 2 (1%). The results of antimicrobial susceptibility test of the isolates show resistance of 77.7%, 66.7% and 55.6% to penicillin, gentamicin and tetracycline, respectively while 66.7% of the isolates were susceptible to erythromycin. Significance: This study has demonstrated the need for food regulatory agency to carry out routine investigation of ready to eat foods sold in the market to ascertain safety of such food for human consumption.

The Impact of COVID-19 Pandemic on Telemedicine

Abstract The COVID-19 pandemic is a public health emergency of international concern. Due to mandatory social distancing and the lack of effective treatments, telemedicine has become the safest interactive system between patients, both infected and uninfected, and clinicians. During this period, all patients, with acute or chronic conditions, need ways to obtain medical attention other than going to Emergency Room (E.R.) or to the clinic. Telemedicine has been an effective approach for COVID-19 management as it allows to reduce the risk of cross-contamination and provides support to remote rural location. The most optimistic forecasts could not anticipate the impact that a pandemic will have on this medical field, whose financial implications exceeded all expectation.

Identifying patterns of utilization of single-use video laryngoscopy and supraglottic airway devices to reduce waste

Introduction: Lean management, a systematic method of reducing waste in manufacturing, is an approach that has led to quality improvement and cost reduction in health systems. Utilization of single-use airway equipment, such as videolaryngoscope (VL) blades and supraglottic airways (SGAs) has helped reduce the risk of cross-contamination and the time and cost associated with reprocessing and sterilization. Methods: We examined the efficiency of single-use VL blades and SGAs within our tertiary care health system. Purchasing data were reviewed and compared with device utilization within the electronic medical record to determine the amount of waste of disposable airway equipment. Direct observations (Gemba walks) were made in the operating rooms to assess device utilization. For SGAs, a diagnostic questionnaire was sent to providers regarding approaches to use. Results: We found the total cost of opened but unused VL blades equaled $29,267. For SGAs, we found that devices were often opened before use and that there is a 16.67% first-time failure rate for placement of SGAs. Conclusions: Through education and organizing device setup, and streamlining communication, there is a possibility to reduce future waste.

Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil.

Listeria monocytogenes and Salmonella spp. are considered important foodborne pathogens that are commonly associated with foods of animal origin. The aim of this study was to perform molecular characterization of L. monocytogenes and Salmonella spp. isolated from biofilms of cattle and poultry slaughterhouses located in the Federal District and State of Goiás, Brazil. Fourteen L. monocytogenes isolates and one Salmonella sp. were detected in poultry slaughterhouses. No isolates were detected in cattle slaughterhouses. All L. monocytogenes isolates belonged to lineage II, and 11 different pulsotypes were detected. Pulsed-field gel electrophoresis analysis revealed the dissemination of two strains within one plant, in addition to the regional dissemination of one of them. The Salmonella isolate was identified via whole genome sequencing as Salmonella enterica serovar Minnesota ST548. In the sequence analysis, no premature stop codons were detected in the inlA gene of Listeria. All isolates demonstrated the ability to adhere to Caco-2 cells, while 50% were capable of invading them. Antimicrobial resistance was detected in 57.1% of the L. monocytogenes isolates, and resistance to sulfonamide was the most common feature. The tetC, ermB, and tetM genes were detected, and four isolates were classified as multidrug-resistant. Salmonella sp. was resistant to nine antimicrobials and was classified as multidrug-resistant. Resistance genes qnrB19, blaCMY-2, aac(6')-Iaa, sul2, and tetA, and a mutation in the parC gene were detected. The majority (78.5%) of the L. monocytogenes isolates were capable of forming biofilms after incubation at 37°C for 24 h, and 64.3% were capable of forming biofilms after incubation at 12°C for 168 h. There was no statistical difference in the biofilm-forming capacity under the different evaluated conditions. Salmonella sp. was capable of forming biofilms at both tested temperatures. Biofilm characterization was confirmed by collecting the samples consistently, at the same sampling points, and by assessing biofilm formation in vitro. These results highlight the potential risk of cross-contamination in poultry slaughterhouses and the importance of surveillance and pathogen control maintenance programs within the meat production industry.

Penanggulangan Sampah Masker Medis Sekali Pakai dengan Pendekatan “Village Health Volunteers” di Kota Surabaya Pada Masa Pandemi Covid-19

This article aims to find out that a Village Health Volunteer approach can be an innovation in handling disposable medical mask waste in the city of Surabaya which incidentally cannot be recycled. This causes it to have the potential to transmit disease if disposable medical masks are reused. The problem in this study is focused on disposable medical mask waste that has not been handled properly from the government side, the community itself and future estimates if left continuously will cause the environment to become worse due to the risk of cross-contamination or biological hazards if polluting the environment. The research method used is literature review. The results of this study explain that the Village Health Volunteer approach can be a way between the government and the community to work together in dealing with health, in this case the handling of disposable medical mask waste. During this pandemic, the use of disposable medical masks by the public has increased so that the waste of disposable medical masks has increased and the risk of cross-contamination or biological hazards by disposable medical mask waste has increased healthy.

Serratia marcescens outbreak in a COVID-19 intensive care unit – Are there any factors specific to COVID-19 units that facilitate bacterial cross-contamination between COVID-19 patients?

COVID-19 dedicated units were created early in the pandemic. Despite their need, factors specific to these units can possibly increase the risk of bacterial cross-contamination between COVID-19 patients. We report a Serratia marcescens outbreak in a COVID-19 intensive care unit, later discussing factors specific to COVID-19 units that may facilitate bacterial cross-contamination in such units, and raising awareness for such an issue.

Cross-Contamination Risk of Dental Tray Adhesives: An In Vitro Study.

Background: The aim of this study was to investigate the risk of cross-contamination in dental tray adhesives with reusable brush systems. Methods: Four dental tray adhesives with different disinfectant components were examined for risk as a potential transmission medium for Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus oralis, and Candida albicans. Bacterial and fungal strains were mixed with artificial saliva. The contaminated saliva was intentionally added to tray adhesive liquid samples. At baseline and up to 60 min, 100 microliters of each sample were collected and cultivated aerobically on Columbia and Sabouraud agar for 24 or 48 h, respectively. Results: At baseline, contamination with Staphylococcus aureus and Candida albicans could be identified in three out of four adhesives. In the subsequent samples, low counts of up to 20 colony-forming units per milliliter could be observed for Staphylococcus aureus. All other strains did not form colonies at baseline or subsequently. Adhesives with isopropanol or ethyl acetate as disinfectant additives were most effective in preventing contamination, while adhesives with hydrogen chloride or acetone as a disinfectant additive were the least effective. Conclusion: Within 15 min, the tested adhesives appeared to be sufficiently bactericidal and fungicidal against all microorganisms tested.

1121 Single Use Nasal Endoscopes – A Review of The Cost and Organisational Implications Based on Department Size

Abstract Aim Single use nasal endoscopes have become increasingly popular since the COVID-19 pandemic. By avoiding the risk of cross contamination and reducing exposure by eliminating the need for re-processing, the disposable scopes have clear safety benefits. Despite ENT UK guidelines recommending that disposable nasal endoscopes be available in every department for use in emergencies, they have often been considered prohibitively expensive. The aim of this study was to analyse the costs associated with traditional nasal endoscopes and compare them to the single use scopes. Method A micro costing exercise was undertaken in three ENT departments: 2 university hospitals, and 1 district general hospital. The outcomes were compared and discussed with relation to the logistics of the departments, as well as organizational considerations. Results Cost per procedure varied according to the reprocessing methods used in the different departments. The cost of the disposable nasal endoscopes appears high, however there are many hidden costs associated with the traditional scopes, which can be difficult to quantify accurately. Conclusions Although disposable endoscopes appear costly, reprocessing and frequent repairs required for re-usable scopes account for the comparable cost per procedure. The high risk of COVID-19 transmission from examining the upper aerodigestive tract means that the safety benefits bear more weight in the current climate. However, concerns regarding environmental impact, image quality and storage of examinations also need to be considered.

Pooling size sorted Malaise trap fractions to maximize taxon recovery with metabarcoding.

BackgroundSmall and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient.MethodsThree Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored.ResultsThe small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample.ConclusionWe demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80–90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.

Bacterial colonisation during regular daily use of a power-driven water flosser and risk for cross-contamination. Can it be prevented?

ObjectiveTo assess whether bacterial colonisation in a power-driven water flosser can be prevented.Materials and methodsTwenty-four patients undergoing supportive periodontal treatment used 2 power-driven water flossers [Sonicare AirFloss (SAF), AirFloss Ultra (SAFU)] for 12 weeks each as follows: (a) with bottled water (BW); (b) with BW and cleaning the device extra-orally twice per week with chlorhexidine gluconate or (c) essential-oil-based (EO) mouth-rinse; (d) with EO only. Water-jet samples were taken after 6 and 12 weeks with the used nozzle and after exchanging to a brand-new nozzle. After 12 weeks, all devices underwent an intensive cleaning procedure. Samples were analysed by PCR-based method for cariogenic and periodontal pathogens and culture for staphylococci, aerobe gram-negative bacteria, and Candida sp.ResultsContamination of SAF/SAFU with Streptococcus mutans was found in > 95% of the samples; periodontal pathogens and aerobe gram-negative bacteria were detected in 19–56% of the samples, while Staphylococcus aureus and Candida sp. were identified only in few samples. Contamination rate was basically unaffected by time-point, device, or way of use. Further, exchanging the nozzle did not prevent transmission of a contaminated water-jet, but the intensive cleaning reduced most of the pathogens significantly, except of S. mutans.ConclusionNeither a specific way of use nor exchanging the nozzle prevented bacterial colonisation and transmission of biofilm components via the water-jet of SAF/SAFU.Clinical relevanceBacterial colonisation in a power-driven water flosser seems impossible to prevent; to restrict the risk of cross-contamination within a household, one device per person should be recommended.

Fate and mitigation of Salmonella contaminated in lettuce (Lactuca sativa) seeds grown in a hydroponic system.

We investigated the fate of Salmonella in lettuce seeds grown in a hydroponic system and the potentials of applying photodynamic inactivation (PDI) to enhance microbial safety of hydroponic farming systems. Lettuce was grown from Salmonella-contaminated seeds, and rose bengal-mediated PDI was applied. Without intervention, Salmonella could persist in plants and hydroponic farming environment throughout 6weeks of lettuce growth. Cross-contamination from Salmonella-inoculated to noninoculated seedlings was observed. PDI significantly decreased Salmonella from 3.90±0.31log colony-forming unit (CFU) per plant to 2.77±0.49log CFU per plant without extra illumination needed (p<0.01) by week six. Salmonella from contaminated seeds could survive for an extended period in lettuce and hydroponic farming environment and posed serious cross-contamination risks. Rose bengal-mediated PDI showed promise in controlling Salmonella contamination in lettuce in a hydroponic farming setting. This study shed light on the serious food safety implications that Salmonella-contaminated lettuce seeds might entail in a hydroponic farming environment and demonstrated rose bengal-mediated PDI as a potential mitigation strategy. These findings contribute to the increasingly relevant field of urban farming systems and their associated food safety concerns.

A simple colorimetric detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification assay using hydroxynaphthol blue metal indicator

A simple reverse transcription loop–mediated isothermal amplification combined with visual detection method (vRT–LAMP) assay was developed for rapid and specific detection of porcine epidemic diarrhea virus (PEDV) in this study, which overcomes the shortcomings of previously described RT–LAMP assays that require additional detection steps or pose a risk of cross-contamination. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubating for 40 min at 62 °C. The assay specifically amplified PEDV RNA and no other viral nucleic acids. The limit of detection of the assay was less than 50 RNA copies per reaction, which was 100 times more sensitive than conventional reverse transcription polymerase chain reaction (RT-PCR) and comparable to real-time RT-PCR (RRT-PCR). In the clinical evaluation, the PEDV detection rate of vRT–LAMP was higher than that of RRT-PCR, showing 99 % concordance, with a kappa value (95 % confidence interval) of 0.97 (0.93–1.01). Considering the advantages of high sensitivity and specificity, simple and direct visual monitoring of the results, no possibility for cross–contamination, and being able to be used as low-cost equipment, the developed vRT–LAMP assay will be a valuable tool for detecting PEDV from clinical samples, even in resource-limited laboratories.

Protective immunity induced by concurrent intradermal injection of porcine circovirus type 2 and Mycoplasma hyopneumoniae inactivated vaccines in pigs

Vaccines against porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are routinely used by intramuscular injection. However, since intramuscular vaccination causes stress and increases the risk of cross-contamination among pigs, research on intradermal vaccination is currently being actively conducted. This study was designed to evaluate the efficacy of intradermally administered inactivated vaccines against PCV2 and Mhp in pigs. Three-week-old specific pathogen-free pigs were divided into three groups (5 pigs per group). Pigs in the two groups were intradermally vaccinated with the PCV2 or Mhp vaccine using a needle-free injector. Pigs in the third group were kept as nonvaccinated controls. At 21 days post-vaccination, pigs in one of these vaccinated groups and the nonvaccinated group were intranasally challenged with PCV2b and Mhp, while the other vaccinated group pigs were maintained as vaccine controls. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes, and humoral or cellular immune responses. As a result, vaccinated pigs revealed significantly higher body weight gain, with lower clinical scores. Vaccinated pigs also showed higher antibody responses but lower PCV2b or Mhp loads in sera, nasal swabs, or lungs than nonvaccinated pigs. Intriguingly, vaccinated pigs upregulated cytotoxic T cells (CTLs), helper T type 1 cells (Th1 cells), and helper T type 17 cells (Th17 cells) after immunization and showed significantly higher levels of CTLs, Th1 and Th17 cells at 14 days post-challenge than nonvaccinated and challenged pigs. This study demonstrated that protective immune responses against PCV2 and Mhp could be efficiently induced in pigs using a relatively small volume of intradermal vaccines, probably due to effective antigen delivery to antigen-presenting cells in the dermis.

The Impact of SARS-CoV-2 on Sperm Cryostorage, Theoretical or Real Risk?

Cryopreservation of human gametes and embryos as well as human reproductive tissues has been characterized as an essential process and aspect of assisted reproductive technology (ART). Notably, sperm cryopreservation is a fundamental aspect of cryopreservation in oncological patients or patients undergoing gonadotoxic treatment. Given that there is a risk of contamination or cross-contamination, either theoretical or real, during the procedures of cryopreservation and cryostorage, both the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) have provided updated guidelines for preventing or reducing the contamination risk of sexually transmitted viruses. Given the ongoing and worldwide COVID-19 pandemic, there is considerable interest in what measures should be taken to mitigate SARS-CoV-2 contamination during cryopreservation and cryostorage of semen samples. The SARS-CoV-2 virus is the virus that causes COVID-19, and whose transmission and infection is mainly aerosol-mediated. Several ART professional societies, including ESHRE and ASRM have proposed measures to mitigate the spread of the SARS-CoV-2 virus. Whether the proposed safety directives are enough to mitigate the possible SARS-CoV-2-contamination of sperm samples during cryopreservation or whether the policies should be re-evaluated will be discussed in this review. Additionally, insights regarding the possible impact of COVID-19 vaccination on the safety of sperm cryopreservation will be discussed.

Cotton and Surgical Face Masks in Community Settings: Bacterial Contamination and Face Mask Hygiene.

During the current COVID-19 pandemic, the use of face masks has become increasingly recommended and even mandatory in community settings. To evaluate the risk of bacterial cross-contamination, this study analyzed the bacterial bioburden of disposable surgical masks and homemade cotton masks, and surveyed the habits and face mask preferences of the Flemish population. Using culture approaches and 16S rRNA gene amplicon sequencing, we analyzed the microbial community on surgical and/or cotton face masks of 13 healthy volunteers after 4 h of wearing. Cotton and surgical masks contained on average 1.46 × 105 CFU/mask and 1.32 × 104 CFU/mask, respectively. Bacillus, Staphylococcus, and Acinetobacter spp. were mostly cultured from the masks and 43% of these isolates were resistant to ampicillin or erythromycin. Microbial profiling demonstrated a consistent difference between mask types. Cotton masks mainly contained Roseomonas, Paracoccus, and Enhydrobacter taxa and surgical masks Streptococcus and Staphylococcus. After 4 h of mask wearing, the microbiome of the anterior nares and the cheek showed a trend toward an altered beta-diversity. According to dedicated questions in the large-scale Corona survey of the University of Antwerp with almost 25,000 participants, only 21% of responders reported to clean their cotton face mask daily. Laboratory results indicated that the best mask cleaning methods were boiling at 100°C, washing at 60°C with detergent or ironing with a steam iron. Taken together, this study suggests that a considerable number of bacteria, including pathobionts and antibiotic resistant bacteria, accumulate on surgical and even more on cotton face masks after use. Based on our results, face masks should be properly disposed of or sterilized after intensive use. Clear guidelines for the general population are crucial to reduce the bacteria-related biosafety risk of face masks, and measures such as physical distancing and increased ventilation should not be neglected when promoting face mask use.

Listeria monocytogenes Survival on Peaches and Nectarines under Conditions Simulating Commercial Stone-Fruit Packinghouse Operations.

Recent recalls of stone fruit due to potential Listeria contamination and associated foodborne outbreaks highlight the risk for pathogen transmission through stone-fruit consumption. Particularly, surface contamination of fruits increases the risk for cross-contamination of produce during processing and storage. This highlights the need for quality control in stone fruits intended for consumption. To develop effective food safety practices, it is essential to determine the critical factors during stone-fruit processing that influence Listeria survival. Therefore, this study evaluated the ability of Listeria to survive on peaches and nectarines under simulated stone-fruit loading and staging, waxing and fungicide application and storage conditions. The results of our study indicate that current stone-fruit handling conditions do not favor Listeria growth. However, once fruit is contaminated, Listeria can survive on the fruit surface in significant numbers under current processing conditions. Therefore, there is a need to develop and implement preventive controls at the stone-fruit packinghouse to prevent Listeria contamination and deter pathogen persistence.

Reduction of SARS-CoV-2 Infectious Titers by Direct Contact with Cuprous-Oxide Impregnated Face Masks External Layers

SARS-CoV-2 can remain viable on the protective face masks surface for several days. Mask touching, reuse and disposal occurs frequently, leading to increased risk of cross-contamination, infection and further transmission. Cuprous-oxide has potent virucidal properties. We determined the capacity of surgical face masks (type IIR) made with nonwoven fabric impregnated with cuprous-oxide microparticles (Test Fabric), to inactivate SARS-CoV-2 when in direct contact with the virus. The Test Fabric reduced the infectious titers of SARS-CoV-2 by 0.73, 3.02 and 4.19 log10 within 5, 30 and 60 minutes, respectively. In contrast, the infectious titers of the virus were reduced by Control Fabric by 0.24, 0.67 and 0.97 within 5, 30 and 60 minutes, respectively. The reductions were significantly higher in the Test Fabric than in the Control Fabric (0.49, 2.35 and 3.22 log difference, accordingly), reaching a statistically significant difference after 5 minutes (p&lt;0.01). The mask filtration properties were not affected by the presence of the cuprous oxide microparticles. We conclude that the use of cuprous-oxide containing face masks in the external layers of respiratory face masks may significantly reduce the risk of SARS-CoV-2 cross-contamination, transmission and infection, due to masks handling and disposal, especially when used by the general population.

Fabrics Attached with Highly Efficient Aggregation-Induced Emission Photosensitizer: Toward Self-Antiviral Personal Protective Equipment.

Personal protective equipment (PPE) is vital for the prevention and control of SARS-CoV-2. However, conventional PPEs lack virucidal capabilities and arbitrarily discarding used PPEs may cause a high risk for cross-contamination and environmental pollution. Recently reported photothermal or photodynamic-mediated self-sterilizing masks show bactericidal-virucidal abilities but have some inherent disadvantages, such as generating unbearable heat during the photothermal process or requiring additional ultraviolet light irradiation to inactivate pathogens, which limit their practical applications. Here, we report the fabrication of a series of fabrics (derived from various PPEs) with real-time self-antiviral capabilities, on the basis of a highly efficient aggregation-induced emission photosensitizer (namely, ASCP-TPA). ASCP-TPA possesses facile synthesis, excellent biocompatibility, and extremely high reactive oxygen species generation capacity, which significantly outperforms the traditional photosensitizers. Meanwhile, the ASCP-TPA-attached fabrics (ATaFs) show tremendous photodynamic inactivation effects against MHV-A59, a surrogate coronavirus of SARS-CoV-2. Upon ultralow-power white light irradiation (3.0 mW cm-2), >99.999% virions (5 log) on the ATaFs are eliminated within 10 min. Such ultralow-power requirement and rapid virus-killing ability enable ATaFs-based PPEs to provide real-time protection for the wearers under indoor light irradiation. ATaFs' virucidal abilities are retained after 100 washings or continuous exposure to office light for 2 weeks, which offers the benefits of reusability and long-term usability. Furthermore, ATaFs show no toxicity to normal skin, even upon continuous high-power light illumination. This self-antiviral ATaFs-based strategy may also be applied to fight against other airborne pathogens and holds huge potential to alleviate global PPE supply shortages.

Serial crystallography using automated drop dispensing.

Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.

Wet grinding of invertebrate bulk samples – a scalable and cost-efficient protocol for metabarcoding and metagenomics

Most metabarcoding protocols for invertebrate bulk samples start with sample homogenisation, followed by DNA extraction, amplification of a specific marker region, and sequencing. Many of the above-mentioned laboratory steps have been verified thoroughly and best practice strategies exist, yet, no clear recommendation for the basis of almost all metabarcoding studies exists: the homogenisation of samples itself. Two different categories of devices are typically used for homogenisation: bead mills or blenders. Both have upsides and downsides. Bead mills rely on single-use plastics and therefore produce a lot of waste and are expensive. In addition to that, processing times can go up to 30 minutes making them unsuitable for large-scale studies. Blenders can handle larger sample volumes in a shorter time, and be cleaned – yet suffer from an increased risk of cross-contamination. We aimed to develop a fast, robust, cheap, and reliable sample homogenisation protocol that overcomes limitations of both approaches, i.e. does not produce difficult to discard waste and avoid single-use plastics while reducing overall costs. We tested the performance of the new protocol using six size-sorted Malaise trap samples and six unsorted stream macroinvertebrate kick-net samples. We used 14 replicates per sample and included many negative controls at different steps of the protocol to quantify the impacts of i) insufficient homogenisation and ii) cross-contamination. Our results show that 3-min homogenisation is sufficient to recover about 80% of OTUs per sample in each replicate and that a non-hazardous DIY cleaning solution provides an effective and efficient way of cleaning. The improvements of the protocol in terms of speed, ease of handling, an overall reduction of costs as well as the documented reliability and robustness make it an important candidate for sample homogenisation after sampling in particular for large-scale and regulatory metabarcoding but also metagenomics biodiversity assessments and monitoring.