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This poster describes the use of short videos demonstrating basic microbiological techniques in a second semester course in Biological Chemistry at the Technical University of Denmark. Videos were a useful complement to the laboratory compendium, allowing students to focus on conceptual understanding of the exercise and the related theory.

Due to logistic and economic benefits, the animal industry has an increased interest in using environmental samples to classify herds free of infections. For a valid interpretation of results obtained from environmental samples, the performance of the diagnostic method using these samples must be assessed. In this abstract we present an approach to evaluate the sensitivity of environmental samples used for herd classification. This is illustrated using results from samples for isolation of methicillin resistant Staphylococcus aureus (MRSA) in 48 pig herds that had previously been tested positive either at farm or slaughter. Three sample matrices were collected; dust samples (5 environmental swabs), nasal swabs (10 pools with 5 animals per pool) and air samples (1 filter). Based on the assumption that MRSA occurred in all 48 herds the overall herd sensitivity was 58% for nasal swabs, 33% for dust samples and 63% for air samples. However, we assumed that the sensitivity of environmental samples varies between herds due to variation in the amount of the analyte. This assumption can be tested by analyzing the association between animal prevalence and the result obtained using environmental samples. In our example, the prevalence of infected pigs in each herd was estimated from the pooled samples of nasal swabs. Logistic regression was used to estimate the effect of animal prevalence on the probability to detect MRSA in the dust and air samples at herd level. The results show a significant increase in the probability of isolating MRSA in the environmental samples by increasing within-herd prevalence. This relationship was strongest for dust where the sensitivity increased from 25% at 1% infected pigs, to 35% at a 25% infected pigs. The air sampling was more sensitive to detect infected herds irrespectively of the within herd prevalence, and performed almost perfectly at a prevalence of 25% infected pigs (sensitivity=99%). In general, the dependence of within herd prevale

Development of a rapid PCR technique is described, which enables slaughterhouses to apply same-day testing for Salmonella in carcasses and fresh meat. The technique is based on a shortened pre-enrichment time and 1-h DNA purification using paramagnetic beads (or an easy-to-use boiling method) followed by Salmonella detection by real-time PCR. Final protocols have been approved for meat testing (fresh meat and carcass swabs) by the Nordval validation organization for Nordic countries.

Viser kødprøven og fortyndingsrørene. Folder stomacherposen ned, viser pipetten og dens indstilling, og udtager 1 ml udenfor filteret. 1 ml overføres og der mixes, gentag.

Vis agarpladen med blandet vækst. Tag den ene rene agarplade og vis hvordan de 3 strøg skal ligge. Tag den anden rene agarplade og renstryg på den.

The aim of this paper was to describe how changes of laboratory training and project based learning were implemented in order to train the students in making a study design, basic laboratory skills, handling of data, technical communication, collaboration and presentation.

Dette er en pipette: håndtag, indstillingshjul og afskydningsknap. 2 modstande, opsugning og afgivning af væske. Sådan påsættes og afskydes en pipettespids korrekt. Sådan afpipetteres væske. På kurset møder du disse 3 pipettetyper; 1000, 200 og 20. Hvilke intervaller dækker de, og hvilke spidser passer til. Sådan indstiller du følgende voluminer: 840, 182 og 18,6.

Mikrobakken vises. Der påsættes 2 l bluejuice i 5 brønde. Pipetten og dens indstilling vises. Pipetten indstilles på 8 l og der tilsættes PCR produkter til de 5 brønde. Gelen vises i elektroforesekaret, og det forklares at vi skal have en markør i brønd 1 og vores prøver i de næste 5 brønde. Pipetten indstilles og der påsættes 6 l markør i gelbrønd 1. Pipetten indstilles til 10 l, prøverne blandes i mikrobakken og påsættes gelen, gentages 3 gange.

Slide show gennemgang af aflæsning.

Pincet åbnes. Filter åbnes. Agarpladens bund vises, og der gøres opmærksom på at pladen skal mærkes med gruppe, øvelse, isolat, dato. Filter overføres til agarpladen, der duppes. 1000 l pipetten vises med korrekt indstilling, mix bakterieblandingen, og der afpipetteres 500 l over på filteret i en samlet dråbe. Låget sættes på agarpladen og skal stå i 10 min før inkubering. ........................... En ny pincet åbnes. Efter endt inkubering overføres filteret til et 50 ml centrifugerør med en ny pincet, stoppes helt ned i bunden, og der mixes grundigt for at få frigjort alle bakterierne i væskefasen.

Mueller-Hinton/bakteireblandingen overføres til reservoirskålen. 8-kanalspipetten introduceres, og dens indstilling vises. Sådan påsættes spidser på 8-kanalspipetten. Der suges bakterieblanding op i pipetten, og der vises at man skal huske at tjekke at der er lige meget væske i alle 8 spidser. Væsken overføres til 1. kolonne i Sensititrepladen, gentages 1 gang mere. Påsætning af film, tryk filmen godt og grundigt fast. Visning af at pladen er mærket med Gruppe, øvelse, isolat, dato. Skrivning af klokkeslæt for inkubering.

Fra 10-1 fortyndingen udtages 100 l, vis pipette og indstilling, og overføres til agarpladen. Bunden af agarpladen vises, og der gøres opmærksom på hvordan de studerende skal huske at mærke pladen. Spred med Drigalskispatlen, gentages 2 gange mere for 10-2 og 10-3 fortyndingen. Pladerne tapes i begge sider. Pladerne pakkes i pose og der bindes knude på posen.

Free-range geese were sampled longitudinally and Salmonella isolates characterized to reveal highly diverging colonization dynamics. One flock was intermittently colonized with one strain of Salmonella enterica serovar Enteritidis from 2 weeks of age, while in another, S. enterica serovar Mbandaka appeared after 9 weeks, without dissemination but with multiple serovars appearing at later stages. ©American Society for Microbiology. All rights reserved.

Background: One of the major sources of human Salmonella infections is meat. Therefore, efficient and rapid monitoring of Salmonella in the meat production chain is necessary. Validation of alternative methods is needed to prove that the performance is equal to established methods. Very few of the published PCR methods for Salmonella have been validated in collaborative studies. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, non-commercial real-time PCR method for detection of Salmonella in meat and carcass swabs. Results: The comparative trial was performed against a reference method (NMKL-71:5, 1999) using artificially and naturally contaminated samples (60 minced veal and pork meat samples, 60 poultry neck-skins, and 120 pig carcass swabs). The relative accuracy was 99%, relative detection level 100%, relative sensitivity 103% and relative specificity 100%. The collaborative trial included six laboratories testing minced meat, poultry neck-skins, and carcass swabs as un-inoculated samples and samples artificially contaminated with 1–10 CFU/25 g, and 10–100 CFU/25 g. Valid results were obtained from five of the laboratories and used for the statistical analysis. Apart from one of the non-inoculated samples being false positive with PCR for one of the laboratories, no false positive or false negative results were reported. Partly based on results obtained in this study, the method has obtained NordVal approval for analysis of Salmonella in meat and carcass swabs. The PCR method was transferred to a production laboratory and the performance was compared with the BAX Salmonella test on 39 pork samples artificially contaminated with Salmonella. There was no significant difference in the results obtained by the two methods. Conclusion: The real-time PCR method for detection of Salmonella in meat and

We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 mu l) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 mu l rather than 100 mu l), and (iv) increasing the PCR template volume (from 5 to 20 mu l) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 mu l were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 mu l) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mu l improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.

As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P = 0.32) when compared to a standard bacteriological method (ISO 10272).

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.

As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10(2) to 10(7) CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of I x 10(3) and 2 x 10(3) CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 x 10(1) to 1 x 10(7) (R-2 = 1.00) for the RotorGene and 10(3) to 10(7) (R-2 = 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (R-2) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was

In a previous study, we reported the performance of a PCR assay amplifying 287-bp of the 16S rRNA gene of thermo-tolerant Campylobacter (C. jejuni, C. lari, C. coli) through an international ring-trial involving 12 participating laboratories. Based on the validated set of primers, a LightCycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving 39 Campylobacter and nine strains of other species indicated that the LC-PCR test was highly specific, giving cross-reactivity with only one strain of C. upsaliensis (CCUG 19559). The sensitivity of the LC-PCR assay, evaluated in 32 spiked poultry-rinse or pork carcass-swab samples, was determined at 10 CFU/ml carcass-rinse. The prevalence of samples positive for thermo-tolerant Campylobacter was 58.8 % in 68 naturally contaminated poultry rinse samples tested by LC-PCR and the data were in good concordance with those of bacteriological method. The Ct values of the three replicates obtained for each sample tested in three different runs demonstrate that the LC-PCR was highly reproducible and afford a powerful tool for rapid detection of the thermotolerant Campylobacter strains.

Human campylobacteriosis has become the major cause of foodborne gastrointestinal diseases in several European countries. In order to implement effective control measures in the primary production, and as a tool in risk assessment studies, it is necessary to have sensitive and quantitative detection methods. Thus, semi-quantitative detection of thermophilic Campylobacter spp. in 20 naturally contaminated chicken rinse samples was carried out using the two most common standard protocols: Preston and Park-Sanders, as proposed by Nordic Committee on Food Analysis (NMKL) and International Standard Organization (ISO), respectively. For both protocols, the chicken rinse samples were prepared in 500 ml buffered peptone water, as recommended in the ISO protocol no. 6887-2. The results indicated that the Preston protocol was superior to the Park-Sanders protocol in supporting growth of Campylobacter spp. In conclusion, the established semi-quantitative assessment using Preston broth could be useful in monitoring the outcome of control programs or quantitative risk assessments.