A fully integrated bacterial pathogen detection system based on count-on-a-cartridge platform for rapid, ultrasensitive, highly accurate and culture-free assay.

Rapid, sensitive and accurate point-of-care-testing (POCT) of bacterial load from a variety of samples can help prevent human infections caused by pathogenic bacteria and mitigate their spreading. However, there is an unmet demand for a POCT device that can detect extremely low concentrations of bacteria in raw samples. Herein, we introduce the 'count-on-a-cartridge' (COC) platform for quantitation of the food-borne pathogenic bacteria Staphylococcus aureus. The system comprised of magnetic concentrator, sensing cartridge and fluorescent image reader with a built-in counting algorithm facilitated fluorescent microscopic bacterial enumeration in user-convenient manner with high sensitivity and accuracy within a couple of hours. The analytical performance of this assay is comparable to that of a standard plate count. The COC assay shows a sensitivity of 92.9% and specificity of 100% performed according to global microbiological criteria for S. aureus which is acceptable below 100 CFU/g in the food matrix. This culture-independent, rapid, ultrasensitive and highly accurate COC assay has great potential for places where prompt bacteria surveillance is in high demand.

Lab in a Pasteur pipette: low-cost, rapid and visual detection of Bacillus cereu using denaturation bubble-mediated strand exchange amplification.

Driven by a bright prospect for rapid, portable and cost-effective point-of-care testing, an assembled Pasteur pipette device to integrate nucleic acid extraction, amplification and detection was developed to detect B. cereus in a sample-to-answer format. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was chosen to perform isothermal amplification because it requires only a pair of primers and one Bst DNA polymerase. The established SEA can detect as low as 1.0 × 10-13 M genomic DNA of B. cereus, which was comparable with the previously reported method for B. cereus detection. The assembled Pasteur pipette allows sample-to-answer diagnostic in a simple, low-cost, portable, and disposable format. The inherent function of Pasteur pipette enables direct liquid handling without the need of extra pipettes, syringes or pumps. Visual readout was achieved by using a pH sensitive dye, further simplifying result judgment process. The detection limit for B. cereus is 1.0 × 104 CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 × 105 CFU/mL without enrichment and 1.0 × 100 CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 × 105 to 1.0 × 108 CFU per gram/milliliter B. cereus, our Pasteur pipette is enough sensitive for answer-to-sample diagnosis of B. cereus even directly from foods without enrichment. The whole diagnostic procedure could be completed within 50 min, dramatically decreasing the detection time. In a word, the assembled Pasteur pipette device, combined with a homemade metal bath, possesses great potential for sample-to-answer application in resource-limited settings.

A portable microfluidic platform for rapid determination of microbial load and somatic cell count in milk.

Microfluidics systems that have been emerged in the last 20 years and used for processing the fluid in a microchannel structure at microliter levels are alternative to the conventional methods. The objective of the study is to develop a microfluidic platform for determination of the microbial load and the number of somatic cells in milk. For this purpose, a polydimethylsiloxane (PDMS) chip with a channel size of 300 µm × 60 µm was produced. Cells/bacteria labeled with fluorescent stain in milk were counted with the proposed microfluidic platform and the results were compared with the reference cell concentration/the bacterial counts by conventional method. It was found that our platform could count somatic and bacterial cells with an accuracy above 80% in 20 min run for each analysis. The portable overall platform has an overall dimension of 25x25x25 cm and weighs approximately 9 kg.

Cost-Effective Live Cell Density Determination of Liquid Cultured Microorganisms.

Live monitoring of microorganisms growth in liquid medium is a desired parameter for many research fields. A wildly used approach for determining microbial liquid growth quantification is based on light scattering as the result of the physical interaction of light with microbial cells. These measurements are generally achieved using costly table-top instruments; however, a live, reliable, and straight forward instrument constructed using parts that are inexpensive may provide opportunities for many researchers. Here, such an instrument has been constructed and tested. It consists of modular test tube holding chambers, each with a low power monochromatic light-emitting diode, and a monolithic photodiode. A microcontroller connects to all modular chambers to control the diodes, and send the live data to either an LCD screen, or a computer. This work demonstrate that this modular instrument can determine precise cell concentrations for the bacteria Escherichia coli and Pseudomonas syringae pv. tomato DC3000, as well as Saccharomyces cerevisiae yeast.

A field study evaluation of Petrifilm™ plates as a 24-h rapid diagnostic test for clinical mastitis on a dairy farm.

Clinical mastitis is one of the most common and expensive diseases of dairy cattle. To make an informed treatment decision, it is important to know the causative pathogen. However, no detection of bacterial growth can be made in approximately 30% of all clinical cases of mastitis. Before selecting the treatment regimen, it is important to know whether the mastitis-causing pathogen (MCP) is Gram-positive or Gram-negative. The aim of this field study was to investigate whether using two 3M Petrifilm™ products on-farm (which conveys a higher degree of sample freshness but also bears a higher risk for contamination than working in a lab) as 24-h rapid diagnostic of clinical mastitis achieved results that were comparable to the conventional microbiological diagnostic method. AerobicCount (AC)-Petrifilm™ and ColiformCount (CC)-Petrifilm™ were used to identify the total bacterial counts and Gram-negative bacteria in samples from clinical mastitis cases, respectively. Missing growth on both plates was classified as no bacterial detection. Growth only on the AC-Petrifilm™ was assessed as Gram-positive, and growth on both Petrifilm™ plates was assessed as Gram-negative bacterial growth. Additionally, milk samples were analysed by conventional microbiological diagnostic method on aesculin blood agar as a reference method. Overall, 616 samples from clinical mastitis cases were analysed. Using the reference method, Gram-positive and Gram-negative bacteria, mixed bacterial growth, contaminated samples and yeast were determined in 32.6%, 20.0%, 2.5%, 14.1% and 1.1% of the samples, respectively. In 29.7% of the samples, microbiological growth could not be identified. Using the Petrifilm™ concept, bacterial growth was detected in 59% of the culture-negative samples. The sensitivity of the Petrifilm™ for Gram-positive and Gram-negative MCP was 85.2% and 89.9%, respectively. The specificity was 75.4% for Gram-positive and 88.4% for Gram-negative MCP. For the culture-negative samples, sensitivity was 41.0% and specificity was 91.0%. The results indicate that the Petrifilm™ concept is suitable for therapeutic decision-making at the farm level or in veterinary practice. As this concept does not allow any statement about the genus or species of microorganisms, relevant MCP should be assessed periodically at the herd level with conventional microbiological diagnostics.

Assessment of the application of an automated electronic milk analyzer for the enumeration of total bacteria in raw goat milk.

Automated electronic milk analyzers for rapid enumeration of total bacteria counts (TBC) are widely used for raw milk testing by many analytical laboratories worldwide. In Ontario, Canada, Bactoscan flow cytometry (BsnFC; Foss Electric, Hillerød, Denmark) is the official anchor method for TBC in raw cow milk. Penalties are levied at the BsnFC equivalent level of 50,000 cfu/mL, the standard plate count (SPC) regulatory limit. This study was conducted to assess the BsnFC for TBC in raw goat milk, to determine the mathematical relationship between the SPC and BsnFC methods, and to identify probable reasons for the difference in the SPC:BsnFC equivalents for goat and cow milks. Test procedures were conducted according to International Dairy Federation Bulletin guidelines. Approximately 115 farm bulk tank milk samples per month were tested for inhibitor residues, SPC, BsnFC, psychrotrophic bacteria count, composition (fat, protein, lactose, lactose and other solids, and freezing point), and somatic cell count from March 2009 to February 2010. Data analysis of the results for the samples tested indicated that the BsnFC method would be a good alternative to the SPC method, providing accurate and more precise results with a faster turnaround time. Although a linear regression model showed good correlation and prediction, tests for linearity indicated that the relationship was linear only beyond log 4.1 SPC. The logistic growth curve best modeled the relationship between the SPC and BsnFC for the entire sample population. The BsnFC equivalent to the SPC 50,000 cfu/mL regulatory limit was estimated to be 321,000 individual bacteria count (ibc)/mL. This estimate differs considerably from the BsnFC equivalent for cow milk (121,000 ibc/mL). Because of the low frequency of bulk tank milk pickups at goat farms, 78.5% of the samples had their oldest milking in the tank to be 6.5 to 9.0 d old when tested, compared with the cow milk samples, which had their oldest milking at 4 d old when tested. This may be one of the major factors contributing to the larger goat milk BsnFC equivalence. Correlations and interactions between various test results were also discussed to further understand differences between the 2 methods for goat and cow milks.