scholarly journals Fast and inexpensive detection of bacterial viability and drug effectiveness through metabolic monitoring

2016 ◽  
Author(s):  
Sondos Ayyash ◽  
Wen I Wu ◽  
Ponnambalam Ravi Selvaganapathy
Keyword(s):  
Sample Preparation ◽  
Bacterial Infections ◽  
Low Cost ◽  
Turnaround Time ◽  
Resource Poor Setting ◽  
Culture Methods ◽  
Drug Effectiveness ◽  
Minimal Sample ◽  
Conventional Culture ◽  
Oxygen Sensitive

Conventional methods for the detection of bacterial infection such as DNA or immunoassays are either expensive, time consuming, or not definitive; thus may not provide all the information sought by the medical professionals. In particular, it is difficult to obtain information about viability or drug effectiveness, which are crucial to formulate a treatment. Bacterial culture test is the gold standard because it is inexpensive and does not require extensive sample preparation, and most importantly, provides all the necessary information sought by healthcare professionals, such as bacterial presence, viability and drug effectiveness. These conventional culture methods, however, have a long turnaround time: anywhere between 1 day to 4 weeks. Here, we solve this problem by monitoring the growth of bacteria in thousands of nanowells simultaneously to identify its presence in the sample and its viability, faster. The segmentation of a sample with low bacterial concentration into thousands of nanoliter wells digitizes the samples and increases the effective concentration in those wells that contain bacteria. We monitor the metabolism of aerobic bacteria by using an oxygen sensitive fluorophore, ruthenium tris (2,2-diprydl) dichloride hexahydrate (RTDP) that allows us to monitor the dissolved oxygen concentration in the nanowells. Using E.Coli K12 as a model pathogen, we demonstrate that the detection time of E.coli can be as fast as 35-60 minutes with sample concentrations varying from 104(62 minutes for detection), 106 (42 minutes) and 108 cells/mL (38 minutes). More importantly, we also demonstrate that reducing the well size can reduce the time of detection. Finally we show that drug effectiveness information can be obtained in this format by loading the wells with the drug and monitoring the metabolism of the bacteria. The method that we have developed is low cost, simple, requires minimal sample preparation and can potentially be used with a wide variety of samples in resource poor setting to detect bacterial infections such as Tuberculosis.

scholarly journals A Simple Off-Grid Incubator for Microbiological Water Quality Analysis

Water ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 240 ◽  
Author(s):  
Carolina Bernardes ◽  
Ricardo Bernardes ◽  
Camille Zimmer ◽  
Caetano C. Dorea
Keyword(s):  
Water Quality ◽  
Low Cost ◽  
Expanded Polystyrene ◽  
Hot Water ◽  
Quality Analysis ◽  
Health Concern ◽  
Culture Methods ◽  
Conventional Culture ◽  
Quality Testing ◽  
Microbiological Water Quality

There is a need for accessible and low-cost microbiological water quality testing in contexts where diarrheal illness is a major public health concern. In most cases, the quantification of Escherichia coli and other microbial indicators by conventional culture methods requires an incubation step for processed samples at specific temperatures for bacterial growth over a prescribed time. However, incubators can be the most expensive equipment required for such microbial analyses, limiting the number and scope of water quality testing available in low-resource contexts. In this study, a low-cost incubator was developed using a locally available expanded polystyrene (EPS) foam cooler, with two water bottles filled with hot water to heat incubator to a target of 35 °C. The EPS incubator performance was validated by processing 150 water samples in duplicates using the Colilert Quanti-tray/2000 system, incubated in either the EPS incubator or a standard laboratory incubator set at 35 °C. Statistically significant correlations of results indicated that the quantification of E. coli was comparable between both methods. Risk categorizations from standard and EPS incubation results agreed for 141 of 150 (94%) samples, with zero false negatives. In addition to being reasonably mobile the EPS incubator would reduce the cost of such water quality testing, thus potentially increasing the scope of water quality testing coverage.

Advanced TEM sample preparation techniques for submicron Si devices

1995 ◽  
Vol 53 ◽  
pp. 516-517 ◽  
Author(s):  
P. B. Basham ◽  
H. L. Tsai
Keyword(s):  
Sample Preparation ◽  
Process Development ◽  
Light Transmission ◽  
Specific Area ◽  
Turnaround Time ◽  
Small Hole ◽  
Area Of Interest ◽  
Transmission Electron ◽  
Polished Side ◽  
Preparation Techniques

The use of transmission electron microscopy (TEM) to support process development of advanced microelectronic devices is often challenged by a large amount of samples submitted from wafer fabrication areas and specific-spot analysis. Improving the TEM sample preparation techniques for a fast turnaround time is critical in order to provide a timely support for customers and improve the utilization of TEM. For the specific-area sample preparation, a technique which can be easily prepared with the least amount of effort is preferred. For these reasons, we have developed several techniques which have greatly facilitated the TEM sample preparation.For specific-area analysis, the use of a copper grid with a small hole is found to be very useful. With this small-hole grid technique, TEM sample preparation can be proceeded by well-established conventional methods. The sample is first polished to the area of interest, which is then carefully positioned inside the hole. This polished side is placed against the grid by epoxy Fig. 1 is an optical image of a TEM cross-section after dimpling to light transmission.

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

No lyse no wash flow cytometry for maximizing minimal sample preparation

Methods ◽  
2018 ◽  
Vol 134-135 ◽  
pp. 149-163 ◽  
Author(s):  
Jordi Petriz ◽  
Jolene A. Bradford ◽  
Michael D. Ward
Keyword(s):  
Flow Cytometry ◽  
Sample Preparation ◽  
Minimal Sample

Amplified Migration Inhibition Effect

1980 ◽  
Vol 29 (2) ◽  
pp. 609-616 ◽  
Author(s):  
J. R. Philp ◽  
A. L. Huffman ◽  
L. R. DeChatelet ◽  
J. E. Johnson
Keyword(s):  
Molecular Weight ◽  
Culture Medium ◽  
Peritoneal Exudate ◽  
Macrophage Migration ◽  
Culture Dish ◽  
Culture Methods ◽  
Peritoneal Exudate Cells ◽  
Inhibition Factor ◽  
Conventional Culture ◽  
Heat Labile

When tuberculin-sensitive peritoneal exudate cells are incubated in a culture flask with tuberculin purified protein derivative, macrophage inhibition factor and other lymphokines are released into the culture medium. We have described how, if incubation is carried out in a stationary conical culture tube, intercellular contact between the peritoneal exudate cells is facilitated as the cells sediment into a pellicle at the bottom of the tube. This results in augmented release of inhibitory lymphokines into the supernatant culture medium with titers up to 10 9 times greater than those obtained by conventional culture methods using a flatbottomed culture dish or flask. When such high-titered inhibitory supernatants were subjected to fractionation by sequential Amicon ultrafiltration, two clearly distinct macrophage-inhibitory lymphokines were found. The first was present, after fractionation, in a titer of 10 12 , had a molecular weight in the range of 50,000 to 100,000, and was heat stable at 56°C for 1 h. This moiety is probably identical to guinea pig macrophage inhibition factor. Unexpectedly, a second heat-labile inhibitory substance with a molecular weight between 500 and 1,000 was found in a titer of 10 4 after fractionation. This low-molecular-weight, heat-labile material may represent a new lymphokine with a direct inhibitory action on macrophage migration. Theoretically, the data are also consistent with the possibility that it could act as a chemical immunotransmitter which stimulates amplified production of macrophage inhibition factor by lymphocytes within the cell pellicle and leads indirectly to inhibition of macrophage migration.

A comparative study of qualitative immunochemical screening assays for the combined measurement of T-2/HT-2 in cereals and cereal-based products

2011 ◽  
Vol 4 (4) ◽  
pp. 385-394 ◽  
Author(s):  
J. Meneely ◽  
F. Ricci ◽  
S. Vesco ◽  
M. Abouzied ◽  
M. Sulyok ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Low Cost ◽  
Ease Of Use ◽  
The Other ◽  
Accuracy And Precision ◽  
Cost Implications ◽  
Electrochemical Array ◽  
The Cost ◽  
Preparation Techniques ◽  
Speed Of Analysis

Many different immunochemical platforms exist for the screening of naturally occurring contaminants in food from the low cost enzyme linked immunosorbent assays (ELISA) to the expensive instruments such as optical biosensors based on the phenomenon of surface plasmon resonance (SPR). The primary aim of this study was to evaluate and compare a number of these platforms to assess their accuracy and precision when applied to naturally contaminated samples containing HT-2/T-2 mycotoxins. Other important factors considered were the speed of analysis, ease of use (sample preparation techniques and use of the equipment) and ultimately the cost implications. The three screening procedures compared included an SPR biosensor assay, a commercially available ELISA and an enzymelinked immunomagnetic electrochemical array (ELIME array). The qualitative data for all methods demonstrated very good overall agreements with each other, however on comparison with mass spectrometry confirmatory results, the ELISA and SPR assay performed slightly better than the ELIME array, exhibiting an overall agreement of 95.8% compared to 91.7%. Currently, SPR is more costly than the other two platforms and can only be used in the laboratory whereas in theory both the ELISA and ELIME array are portable and can be used in the field, but ultimately this is dependent on the sample preparation techniques employed. Sample preparative techniques varied for all methods evaluated, the ELISA was the most simple to perform followed by that of the SPR method. The ELIME array involved an additional clean-up step thereby increasing both the time and cost of analysis. Therefore in the current format, field use would not be an option for the ELIME array. In relation to speed of analysis, the ELISA outperformed the other methods.

A review of plant analysis in Australia

1993 ◽  
Vol 33 (8) ◽  
pp. 1029 ◽  
Author(s):  
PD Handson ◽  
BC Shelley
Keyword(s):  
Quality Assurance ◽  
Sample Preparation ◽  
Turnaround Time ◽  
Plant Analysis ◽  
Nutrient Analysis ◽  
Testing Laboratories ◽  
Australian Plant ◽  
Analytical Accuracy ◽  
Plant Sap

This review of plant analysis in Australia examines sample preparation, instrumentation, problem analytes, calibration, detection limits, and quality assurance. The issue of turnaround time v. analytical accuracy is discussed and the role of 'plant sap quick tests' in nutrient analysis is assessed. Results of a survey of Australian plant-testing laboratories are included.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

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