Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

The overall goal of this procedure is to detect foodborne pathogens in large volumes of agricultural water. To do this, a modified more swab referred to hereafter as an MMS is assembled by placing a folded piece of gauze or cheesecloth inside an MMS cassette. Next, at the sampling site, target bacteria are concentrated by passing large volumes of water through the MMS using a peristaltic pump.

The ends of the MMS are then tightly capped and transported back to the lab. Selective or non-selective enrichment broth is added to the MMS, which is incubated for 18 to 24 hours. The enriched cultures are then collected and for e coli detection lysates are prepared.

These are added to micro pad micro spots impregnated with substrates for colormetric enzymatic detection and incubated for up to three hours. The micro pads are then inspected and scanned. Ultimately, visual and quantitative analysis of the micro spots reveal the presence of e coli, salmonella species, and L monocytogenes at levels as low as 0.1 CFU per milliliter.

The main advantage of this technique over membrane filtration and culture on solid media is that larger volumes of water sample. This increases the sensitivity of the assay and also decreases the enrichment time. The time to detection is also decreased through within 24 hours as compared to 24 to 48 hours for the membrane filtration method.

In addition, the micro pads can be used to simultaneously detect three different foodborne pathogens, which cannot be done on the traditional solid bacterial medium. The implications of this technique extend toward analysis of agricultural water and also after extreme events such as wildfires or extreme rainfall events, both of which can lead to an increase in microbial contamination of surface water, which is used for crop irrigation. And we first had this idea for this method when we needed to rapidly screen large volumes of water for presence of pathogens following the wildfires in Colorado during the summers of 2012 and 2013, the method needed to be adaptable to use in the field due to the remote sampling areas, which made it difficult to collect and transport large volumes of water back to the lab for analyses.

Begin this protocol by preparing a modified more swab or MMS cut a rectangular section of four ply cheesecloth measuring 40 by 12 centimeters. Fold the cheesecloth along both AEs to obtain a rectangle of 20 by six centimeters. Next, roll the cheesecloth tightly along its lengthier axis to form a cylindrical swab approximately six centimeters tall and three centimeters in diameter.

Insert the swab into the tinfoil packet and autoclave it for 15 minutes. At 15 PSI using a dry cycle after autoclaving, the device is ready to be taken to the field. Once on location, attach vinyl tubing to spigots on both sides of the MMS, ensuring the tubing fully adheres to the spigots.

Then fix the tubing into the peristaltic pump. Ensure that the tubing upstream of the peristaltic pump is of sufficient length for sampling to concentrate the bacteria. Place the peristaltic pump inlet in the agricultural water to be sampled.

Run the peristaltic pump at a flow rate of 300 milliliters per minute and sample at least 10 liters After sampling is complete. Withdraw the peristaltic pump inlet from the water sample and continue running the pump until no effluent water is observed. Exiting the MMS when concentration has been completed.

For all modified more swabs, carefully removed the vinyl tubing from the MMS spigots and capped the spigots on both sides of the MMS stored the tightly capped MMS for up to 12 hours at refrigeration temperature before adding enrichment. Medium here, enrichment of e coli, salmonella species, and l monocytogenes will be demonstrated. One MMS is used for each selective enrichment.

Unscrew the lid of the MMS and for enrichment of e coli. Add 20 milliliters of sterile buffered peptide water or BPW supplemented with eight milligrams per liter of vancomycin hydrochloride. Screw the lid of the MMS back on tightly.

For enrichment of salmonella species, add 20 milliliters of sterile BPW supplemented with four milliliters per liter. Salmonella supplement for enrichment of L monocytogenes, add 20 milliliters of sterile up list area broth. Incubate the MMS for 18 to 24 hours in a shaking incubator at 150 RPM when the incubation has completed.

Remove the MMS from the incubator. Unscrew the lid. Gently pour the media into a Falcon tube and using a pipette gently transfer 0.5 milliliters into a 1.5 milliliter micro centrifuge tube.

In preparation for colormetric analysis of e coli selective enrichment, or for the analysis of the non-selective enrichments sonicate 0.5 milliliters of enrichment at five watts 22 kilohertz for 20 seconds. Using a probe ator, prepare stock solutions of the substrates C-P-R-G-X glue, magenta cap relate, and XINP in pH adjusted heebies buffer. Protect the stock solutions from light five promo six.

CHLORO three in allele, capite, or magenta capite is light sensitive and could potentially produce false positive results through auto degradation. For each sample and target tested, place one micro pad inside a sterile Petri dish. For e coli detection, prepare CPRG and xgl micro pads.

For salmonella species detection, prepare magenta capite micro pads, and for l monocytogenes detection, prepare XINP micro pads. Using a pipetter, embed the micro padd micro spot with 24 microliters of each substrate stock solution. In preparation for detection, add six microliters of the enriched sample to each appropriate micro pad.

Place the cover back on the Petri dish and seal it with para film. Incubate the sample with a micro pad at 37 degrees Celsius for up to three hours to allow for color development and drying of the micro spots. After the incubation, perform detection by simple visual inspection of the color changes of the micro pads.

Note that strong reactions which produce definitive color changes are typically expected following 18 to 24 hours of enrichment. These are considered positive and do not require further clarification. If e coli is present, micro spots embedded with CPRG will change from yellow to red.

Violet and micro spots. Embedded with xlu will change from colorless to blue green as shown here. If l monocytogenes is present, micro spots embedded with XINP will change from colorless to indigo.

If salmonella species are present, micro spots embedded with magenta capite will change from colorless to purple M.In cases where the reactions are weekly positive, use software to facilitate a semi-quantitative analysis of the results. To do this, allow the micro pads to fully dry. Scan the micro pad using a flatbed scanner.

Use image J software to process the image. Next in image J, open the scanned image to be analyzed. Then to invert the image, click on the edit tab on the main menu and select invert.

Use the analyze tab on the main menu and select set measurements to select the reported measurements to analyze. Make sure that the mean gray intensity limit to threshold and display label boxes are checked and press okay. This sets how the software analyzes each area selected and how it reports it.

Next, using the oval tool outline the area to be analyzed under the analyze tab, select measure. The software will measure the average gray intensity within the area defined and report it in a pop-up screen that can be copied and pasted to excel for further analysis. Micro pad assays are based on the detection of bacteria indicative enzymes, which react with chole metric substrates.

Assays detecting e coli produce a blue green product and a red violet product. Assays detecting salmonella species and L monocytogenes produce purple mov and indigo colors respectively. Alternatively, digitized images can be manipulated using Image J software to allow for more objective and standardized data interpretation.

Here, digitized color and metric images for each assay with both a negative and positive tests are shown. Negative tests were performed using bacterial species that do not encode the target Enzymes and positive tests were performed with target bacteria. The first row shows unmodified scanned images in row two.

The scanned images have been converted to gray scale using image J software. And in row three, the color has been inverted. For subsequent interpretation of gray intensity.

Row four shows the average gray intensity measured using image J within each micro spot of the micro pad. An example micro spot is indicated by the yellow arrow and circle. This graph shows the average gray intensities from four replicates determined by image J.For each color, I metric micro pad positive and negative test.

These data demonstrate that ambiguous detection of the three target microorganisms concentrated from large volumes of agricultural water can be performed via this procedure. After watching this video, you should have a good understanding of how to assemble a modified Moore swab. Concentrate microorganisms of interest from large volume of agricultural water, as well as perform color metric enzymatic detection and analysis on paper-based analytical devices.

While attempting This procedure, it's important to remember to include a sonication step for detection of e coli. It's also important to remember to protect the magenta capite substrate from light as much as possible. Proper ceiling and assembly of the MMS prior to incubation is important for the procedure to be complete with additional development.

This technique promises to become very useful as a simple, inexpensive, and rapid approach to point of need diagnostics for microbial pathogens and indicator microorganisms beyond food safety applications. This technique can find the poten, find potential use in environmental and clinical microbiology. Don't forget that working with pathogenic microorganisms can be extremely hazardous, and therefore it is important to avoid spilling the bacterial enrichments and also the formation of bio aerosols.

Standard precautions for handling pathogenic microorganisms should always be observed when performing this procedure.