A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

The overall goal of this procedure is to create a bio reporter assay to directly monitor the influence of mycelial networks on the bioavailability of organic contaminants. This is accomplished by immobilizing bio reporter bacteria that express enhanced green fluorescent protein or EGFP as a function of contaminant bioavailability in auger, and applying them on a suitable glass slide. Next, the contaminant source is added to the setup inside of an auger ring, and the mycelial organism is inoculated on an auger patch on top of it.

Then the microcosm is incubated until the mycelium has overgrown the whole setup and the contaminant may be trans located along the network. Finally, the bio reporter cells are visualized using confocal laser scanning microscopy. Ultimately, the influence of mycelial networks on fluorine bioavailability is evaluated by quantification of EGFP expression of the bio reporter cells using image analysis software.

The main advantage of this technique over existing methods like chemical PAH extraction, is that contaminant bioavailability can be assessed directly in a biological system and with only very little consumption of material. This method can provide insight into the bioavailability of p hs in a water unsaturated environment like soil in the future. It also may be used for other chemicals such as pesticides and or by using other bacterial and fungal strains.

To begin, prepare the following material for each microcosm. One 10 centimeter Petri dish, bottom one modified Petri dish bottom that has been cut at the brim to fit a slide with a 26 millimeter edge length, one counting chamber slide with three cavities and a small Petri dish lid. After preparing media growing Pythium Ultima on PDA plates and culturing, behold area sarti soli RP O 37 mg in minimal medium, according to the text protocol, measure the OD 5 78 of the behold area sarti soli culture.

The expected OD is about 0.2 centrifuge the culture at 2000 G for 10 minutes and resuspend the cells in an appropriate amount of minimal medium to reach an OD of 0.4. Mix 50 microliters of the cell suspension with 500 microliters of warm liquid, minimal medium auger or MMA in two milliliter tubes and store at 50 degrees Celsius until ready to use under a flow cabinet. Expose the desired number of Petri dishes and slides to UV light for 30 minutes.

Then place the small Petri dish inside the larger one and fit the slide through the gap of the small Petri dish. Add 150 microliters of the warm cell suspension into the middle cavity of each slide, and allow them to solidify for five minutes. Use a one centimeter cork borrower to punch out circular patches from a blank PDA plate to promote mycelial growth.

Place one patch at a distance of two millimeters from the MMA inside the small Petri dish. Next to cut curved PDA patches as mechanical barriers. Use a clean and sterile small Petri dish bottom part and press it into a PDA plate.

Then use a spatula to cut another circle about 0.5 centimeters in diameter smaller to produce a PDA ring. Cut out a piece of the auger ring that exactly fits into the gap of the small Petri dish in the microcosm set up and place it inside the gap on top of the slide. Ensure that the distance between the MMA and the circular PDA barrier is about two millimeters.

Close the lid of the small Petri dish gently pressing it into the PDA barrier as a control setup for airborne transport towards bio reporter cells, prepare a mechanical PDA barrier using a PDA plate containing two grams per liter of cyclo heide. Use a one centimeter cork borrower to punch out circular patches of a blank PDA plate. Use another 0.5 centimeter cork boer to cut out the middle of the first patch.

Place the resulting small PDA ring at a distance of one millimeter from the PDA barrier. Now add two milligrams of PAH fluorine into the hole of PDA one. Then use a one centimeter cork boer to cut out circular patches from PDA plates overgrown with pythium ultimo, and place one patch bottom down onto the PDA ring so that the mycelial mat is facing the fluorine crystals.

Prepare a negative control without fluorine crystals to determine background fluorescence of bio reporter cells in a similar manner. Cut out one centimeter circular patches from auger containing activated carbon, and place four patches into each microcosm set up to further decrease the gaseous fluorine concentration. To prepare a positive control add fluorine crystals to 200 microliters of MMA cell suspension and put it in one cavity of an empty slide.

Place a Petri dish with some activated carbon powder on the bottom of the incubation chamber to minimize gaseous fluorine concentration in the chamber. Then place four or five 50 milliliter beakers with sterile water on the bottom to maintain high humidity in the chamber. Transfer the microcosm setups into the incubation chambers, placing samples randomly in the different growth chambers to exclude location effects for pythium Ultimo.

Incubate at 25 degrees Celsius for 96 hours after setting up the confocal microscope. Using the guidelines outlined in the text protocol, open a microcosm setup. Use a scalpel to remove PDAs 1, 2, 3, and the PDA barrier.

Place a glass cover slip on top of the MMA and press it gently to remove air bubbles. Mount the slide on the microscope stage. Add a droplet of water on top of the cover slip and use m cherry settings to find bio reporter cells in the sample.

Next, choose a random position on the sample to get a quick overview of it. Then use a glow over under lookup table for the red and green channels and optimize the signal to noise ratio. Then define and record Z stacks for each position in the green and red channels.

Repeat the Zack acquisition for 10 random evenly distributed positions using image J According to the text protocol, open a stack of images, select split channels, and close the green channel. Use the macro M cherry area provided as a supplementary code file to quantify the area of red fluorescence in the Z stack and output table will be generated with the measured pixels above threshold in the Z stack with the sum of all values representing the total number of red pixels in the stack. Next with the file open close the red channel.

Use the macro E-G-F-P-I-N-T provided as a supplementary code file to quantify the intensity of green fluorescence in the Z stack. The output is a table with the mean intensities and area of all green objects above threshold in the Z stack. To calculate the total intensity of green pixels in the stack, multiply each mean intensity by the corresponding area.

Calculate the relative EGFP induction using the following formula, and refer to the text protocol for additional options to chemically quantify fluorine amounts. After preparing microcosms with and without bio reporter cells, place three previously cleaned PDMS coated glass fibers as artificial and quantifiable contaminant sinks inside the MMA of the Samin microcosms. After incubating for 96 hours, use forceps to remove the glass fibers and transfer each one into a gas chromatography mass spectrometry or GCMS vial with insert add toluene to the samples and incubate overnight.

Finally, use GCMS to analyze the samples. A maximum intensity projection can be generated to gain a first visual impression of the sample and the controls as demonstrated here. The positive control con pause shows distinct EGFP induction, whereas the Airborne Control Con Air exhibits only background EGFP fluorescence, these panels show that EGFP fluorescence in the test sample Sam, appears to be elevated compared to con error, yet not as marked as con ps.

The M cherry fluorescence is independent from PAH degradation and therefore similar for all samples for the tested microcosms. Visual inspection was complimented by image analysis and calculation of relative EGFP induction as demonstrated here under the given conditions. No significant difference could be detected between coneg and con air, thus excluding any vapor phase fluorine transport towards the bio reporter cells in the presence of mycelia and fluorine.

EGFP was induced significantly in the cells compared to the negative controls. However, the EGFP induction is relatively low compared to the positive control. This figure shows that Mycelia Trans located about 25 nanograms of fluorine within 96 hours controls in the absence of mycelia Conair minus however revealed gaseous fluorine transport into the MMA in the range of only two nanograms within 96 hours.

While attempting this procedure, it is very important to remember that BioPort bacteria are highly sensitive to gas phase concentrations of fluorine. Hence, it is very important to minimize gas phase transport as much as possible, and to verify the absence of gas phase transport by chemical extraction of MMA. After watching this video, you should have a good understanding of how to assemble the microcosms and how to perform visual inspection using confocal laser scanning microscopy.

You can also see that the microcosms are suitable for different microbial strains and various chemicals.