This method can help answer key questions regarding the biogeochemical cycling of cadmium and mercury such as how different environmental variables affect their bioavailability to bacteria. The main advantage of this technique is that it offers quasi real time bioavailability data and viable cells irrespective of the presence of oxygen. Generally individuals new to this method will struggle because there are many time sensitive steps that require very meticulous attention to detail.
Demonstrating the procedure will be Ben, a grad student from my lab. To prepare for the assay, retrieve the mercury inducible biosensor and the constitutively expressed biosensor from a minus 80 degree Celsius cryo stock. Plate the cells onto Lysogeny broth plates containing 120 micrograms per milliliter of ampicillin.
Grow the plate cultures in an incubator at 37 degrees Celsius overnight. Between 4:30 and 5 p.m. inoculate a culture in ten milliliters of LB with ampicillin and grow overnight at 37 degrees Celsius with shaking at 220 rpm.
At between nine and ten a.m. the next morning bring the culture as well as the previously prepared growth medium into the anaerobic chamber. Then add eight milliliters of fresh growth medium and 210 micrograms per milliliter of ampicillin into a sterile balch tube.
Now collect two milliliters of the overnight grown culture and transfer into a two milliliter micro centrifuge tube. After centrifuging at 10, 000 RCF for 90 seconds, remove the supernatant and resuspend in two milliliters of fresh growth medium. Then add the resuspended culture to the balch tube containing eight milliliters of fresh growth medium and ampicillin.
Using sterile technique, carefully place a rubber stopper on the balch tube before removing it from the anaerobic chamber. Then place the tube into an incubator and grow anaerobically at 37 degrees Celsius with shaking at 220 rpm. Between three and five p.m.
bring the balch tube with the culture, a new sterile balch tube, and the growth medium into the anaerobic chamber. Add 100 microliters of the culture to 10 milliliters of fresh growth medium with ampicillin in the sterile balch tube. Using sterile technique, carefully place a rubber stopper on the balch tube.
Remove the tube from the anaerobic chamber before growing it overnight at 37 degrees Celsius with shaking at 220 rpm. Between nine and ten a.m. the next day, transfer the culture and the growth medium back to the anaerobic chamber.
Again add eight milliliters of growth medium and ampicillin into a sterile balch tube. Transfer two milliliters of the overnight grown culture into a two milliliter micro centrifuge tube. Following centrifugation as before, remove the supernatant and resuspend the cells in two milliliters of fresh growth medium.
Then add the resuspended culture to the balch tube containing the fresh growth medium and ampicillin. Using sterile technique, carefully place a rubber stopper on the balch tube. Remove it from the chamber and grow anaerobically at 37 degrees Celsius with shaking at 220 rpm.
Monitor the growth of the culture using a spectrophotometer by vortexing the culture and then measuring the optical density at 600 nanometers or OD600. After three to four hours of expected growth, the culture should reach an OD600 of 6. Now bring the tube in the anaerobic chamber and transfer the culture into two two milliliter micro centrifuge tubes.
Following centrifugation as before remove the supernatant and resuspend each cell palate in two milliliters of fresh exposure medium. Repeat this washing step once to remove any trace of the growth medium. Now combine both micro centrifuge tubes of cell culture into a seven milliliter PTFE standard vial to obtain the biosensor stock for use in the exposure assay.
Before starting the experiment check the anaerobic monitor to ensure that there is no oxygen in the anaerobic chamber. Design the plate layout according to a 96 well template. To run experiments in technical replicates of three this will allow for 32 different treatments, which is best represented with a four by eight grid to set up the vials.
Set up the four by eight grid according to the assay plate layout. Then place seven milliliter PTFE standard vials in the tray. Vials should only be handled by manipulating the outside of the vial.
Add the exposure medium volume into each vial corresponding to each treatment. To each vial add the corresponding volume of the chemical variable to be tested according to the plate layout. Now add nitrate to each vial so that the concentration is 200 micromolar.
Exclude this step for constitutive biosensor treatment blanks. To add mercury to the vials, first take the four to eight micromolar stock and shake well. Dilute the solution and exposure medium in a seven milliliter PTFE vial to a concentration of 100 to 250 nanomolar to make a working mercury solution.
From this working solution, add mercury to the required vials according to the plate layout. After adding the mercury or cadmium manually shake the plate in an orbital motion. The experiment may be paused now depending on the time required for mercury or cadmium to speciate in solution.
When the experiment is resumed, gently pipette biosensor stock back and forth to ensure homogeneity. Then add 100 microliters of the biosensor stock to each vial and manually shake the plate as before. Warm up the plate reader to 37 degrees Celsius and set up a kinetic run for ten hours with reads every two point five to five minutes.
An orbital shaking in between readings. Set up the run to take fluorescence measurements with a fluorescence excitation of 440 nanometers and an emission of 500 nanometers. Now pipette 200 microliters from each PTFE vial in the four by eight grid into the corresponding wells of the 96 well plate.
Pipette back and forth five times before transferring each 200 microliters. Instead of discarding the pipette tip, leave the pipette tip in the PTFE vial to keep track of pipetting progress. Place the 96 well plate into the tray of the plate reader.
Then place the lid on the 96 well plate and begin the assay. Here are representative results showing corrected fluorescence data as a function of time. Fluorescence is shown with increasing concentrations of mercury to the inducible biosensor.
All values are blank to the no mercury control. The fluorescent peaks can be quantified to show the fluorescent signal as a function of mercury or cadmium concentration. For both the inducible and the constitutive biosensors, the mercury or cadmium concentration in the middle of the inducible sensors linear range should be used for testing variables.
Here are examples of an inconclusive result with zinc. And a conclusive result with magnesium and manganese on mercury bioavailability to the biosensor. The result with zinc is inconclusive because the constitutive fluorescence decays with inducible fluorescence indicating toxicity.
While attempting this procedure it's important to remember to enter the concentrations of either cadmium or mercury as these will be used to calibrate the biosensor. Following this procedure, other methods like thermodynamic modeling of aqueous systems can be performed to answer additional questions such as how do specific chemical species of mercury or cadmium affect their bioavailability. Don't forget that working with cadmium and mercury and strong acids such as sulfuric acid can be extremely hazardous and precautions such as the wearing of personal protective equipment should always be taken while performing this procedure.
After its development this technique paved the way for researchers in the field of environmental microbiology to explore quasi real time anaerobic mercury or cadmium bioavailability and genetically tractable microbes.
