The overall goal of this experiment is to characterize the evolutionary pathway of antibiotic resistance using a Morbidostat that continuously monitors bacterial growth and dynamically adjusts the antibiotic concentration as bacteria acquire drug resistance. The main advantage of this technique is that the set up is made of low cost easily available electronic components and can be easily reconfigured for specific means. Demonstrating the procedures will be Po Cheng Liu and Yi Tai Lee, both are grad students of my laboratory.
To assemble the Morbidostat, begin by using an 18 gauge needle to punch three holes into the cap of the culture vial. Cut three pieces of polyethylene tubing approximately seven centimeters in length and insert them into the holes in the cap. Use tape to wrap the edge of the cap to serve as the cast for the Polydimethylsiloxane, or PDMS, mixture.
Then add five grams of component A and 0.5 grams of component B of the PDMS in a 150 milliliter plastic container and use a toothpick to manually stir it. Load the mixture into a 10 milliliter syringe. Then, with the syringe, inject the PDMS mixture onto the cap.
Bake the entire cap at 70 degrees Celsius for eight hours to cure the PDMS. Next, using a soldering iron, solder the LED anode with the 24 gauge wire and the LED cathode with the carbon resistor. Then solder a 680 Ohm carbon resistor with a 24 gauge wire.
Use electrical tape to insulate the wire connection. Solder the collector of the photo detector with the 24 gauge wire and the emitter of the photo detector with a 100 Ohm carbon resistor. Then use electrical tape to insulate the wire connection.
Place the light emitting diode in the culture vial holder. By following the circuit diagram shown here, connect the light emitting diode and use a five volt power supply to power it. Ensure that the LED works by using a digital camera that can detect IR.Now place the photo detector in the culture vial holder.
Then following the diagram, connect the photo detector and use a five volt power supply to power it. Connect the wire to both sides of the photo detector resistors o measure the voltage across the electronic control board. Glue a magnet on the shaft of the cooling fan which serves as the magnetic stirrer unit.
Because the magnetic stirring unit is formed from the magnet and the cooling fan, it's crucial to align the shelf of the fan to culture vial. Also the distance between the culture vial and the magnet is very critical to ensure the stable operation. Connect each piece of polyethylene tubing of the culture vial into the corresponding piece of silicone tubing for the micro pumps, medium bottles, and waste bottle.
Turn on the pump for one hour and measure the total volume being pumped from the medium bottle to the culture vial to determine the pump rate. The typical pumping rate should be approximately four milliliters per hour, which corresponds to the dilution rate of D equals 0.33 per hour for a 12 milliliter working volume of the culture vial. When the set up is complete, place the entire assembly on a mid sized shaking incubator.
To pre test the Morbidostat, set the power supply voltage of the cooling fan to five volts to start its motor to test the magnetic stir bar. After preparing a series of E-coli samples and plotting a calibration curve according to the text protocol, prepare the culture vial with three seven centimeter lengths of ultra chemical resistant Tygon tubing with an inner diameter of 132nd of an inch and an outer diameter of 332nds of an inch for inlets to form the device. Prepare M9 minimal medium with 0.2%glucose and M9 containing the drug Trimethoprim or TMP.
Then sterilize the medium, medium bottle, the drug medium bottle, the waste bottle, and the culture vial in an autoclave at 121 degrees Celsius. On the first day of the experiment, thaw one milliliter of frozen wild type E-coli MG1655 cells at room temperature for five minutes and transfer 120 microliters of the cells to a culture vial containing approximately 12 milliliters of M9 growth medium. Turn on the shaking incubator and set the temperature to 30 degrees Celsius with no shaking.
Start the Morbidostat operation by turning on the micro pump, the magnetic stirring unit, and the optical density measurement. After approximately 23 hours, stop the micro pump by turning off its supply voltage and take out the culture vial. Add 15%glycerol into the culture vial and freeze the samples at negative 80 degrees Celsius for storage.
Switching to a new culture vial in the beginning of each of these experiments is also important to avoid biotin formations. Otherwise the biotin formation can occur within two or three days. After thawing the daily frozen sample at room temperature for five minutes, inoculate a new test tube with fresh M9 medium.
Place the sample in a shaking incubator at 37 degrees Celsius overnight. The following day, use M9 medium to dilute the microbial sample tenfold and load it into the microfluidic device by pressuring the microbial sample container at five PSI. Then load the TMP containing medium into the chip by pressuring the M9 with TMP container at five PSI.
Execute the mixing between the microbe and drug by actuating the micro mechanical valve between the two chambers on the microfluidic chip for 15 minutes. Finally place the microfluidic chip in the microscope incubator mounted on the inverted microscope. With a CCD camera, acquire cell images in the growth chamber every hour for eight hours.
Calculate cell number data according to the text protocol. The common Morbidostat operations including experimental evolution, antibiotic susceptibility test, and cell morphology checking were invalidated using an E-coli MG1655 culture exposed to the antibiotic TMP, which induces a distinctive step wise increase in drug resistance with mutations clustered around the dihydrofolate reductase, or DHFR, gene. Each day, the microbe is expected to grow above the pre set optical density threshold and trigger the drug injection as shown here.
The drug injection is expected to inhibit the growth, and as a result, decrease the optical density. This plot shows the temporal increase in the antibiotic drug resistance in an experiment to determine the IC50. The drug resistance increased approximately 1500 fold to 1, 000 micrograms per milliliter over about 12 days.
Consistent with previous findings. The DHFR point mutations in the last day mutant were measured by Sanger sequencing and are listed in this table. The acquisition of drug resistance with multiple mutations proves the usefulness of the Morbidostat.
As the selection on drug containing agar plates tends to confer only the single mutation. This figure displays the on chip growth curves at various concentrations of TMP. The mutant samples shows a significant increase in antibiotic drug resistance.
After its develop, this technique paved the way for the researchers to study the position of antibiotic drug resistance in a real control laboratory environment. Following this procedure, other measure like microfluidic single cell measurement can be performed in order to answer additional questions like it still morphology to change due to the exposure of antibiotic drugs. Once mastered, this device can be easily reconfigured for all the bacterial culture experiment such as the cultivation of a sign of bacteria or increasing the costing tolerance level of a bacterial strain.
After watching this video, you should have a good understanding of how to study the acquisition of antibiotic drug resistance in a real control adaptive laboratory evolutionary experiment. Don't forget to handle the drug resistant bacteria according to the bio safety rules for microbiological practice. Special permission may be required for an experiment involving basic training of bacteria.
