This method allows growing three different species of magnetotactic bacteria called MSR-1, AMB-1, and MS-1. All freshwater species, and all from the genus magnetospirillum. It is an essential first step for any kind of reproducible research involving these organisms.
Magnetotactic bacteria have very specific oxygen requirements, which is why they developed a magnetic navigation system. It is thus very important to control the amount of oxygen available to them. All of this protocol has been optimized for a few specific strains of freshwater magnetospirilla.
Its details can be optimized in order to accommodate the oxygen and nutrient requirements of other magnetotactic bacteria. Safely install a nitrogen gas tank close to a bench on which there is enough space to set-up the nitrogen station. Connect a piece of tubing to the tank that is long enough to reach the area where the station will be built.
If necessary, apply Teflon tape at the output of the tank to avoid any leakage. To build a station capable of bubbling five bottles of medium at the same time, cut four pieces of tubing of aproximately five centimeters in length. Assemble the pieces of tubing in a line with three 3-way T-shaped plastic fittings.
Connect one end of this line to the piece of tubing at the output of the nitrogen tank through an extra T-shaped fitting. Add a 90-degree elbow fitting at the other end. Use tape to attach the structure to a horizontal metal rod placed aproximately 30 centimeters above the bench.
Connect five pieces of tubing of aproximately 20 centimeters in length to the three outputs of the installed fittings. Remove the pistons from five one-milliliter plastic syringes, and cut the larger end of these syringes, keeping only the graduated part. Fill these syringes with cotton, taking care not to pack too tightly.
Insert the syringes in the 20-centimeter long vertical pieces of tubing. Turn the gas on, and use soapy water to ensure that there is no leakage when nitrogen is flowing. Remove the caps of five 25 gauge needles and insert these needles into 10-centimeter pieces of thin tubing.
Attach the prepared needles to the syringes of the nitrogen station. Ensure that nitrogen is flowing through all five lines, and keep the station on standby. Prepare a 10-millimolar ferric citrate solution by adding 0.245 grams of ferric citrate to 100 milliliters of distilled deionized water.
Heat and stir to dissolve until an orange clear solution is obtained. Autoclave the solution using a standard cycle of at least 15 minutes exposure at 121 degrees Celsius before storing the sterilized stock solution at room temperature in the dark. To prepare five bottles of liquid growth medium for MSR-1, add the chemicals listed in a text protocol to a beaker containing 300 milliliters of distilled deionized water.
Addition of the sterile ferric citrate and mineral solutions must be performed under the flame of a Bunsen burner using steril techniques. Sterile conditions are essential in order to avoid contamination of the culture by other bacterial species. After the addition of all chemicals, adjust the pH to 7.0 with one molar sodium hydroxide solution.
Dispense the freshly prepared medium into 125-milliliter serum bottles, pouring 60 milliliters of medium in each bottle. Bubble nitrogen into the medium for 30 minutes to remove the dissolved oxygen using the small tubing connected to the nitrogen station. Place a butyl rubber stopper on top of each bottle, leaving a small opening to allow the excess gas to exit the bottle.
After 30 minutes, crimp seal each bottle with the prepared stopper and an aluminum seal. The aluminum seal ensures that the bottle remains sealed during the rest of the protocol. Disconnect the needles and the thin tubing from the nitrogen station and replace them with clean needles.
Adjust the valves of the nitrogen tank so that a gentle, continuous flow of gas exits the tank. Now, insert one of the needles connected to the nitrogen tank into a bottle of medium through the rubber stopper. Immediately insert another clean needle into the same bottle.
Repeat this step for the other bottles, and let nitrogen flow for about 30 minutes to replace the air in the bottles by nitrogen. After 30 minutes, disconnect one bottle from the nitrogen station by removing the corresponding needle. Wait for a few seconds until the pressure in the bottle of medium decreases to atmospheric pressure, and remove the second needle.
Repeat this step for all remaining bottles. To prepare 120 milliliters of semisolid growth medium for MSR-1, cover the beaker with aluminum foil and autoclave the solution prepared as described in the text protocol. Just before the end of the autoclave cycle, prepare a fresh 4%cysteine solution and adjust the pH to 7.0 with a five molar sodium hydroxide solution, as described in the text protocol.
After autoclaving, let the medium cool down to 50 to 60 degrees Celsius, and bring the beaker under the flame of a Bunsen burner. Remove the aluminum foil and quickly add the chemicals listed in the text protocol using sterile techniques while gently stirring. Add 1.2 milliliters of filter sterilized cysteine solution.
After the addition of all chemicals, transfer the warm medium into 16 milliliters sterile screw cap Hungate tubes. Transfer 12 milliliters of the medium into each tube, and seal the tubes. To inoculate strains MSR-1, AMB-1, and MS-1 in liquid medium, flame the tops of the oxygen and fresh medium bottles by applying ethanol on the stoppers and passing them through the flame of a Bunsen burner.
Using a sterile syringe and a needle, extract one milliliter of oxygen from the oxygen bottle and transfer it into the fresh medium bottle. If using the inoculum from another culture grown in a glass bottle, flame both bottles. Then inoculate one milliliter of the older culture into the fresh medium.
Incubate the culture at 32 degrees Celsius, and inoculate it into fresh medium after four to seven days. To inoculate MSR-1 in oxygen gradient, semisolid medium, verify that the tube of fresh medium displays a well-defined OAI, materialized by a pink to colorless interface about one to three centimeters below the surface of the medium. If the inoculum is coming from another oxygen concentration gradient semisolid culture, harvest the bacteria by aspirating 50 microliters of the culture with a sterile pipette tip placed on the band formed by the bacteria.
Slowly inoculate these bacteria at the OAI in the fresh medium, avoiding disturbing the interface. Seal the tube and let the bacteria grow between 25 degrees Celsius and 30 degrees Celsius. To ensure that the bacteria are both magnetic and modal, place a drop of culture on microscope cover slip.
Flip the cover slip, and install it on an O-ring resting on a glass slide. Ensure that the drop does not touch the bottom slide. Place the hanging drop under a microscope and focus on an edge of the droplet.
Place the south pole of a magnet close to that edge, and watch the bacteria swim towards the edge. Flip the magnet to make them swim in the opposite direction. Shown here are the bottles of liquid media before inoculation and after bacterial growth.
Turbidity in the second bottle indicates that bacteria are growing. This two times accelerated movie shows the expected result of a hanging drop experiment for a modal and magnetic magnetotactic bacteria culture. The bacteria initially swam towards the edge of the droplet when a magnetic field is applied.
When the field direction of the magnet is reversed, the bacteria swam away from the edge where the magnetic field is applied. Shown here are tubes of semisolid medium before inoculation and after bacterial growth. As expected, a band of bacteria forms at the oxic-anoxic interface and migrates over time as oxygen is consumed and the interface moves up.
Shown here is a representative electron micrograph of a magnetic spirillum. The flagella are indicated by the black arrows, while the red arrows show the magnetosomes. When attempting this procedure, it's very important to remember that magnetotactic bacteria have very specific oxygen level requirements in order to both grow properly and produce magnetosome.
This protocol shows how to control and monitor the oxygen levels in the growth media in order to achieve the micro conditions preferred by these organisms. Following this method, high concentrations of healthy and highly magnetic cells can be obtained. This way, experiments that require large quantities of bacteria can be performed, such as magnetosome extraction, genomic and genetic studies, or observation of collective motion by microscopy.
The possibility to grow magnetotactic bacteria in the lab is really what has allowed scientists exploring in a systematic way the fascinating biochemical and physical properties of these organisms.
