Growing Magnetospirillum Bacteria and Assessing Their Magnetotactic Behavior

Samenvatting

Source: Le Nagard, L., et al. Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1. J. Vis. Exp. (2018).

This video demonstrates how to culture Magnetospirillum bacteria under microaerobic conditions using both liquid and semi-solid media. It highlights the formation of a visible bacterial band at the oxic-anoxic interface and its upward migration as oxygen is depleted. The video also shows how to assess magnetotactic behavior by observing directional swimming in response to a magnetic field.

Protocol

1. Inoculation of magnetotactic bacteria (MTB)

NOTE: The cultures of strains AMB-1, MS-1, and MSR-1 can be obtained commercially (Table of Materials).

CAUTION: Perform all the following steps in sterile conditions, under the flame of a Bunsen burner.

1. Inoculation of strains MSR-1, AMB-1 and MS-1 in liquid medium
   1. Seal an empty 125 mL serum bottle with a butyl-rubber stopper and an aluminum crimp seal. Insert two needles in the bottle through the stopper, and connect a syringe to one of them. Connect the syringe to a cylinder of O₂ through the same type of tubing as the one used for the N₂ station.
   2. Let O₂ flow through the bottle for about 30 min to ensure that all air in the bottle is replaced by O₂. Remove both needles, allowing for a slight overpressure in the bottle, and then autoclave. Allow the bottle to cool down to room temperature before use.
      NOTE: To save time, perform Steps 1.1.1 and 1.1.2 during the medium preparation and autoclave the bottle along with the medium or the stock solutions.
   3. Sterilize the tops of the stoppers of both the fresh medium bottle and the O₂ bottle by applying a few droplets of 70% ethanol solution on top of them and passing them through the flame of a Bunsen burner.
   4. Using a sterile syringe and a needle, extract 1 mL of O₂ from the O₂ bottle and transfer it into the fresh medium bottle. Make sure that the needle tightly fits on the syringe during this step to avoid any air in the syringe.
   5. If using the inoculum from another culture grown in a glass bottle, sterilize the stoppers of both the fresh medium bottle and the older culture bottle by applying a few droplets of 70% ethanol solution on top of them and passing them through the flame of a Bunsen burner. If using the inoculum from a tube of frozen culture, just let it warm up to room temperature with the tube sealed under the flame of a Bunsen burner.
   6. If using the inoculum from another culture grown in a glass bottle, inoculate 1 mL of the older culture into the fresh medium. If using the inoculate from a frozen stock, inoculate only 0.1 mL to dilute the glycerol or dimethyl sulfoxide (DMSO) used in the freezing process. In both cases, use a sterile needle and a sterile syringe.
   7. Incubate the culture at 32 °C and inoculate it into fresh medium after 4 to 7 days.
2. Inoculation of MSR-1 in an O₂ gradient semi-solid medium
   1. Verify that the tube of fresh medium displays a well-defined oxic-anoxic interface (OAI), materialized by a pink-to-colorless interface.
   2. If the inoculum is coming from another O₂ concentration gradient semi-solid culture, harvest the bacteria by pipetting 50 µL 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 (pink/colorless interface), avoiding disturbing the interface. If the inoculum is coming from a frozen culture, proceed in the same way with 100 µL of inoculum instead.
   3. Seal the tube and let the bacteria grow between 25 °C and 30 °C. Transfer into fresh medium using the same procedure before the band of bacteria reaches the surface of the medium.

2. Observation of the Bacteria

1. Sterilize the stopper of the culture bottle by applying 70% ethanol solution on top of it and passing it through the flame of a Bunsen burner. For a semi-solid medium, open the tube under the flame of a Bunsen burner. Use a sterile needle and a sterile syringe to extract the bacteria.
2. Use the hanging drop method8 to ensure that the bacteria are both magnetic and motile.
   NOTE: Phase contrast microscopy gives great results, but is not mandatory. Magnifications ranging from 10X to 60X are suitable.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
AMB-1	American Type Culture Collection (ATCC)	ATCC 700264
MS-1	ATCC	ATCC 31632
MSR-1	Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)	DSM 6361
L-cysteine.HCl.H₂O	Sigma-Aldrich	C7880-100G
1.0 mL syringes	Fischer scientific	B309659
25G x 1 needles	BD	305125
125 mL serum bottles	Wheaton	223748
20 mm aluminum seals	Wheaton	224223-01
20mm E-Z Crimper	Wheaton	W225303
Butyl-rubber stoppers	Bellco Glass, Inc.	2048-11800
Hungate tubes	Chemglass (VWR)	CLS-4208-01
Septum stopper, 13mm, Hungate	Bellco Glass, Inc.	2047-11600
Glass culture Tubes	Corning (VWR)	9826-16X