Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Summary

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O₂ concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Abstract

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe₃O₄) or greigite (Fe₃S₄) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O₂ concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O₂ concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Introduction

Magnetotactic bacteria (MTB) represent a wide range of Gram-negative prokaryotes ubiquitous in freshwater and marine aquatic habitats¹. These bacteria share the ability to produce magnetic crystals made of either magnetite (Fe₃O₄) or greigite (Fe₃S₄), which are in most cases assembled into chains inside the cells. This particular structural motif is due to the presence of several specific proteins acting both in the cytoplasm of the bacteria and on the lipid membrane that surrounds each crystal². Each individual crystal and its surrounding membrane vesicle is called a magnetosome and is ranging in size from about 30 to 50 nm in Magnetospirillum species³. Because of the chain arrangement of magnetosomes, these bacteria possess a permanent magnetic dipole moment that makes them align passively with externally applied magnetic fields. Therefore, these bacteria actively swim along magnetic field lines, acting as self-propelled micro-compasses presumably to more effectively locate the most favorable conditions (e.g., O₂ concentration) for growth.

An interesting property of MTB is their ability to regulate both the chemistry and the crystallography of their magnetosome crystals. Most strains produce relatively high purity crystals of either magnetite or greigite, although some biomineralize both minerals⁴. In all cases, the bacteria are able to precisely control the size and the shape of their single magnetic domain crystals. This explains why a great amount of research is undertaken to develop a better understanding of how MTB perform this biomineralization process. Understanding this process might allow the researchers to tailor-make magnetic nanocrystals for many commercial and medical applications.

A substantial obstacle to extensive research on MTB has been the difficulty of growing them in the laboratory. Most species, including the strains used in this work, are obligately microaerophilic when grown with O₂ as a terminal electron acceptor. This explains why these bacteria are most often found at the transition zone between oxic and anoxic conditions (the oxic-anoxic interface, OAI). This clearly shows that MTB have precise O₂ concentration requirements which obviously needs to be taken into account when devising growth media for these organisms. Moreover, the great existing diversity of MTB implies that different strains will need different types of chemical gradients and nutrients to achieve optimal growth.

In this work, we describe the methods for growing three of the most widely studied MTB: Magnetospirillum magneticum (strain AMB-1), M. magnetotacticum (MS-1) and M. gryphiswaldense (MSR-1). These species phylogenetically belong to the Alphaproteobacteria class in the Proteobacteria phylum, are helical in morphology and possess a polar flagellum at each end of the cell. We provide the protocols for growing strain MSR-1 in both liquid and O₂ concentration gradient semi-solid media, based on previously published medium recipes^5,6. We also present a detailed protocol for growing strains AMB-1 and MS-1 in modified Magnetic Spirillum Growth Medium (MGSM)⁷.

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Protocol

1. Installation of the N₂ Station

NOTE: Choose the inner diameter of the tubing so that it can be connected to the gas tank with minimum leakage and so that the cylinder of a 1 mL plastic syringe tightly fits in this tubing. An illustration of the complete N₂ gassing station is provided in Figure 1.

1. Safely install a N₂ gas tank close to a bench on which there is enough space to set up the N₂ station (a length of approximately 50 cm).
2. Connect to the tank a piece of tubing 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.
3. To build a station capable of bubbling five bottles of medium at the same time, cut four pieces of tubing of approximately 5 cm in length.
4. Assemble the pieces of tubing in a line with three three-way T-shaped plastic fittings. Connect one end of this line to the piece of tubing at the output of the N₂ tank through an extra T-shaped fitting. Add a 90° elbow fitting at the other end.
5. Use tape to attach the structure to a horizontal metal rod placed approximately 30 cm above the bench.
6. Connect five pieces of tubing (approximately 20 cm in length) to the free outputs of the fittings installed in Step 1.4.
7. Remove the pistons from five 1.0 mL plastic syringes and cut the larger end of these syringes (i.e., the opposite side to the needle), keeping only the graduated part. Fill these syringes with cotton, not too tightly.
8. Insert the syringes in the 20 cm long vertical pieces of tubing and use soapy water to ensure that there is no leakage when N₂ is flowing.
9. Remove the caps of five 25 G needles (0.5 mm x 25 mm) and insert these needles into the 10 cm pieces of thin tubing. Ensure that the needles tightly fit in the tubing.
   CAUTION: There is a risk of stabbing during this step. Do it slowly and carefully.
10. Attach the needles prepared in Step 1.9 to the syringes of the N₂ station. Ensure that N₂ is flowing through all five lines and keep the station on stand-by.

2. Growth Medium Preparation

NOTE: It is possible to adjust the amount of medium prepared, as explained in Steps 2.1.2, 2.2.2 and 2.3.8. The amount of water and chemicals used just needs to be proportionally adjusted. The role of all medium components is described in Supplementary Table 1.

1. Preparation of liquid growth medium for MSR-1
   1. Prepare a 10 mM ferric citrate solution by adding 0.245 g of ferric citrate to 100 mL of distilled deionized water. Heat and stir to dissolve, until a yellow to orange clear solution is obtained. Autoclave the solution using a standard cycle (at least 15 min exposure at 121 °C) and then store this stock solution at room temperature in the dark.
      NOTE: Discard the ferric citrate solution when a precipitate becomes obvious.
   2. In a beaker containing 1 L of distilled deionized water, add the following in order while stirring: 1.0 mL of the trace mineral supplement solution, 0.1 g of KH₂PO₄, 0.15 g of MgSO₄.7 H₂O, 2.38 g of HEPES, 0.34 g of NaNO₃, 0.1 g of yeast extract, 3.0 g of soy bean peptone, 4.35 mL of potassium lactate (60% w/w solution) and 5 mL of the 10 mM Fe(III) citrate stock solution.
      NOTE: Adjust the quantities proportionally if a smaller amount of growth medium is needed. For a N₂ station capable of bubbling five bottles at the same time, such as the one built in Step 1 of this protocol, prepare 300 mL of medium.
      CAUTION: Trace mineral and Fe(III) citrate stock solutions need to be kept sterile. To avoid contamination, use standard sterile technique when using them (flame open tops of bottles using a Bunsen burner) and use sterile pipette tips for dispensing. Store the mineral solution in a refrigerator at 4 °C.
   3. After the addition of all chemicals, adjust the pH to 7.0 with 1 M NaOH solution. Dispense the freshly prepared medium into 125 mL serum bottles. Pour 60 mL of medium in each bottle.
   4. Bubble N₂ into the medium for 30 min to remove the dissolved O₂, using the small tubing connected to the N₂ station described in Step 1. Place a butyl-rubber stopper on top of each bottle, leaving a small opening to allow the excess gas to exit the bottle.
      CAUTION: Foam might form while bubbling with N₂. Adjust the gas flow accordingly to avoid foam production.
   5. 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.
   6. Disconnect the needles and the thin tubing from the N₂ station and replace them with clean needles (1 inch, ≤ 23G). Adjust the valves of the N₂ tank so that a gentle continuous flow of gas exits the tank (about 50 mL/min).
      Note: Needles larger than 23G may leave permanent unsealable holes in the stopper.
   7. Insert one of the needles connected to the N₂ 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 N₂ flow for about 30 min to replace the air in the bottles by N₂.
   8. Disconnect one bottle from the N₂ 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.
      CAUTION: To prevent O₂ from re-entering the bottles of growth medium after Step 2.1.4, perform Steps 2.1.4 - 2.1.8 in quick succession. If all bottles cannot be connected to the N₂ station at the same time, proceed with Steps 2.1.4-2.1.8 for the first set of bottles and then repeat these steps for the remaining bottles.
   9. Autoclave the bottles. Let them cool down to room temperature overnight and store them at room temperature afterwards.
2. Preparation of liquid growth medium for AMB-1 and MS-1
   1. Prepare a 10 mM ferric quinate solution. First dissolve 0.19 g of quinic acid in 100 mL of distilled deionized water, then add 0.27 g of FeCl₃.6H₂O. Stir to dissolve, until a dark red, clear solution is obtained. Autoclave the solution using a standard cycle (at least 15 min exposure at 121 °C).
      NOTE: Store the ferric quinate solution at room temperature in the dark as a sterile stock solution. Discard the solution when a precipitate becomes obvious.
   2. In a beaker containing 1 L of distilled deionized water, add the following in order while stirring: 10.0 mL of the vitamin supplement solution, 5.0 mL of the trace mineral supplement solution, 0.68 g of KH₂PO₄, 0.848 g of sodium succinate dibasic hexahydrate, 0.575 g of di-Sodium tartrate dihydrate, 0.083g of Sodium acetate trihydrate, 0.45mL of 0.1% aqueous Resazurin, 0.17 g of NaNO₃, 0.04 g of ascorbic acid and 3.0 mL of the 10 mM Fe(III) quinate stock solution.
      NOTE: Adjust the quantities proportionally if a smaller amount of growth medium is needed. For a N₂ station capable of bubbling five bottles at the same time, such as the one built in Step 1 of this protocol, prepare 300 mL of medium.
      CAUTION: Vitamin, trace mineral and Fe(III) quinate stock solutions need to be kept sterile. To avoid contamination, use standard sterile technique and sterile pipette tips when dispensing. Store the mineral and vitamin solutions in a refrigerator at 4 °C.
   3. After the addition of all chemicals, adjust the pH to 6.75 using 1 M NaOH solution.
   4. Refer to Steps 2.1.3-2.1.9 for the rest of the protocol.
3. Preparation of semi-solid growth medium for MSR-1
   1. Prepare a 0.5 M phosphate buffer solution pH 7.0 by dissolving 3.362 g of K₂HPO₄ and 4.178 g of KH₂PO₄ in 100 mL of distilled deionized water. Check if the pH is 7.0 and adjust the pH slightly with KH₂PO₄ or NaOH if needed. Store the solution in a sealed glass bottle (preferable to plastic in order to avoid oxygen exchange).
   2. Prepare 100 mL of a 0.02 M hydrochloric solution. Add 0.2 g of FeCl₂.4H₂O to this solution and stir to dissolve in order to obtain a 10 mM iron chloride solution. Store the solution in the dark in a sealed glass bottle.
   3. Prepare a 0.8 M sodium bicarbonate solution by dissolving 6.72 g of NaHCO₃ in 100 mL of distilled deionized water. Store the solution in a sealed glass bottle.
   4. Autoclave the solutions prepared in Steps 2.2.1 - 2.2.3 using a standard cycle (at least 15 min exposure at 121 °C). Store them as sterile stock solutions in the dark.
   5. In a beaker containing 1 L of distilled deionized water, add the following in order while stirring: 5 mL of the trace mineral supplement solution, 0.2 mL of 1% aqueous resazurin solution, 0.4 g of NaCl, 0.3 g of NH₄Cl, 0.1 g of MgSO₄.7H₂O, 0.05 g of CaCl₂.2H₂O, 1 g of sodium succinate, 0.5 g of sodium acetate, 0.2 g of yeast extract and 1.6 g of agar.
   6. Cover the beaker with aluminum foil and autoclave the solution prepared in Step 2.3.5.
   7. Just before the end of the autoclave cycle, prepare a fresh 4% L-cysteine·HCl·H₂O solution by dissolving 0.8 g of L-cysteine·HCl·H₂O in 20 mL of distilled deionized water. Neutralize the solution to pH 7.0 with 5 M NaOH solution.
      CAUTION: it is important that the cysteine solution is prepared fresh to avoid oxidation of the cysteine. Store the mineral solution in a refrigerator at 4 °C.
   8. After autoclaving, let the medium cool down to 50–60 °C and bring the beaker under the flame of a Bunsen burner. Remove the aluminum foil and quickly add the following in order while gently stirring: 0.5 mL of the vitamin solution, 2.8 mL of the sterile phosphate buffer stock solution, 3 mL of the sterile iron chloride stock solution, 1.8 mL of the sterile sodium bicarbonate stock solution and 10 mL of filter-sterilized cysteine solution.
      NOTE: Adjust the quantities proportionally if a smaller amount of growth medium is needed. It is usually convenient to prepare a batch of 120 mL of medium.
      CAUTION: All solutions must remain sterile for future use. Perform Step 2.3.8 using standard sterile technique and sterile pipette tips when dispensing.
   9. After the addition of all chemicals, transfer the warm medium into 16 mL sterile screw-cap Hungate tubes. Transfer 12 mL of the medium into each tube and seal the tubes.
      CAUTION: To avoid contamination, perform Step 2.3.9 under the flame of a Bunsen burner. Perform it before the agar solidifies, while the medium is at 40 °C or above.
   10. Leave the tubes undisturbed for several hours until the agar solidifies and the OAI becomes apparent, materializing as a pink to colorless interface, approximately 1 to 3 cm below the surface of the medium.
       NOTE: The medium should slowly turn colorless in the tube. The amount of time needed for this transition is variable and depends on the amount of O₂ initially dissolved in the medium.

3. Inoculation of 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 prepared as in Step 1.7 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 3.1.1 and 3.1.2 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 O₂ gradient semi-solid medium
   1. Verify that the tube of fresh medium displays a well-defined 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.

4. 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 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.
3. Use transmission electron microscopy (TEM) to observe the cell structure and the magnetosomes in detail⁸.

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Results

Successful preparation of the growth media can be assessed as follows. At the end of the process, clear solutions (i.e., free of any precipitate) should be obtained (this is true for both the liquid media and the O₂ gradient semi-solid medium). A picture displaying the expected aspect of MSR-1 liquid medium before inoculation can be seen in Figure 2a. A successful O₂ concentration gradient semi-solid medium is signaled by the fo...

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Discussion

The specific O₂ concentration requirements of MTB make them non-trivial to grow in the laboratory. A key step of the protocol for liquid medium is the initial removal of all O₂ from the medium in order to control the final concentration by adding a definite volume of O₂, just before inoculation. It has been shown that MSR-1 grows under almost fully aerobic conditions, however, the magnetism of the cells is drastically reduced. The results from the same study showed that strains AMB-1 and ...

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Materials

Name	Company	Catalog Number	Comments
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
Ferric citrate	Sigma-Aldrich	F3388-250G
Trace mineral supplement	ATCC	MD-TMS
KH2PO4	EMD	PX1565-1
MgSO4.7 H2O	EMD	MX0070-1
HEPES	BioShop Canada Inc	HEP001.250
NaNO3	Sigma-Aldrich	S5506-250G
Yeast extract	Fischer scientific	DF210929
Peptone	Fischer scientific	DF0436-17-5
Potassium L-lactate solution (60%)	Sigma-Aldrich	60389-250ML-F
D-(-)-Quinic acid	Sigma-Aldrich	138622
FeCl3.6H2O	Fischer scientific	I88-100
Vitamin supplement	ATCC	MD-VS
Sodium succinate hexahydrate	Fischer scientific	S413-500
Sodium L-tartrate dibasic dihydrate	Sigma-Aldrich	228729-100G
Sodium acetate trihydrate	EMD	SX0255-1
Resazurin	Difco	0704-13
Ascorbic acid	Sigma-Aldrich	A4544-25G
K2HPO4	Caledon	6620-1-65
FeCl2 .4H2O	Sigma-Aldrich	44939-250G
Sodium bicarbonate	EMD	SX0320-1
NaCl	Caledon	7560-1
NH4Cl	EMD	1011450500
CaCl2.2 H2O	EMD	1023820500
Agar A	Bio Basic Canada Inc	FB0010
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
Hydrochloric acid 36.5-38%, BioReagent	Sigma-Aldrich	H1758-100ML	11.6 - 12 N