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.
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.
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 recipes5,6. We also present a detailed protocol for growing strains AMB-1 and MS-1 in modified Magnetic Spirillum Growth Medium (MGSM)7.
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.
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.
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.
4. Observation of the Bacteria
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 formation of an OAI after a few hours, indicated by the presence of 1–3 cm of pink medium at the top of the tube (Figure 3, tube A). The rest of the medium should be colorless. An all-pink medium before inoculation is a sign of complete oxidation of the resazurin and therefore of an unsuccessful preparation of the O2 concentration gradient.
Successful growth in liquid medium can be checked about 48 h after inoculation (or after a few days if the inoculum is coming from a frozen culture) simply by examining the cultures for turbidity using a light source or a spectrophotometer. Successful growth is confirmed by turbidity proving that the bacteria actually grew (Figure 2b). If the medium remains clear after 48 h, the bacteria have not grown. For a liquid medium culture growing normally, the presence of bacteria synthesizing magnetosomes can be checked after 48 h by placing the bottle of medium on a magnetic stir plate and by illuminating it with a flashlight. At a low stirring speed, the medium should "flash", looking alternately darker and brighter depending on the orientation of the stirring magnet with respect to the position of the experimentalist. For a non-magnetic culture, the intensity of light scattered by cells towards the experimentalist will remain constant.
Successful growth in semi-solid medium is indicated by the formation of a microaerophilic band of bacteria at the pink/colorless interface. Due to the consumption of O2 by the bacteria and the changes in the O2 gradient, the band will slowly migrate to follow the OAI (Figure 3, tube B).
The hanging drop method enables the observer to check that the bacteria are both magnetic and motile. After a few minutes, MTB should concentrate at the edge of the hanging drop, proving that they actively swim along the magnetic field lines (Figure 4a). To ensure that the cells are magnetic, flip the orientation of the magnet sitting next to the drop. The bacteria should start swimming towards the opposite edge (Figure 4b). Finally, the shape of the magnetosomes can be checked with TEM. Cuboctahedral crystals of about 30–45 nm in diameter should be observed9. These crystals are normally arranged in one long chain or multiple shorter chains in the species studied here (Figure 5).
Figure 1: Illustration of the N2 gassing station. (a) Schematic representation. (b) Picture of the setup. A successful station will have the appropriate length of large and thin tubing so that the end of the thin tubing described in Step 1.9 of the protocol remains immersed in the medium during the gas bubbling step. The flow of gas should also be adjustable at all times, to ensure that all O2 is removed and that the medium does not spill. Please click here to view a larger version of this figure.
Figure 2: Bottles of liquid growth medium before and after inoculation. (a) Clear MSR-1 medium before inoculation. (b) Turbid MSR-1 medium after 3 days of successful growth. Please click here to view a larger version of this figure.
Figure 3: Tubes of O2 gradient semi-solid medium before (tube A) and 10 days after (tube B) inoculation. The band of bacteria in tube B is indicated by a red arrow.
Figure 4: Typical results of a hanging drop experiment at 20X magnification using phase contrast microscopy. (a) MTB swim towards the south pole of a magnet and accumulate at the liquid/air interface. (b) 1 min after flipping the magnet, most cells have left the field of view.
Figure 5: TEM observation of an AMB-1 cell. Magnetosome chains are indicated by red arrows. The two flagella are indicated by black arrows.
Supplementary Table 1. Please click here to download this table.
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 MS-1 do not grow under fully aerobic conditions6. For the semi-solid culture of MSR-1, all O2 is first reduced by the cysteine solution and then the O2 concentration gradient appears, leading to the formation of the OAI. If a culture does not grow well or is not magnetic, the O2 concentration should be the first thing to check.
Semi-solid growth medium is an interesting choice for isolating unknown MTB or growing the species that are highly sensitive to both O2 and redox gradients, as it ensures the existence of stable gradients and allows the bacteria to position themselves at the location offering the best growth conditions. Liquid medium is more convenient to work with when higher volumes are needed (e.g., for DNA extraction), or when further analysis needs to be conducted on the cells (e.g., magnetosome studies) as it allows for an easier separation of the cells from the growth medium. The agar in semi-solid medium might indeed interfere with the cell harvesting technique.
In some cultures, the cells sometimes become non-motile, likely due to spontaneous mutations. Non-motile cells survive and grow in the growth medium since they do not need to swim to find nutrients necessary for their survival. The standard race-track method10 can then be used to recover a motile culture since only motile cells can swim down the race-track. The loss of the magnetic phenotype can also happen in the culture even if the O2 concentration is optimal, again due to spontaneous mutations11. The best solution in that case is to start a new culture by inoculating frozen stocks of the wild-type bacteria into fresh medium. Alternatively, the race-track technique can also allow the recovery of a magnetic culture.
It is essential to avoid contamination of the culture by other organisms and therefore most of the protocol must be performed using sterile techniques. Manipulating under the flame of a Bunsen burner usually gives satisfactory results in keeping aseptic conditions. However, if the culture is grown in a contamination-prone environment, additional precautions should be taken, such as working in a laminar flow hood when preparing the medium and inoculating the bacteria. It should be noted that the risk of contamination is higher in semi-solid medium as the tubes need to be opened every time the cells are removed.
These protocols enable the growth of enough bacteria to perform many different types of experiments, such as physical studies based on optical microscopy12,13, electron microscopy imaging14,15, X-ray spectromicroscopy analyses16, genomic and protein studies17,18. If necessary, higher concentrations of bacteria in suspension can be achieved by centrifugation. In addition, magnetosomes can be extracted and purified for further applications from cell pellets or dense cell suspensions obtained by centrifugation19.
Growth media formulations are usually based on the same governing principles (see Supplementary Table 1 for a summary of the list of the role of each component in the growth media described here). A carbon source must be available to the bacteria, via organic acids (e.g., succinate, acetate, tartrate, lactate) or inorganic compounds such as bicarbonate. The choice of a suitable carbon source depends on the metabolism of the bacteria. Nitrogen present in DNA and proteins, and phosphorus (DNA, membranes) are also required and provided by NaNO3, NH4Cl and KH2PO4 in the recipes described here. A buffer (HEPES, phosphate buffer) is necessary to resist pH changes. The trace mineral supplement solution contains cofactors for enzymes, and the vitamins can be used by the bacteria as coenzymes or as functional groups for certain enzymes. Yeast extract and soy bean peptone provide a source of amino acids and salts used for protein production. Since MTB are redox sensitive, one or several reducing agents must often be added to the medium (e.g., cysteine, ascorbic acid, lactate). A major source of iron (e.g., ferric citrate, ferric quinate, iron chloride) is needed in the medium for biomineralization. Finally, a small amount of O2 is required for microaerophilic bacteria as terminal electron acceptor. Obligate anaerobic MTB use other compounds such as nitrate or nitrous oxide instead.
The principal limitation of these protocols is that there is no guarantee that they will work for other species of MTB. Strains AMB-1, MS-1 and MSR-1 are all freshwater MTB and therefore the recipes described in this article cannot be used to grow marine magnetospirilla such as Magnetospira thiophila strain MMS-1, Magnetospira strain QH-2, or other marine MTB, which require higher salt concentrations. However, to grow other strains of MTB, and for the isolation of new strains, the general methods presented in this article for both liquid and semi-solid media can be used to prepare media with different recipes.
The authors have nothing to disclose.
We thank Richard B. Frankel for his help with MTB cultures, Adam P. Hitchcock and Xiaohui Zhu for their support while setting up the MTB cultures at McMaster University, and Marcia Reid for training and access to the electron microscopy facility (McMaster University, Faculty of Health Sciences). This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the US National Science Foundation.
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 |