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.

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.
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#176; elbow fitting at the other end.</li>
	<li>Use tape to attach the structure to a horizontal metal rod placed approximately 30 cm above the bench.</li>
	<li>Connect five pieces of tubing (approximately 20 cm in length) to the free outputs of the fittings installed in Step 1.4.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Preparation of liquid growth medium for MSR-1
	<ol>
		<li>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&#160;clear solution is obtained. Autoclave the solution using a standard cycle (at least 15 min exposure at 121 &#176;C) and then store this stock solution at room temperature in the dark. 
		NOTE: Discard the ferric citrate solution when a precipitate becomes obvious.</li>
		<li>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 &#176;C.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Disconnect the needles and the thin tubing from the N2 station and replace them with clean needles (1 inch,&#160;&#8804; 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Autoclave the bottles. Let them cool down to room temperature overnight and store them at room temperature afterwards.</li>
	</ol>
	</li>
	<li>Preparation of liquid growth medium for AMB-1 and MS-1
	<ol>
		<li>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 &#176;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.</li>
		<li>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 &#176;C.</li>
		<li>After the addition of all chemicals, adjust the pH to 6.75 using 1 M NaOH solution.</li>
		<li>Refer to Steps 2.1.3-2.1.9 for the rest of the protocol.</li>
	</ol>
	</li>
	<li>Preparation of semi-solid growth medium for MSR-1
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Autoclave the solutions prepared in Steps 2.2.1 - 2.2.3 using a standard cycle (at least 15 min exposure at 121 &#176;C). Store them as sterile stock solutions in the dark.</li>
		<li>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.</li>
		<li>Cover the beaker with aluminum foil and autoclave the solution prepared in Step 2.3.5.</li>
		<li>Just before the end of the autoclave cycle, prepare a fresh 4% L-cysteine&#183;HCl&#183;H2O solution by dissolving 0.8 g of L-cysteine&#183;HCl&#183;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 &#176;C.</li>
		<li>After autoclaving, let the medium cool down to 50&#8211;60 &#176;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.</li>
		<li>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 &#176;C or above.</li>
		<li>Leave the tubes undisturbed for several hours until the agar solidifies and the OAI becomes apparent,&#160;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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Inoculation of strains MSR-1, AMB-1 and MS-1 in liquid medium
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Incubate the culture at 32 &#176;C and inoculate it into fresh medium after 4 to 7 days.</li>
	</ol>
	</li>
	<li>Inoculation of MSR-1 in O2 gradient semi-solid medium
	<ol>
		<li>Verify that the tube of fresh medium displays a well-defined OAI, materialized by a pink to colorless interface.</li>
		<li>If the inoculum is coming from another O2 concentration gradient semi-solid culture, harvest the bacteria by pipetting 50 &#181;L 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 &#181;L of inoculum instead.</li>
		<li>Seal the tube and let the bacteria grow between 25 &#176;C and 30 &#176;C. Transfer into fresh medium using the same procedure before the band of bacteria reaches the surface of the medium.</li>
	</ol>
	</li>
</ol>
4. Observation of the Bacteria
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Use transmission electron microscopy (TEM) to observe the cell structure and the magnetosomes in detail8.</li>
</ol>

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The authors have nothing to disclose.

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 ...

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1029px;" width="1028">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">AMB-1</td>
			<td style="width:437px;">American Type Culture Collection (ATCC)</td>
			<td style="width:119px;">ATCC 700264</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">MS-1</td>
			<td>ATCC</td>
			<td>ATCC 31632&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">MSR-1</td>
			<td>Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)</td>
			<td>DSM 6361</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Ferric citrate</td>
			<td>Sigma-Aldrich</td>
			<td>F3388-250G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Trace mineral supplement</td>
			<td>ATCC</td>
			<td>MD-TMS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">KH2PO4</td>
			<td>EMD</td>
			<td>PX1565-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">MgSO4.7 H2O</td>
			<td>EMD</td>
			<td>MX0070-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">HEPES</td>
			<td>BioShop Canada Inc</td>
			<td>HEP001.250</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">NaNO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5506-250G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Yeast extract</td>
			<td>Fischer scientific</td>
			<td>DF210929</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Peptone</td>
			<td>Fischer scientific</td>
			<td>DF0436-17-5</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Potassium L-lactate solution (60%)</td>
			<td>Sigma-Aldrich</td>
			<td>60389-250ML-F</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">D-(-)-Quinic acid</td>
			<td>Sigma-Aldrich</td>
			<td>138622</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">FeCl3.6H2O</td>
			<td>Fischer scientific</td>
			<td>I88-100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Vitamin supplement</td>
			<td>ATCC</td>
			<td>MD-VS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium succinate hexahydrate</td>
			<td>Fischer scientific</td>
			<td>S413-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium L-tartrate dibasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>228729-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium acetate trihydrate</td>
			<td>EMD</td>
			<td>SX0255-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Resazurin</td>
			<td>Difco</td>
			<td>0704-13</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-25G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">K2HPO4</td>
			<td>Caledon</td>
			<td>6620-1-65</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">FeCl2 .4H2O</td>
			<td>Sigma-Aldrich</td>
			<td>44939-250G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium bicarbonate</td>
			<td>EMD</td>
			<td>SX0320-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">NaCl</td>
			<td>Caledon</td>
			<td>7560-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">NH4Cl</td>
			<td>EMD</td>
			<td>1011450500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">CaCl2.2 H2O</td>
			<td>EMD</td>
			<td>1023820500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Agar A</td>
			<td>Bio Basic Canada Inc</td>
			<td>FB0010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">L-cysteine.HCl.H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7880-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">1.0 mL syringes</td>
			<td>Fischer scientific</td>
			<td>B309659</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">25G&#160; x 1 needles</td>
			<td>BD</td>
			<td>305125</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">125 mL serum bottles</td>
			<td>Wheaton</td>
			<td>223748</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">20 mm aluminum seals</td>
			<td>Wheaton</td>
			<td>224223-01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">20mm E-Z Crimper</td>
			<td>Wheaton</td>
			<td>W225303</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Butyl-rubber stoppers</td>
			<td>Bellco Glass, Inc.</td>
			<td>2048-11800</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Hungate tubes</td>
			<td>Chemglass (VWR)</td>
			<td>CLS-4208-01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Septum stopper, 13mm, Hungate</td>
			<td>Bellco Glass, Inc.</td>
			<td>2047-11600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Glass culture Tubes</td>
			<td>Corning (VWR)</td>
			<td>9826-16X</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Hydrochloric acid 36.5-38%, BioReagent</td>
			<td>Sigma-Aldrich</td>
			<td>H1758-100ML</td>
			<td>11.6 - 12 N</td>
		</tr>
	</tbody>
</table>

growing magnetotactic bacteria of the genus magnetospirillum: strains msr-1, amb-1 and ms-1

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.

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...

Watch this Scientific Journal Video about Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1 at JoVE.com

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

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.

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

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

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Research

JoVE Journal

Biology

15.4K Views.  McMaster University. 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. 

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

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...