Editing DSMZ Cells (section)

== Propagation Procedure ==

=== ATCC Method ===

1.&nbsp; Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flaming the top.

<br/>2.&nbsp; If needed exchange the gas in the test tube for 100% N 2 .

<br/>3.&nbsp; If the medium is pink (see discussion about resazurin) add 2.0 mL of reducing agent (3% cysteine, stock solution) per 100 mL of medium.&nbsp; Let the medium sit at room temperature for 10 to 20 minutes  until the resazurin becomes colorless  before inoculating.

4.&nbsp; When the Balch tube is ready to be inoculated let the frozen vial thaw at room temperature under a<br/>gentle stream of sterile oxygenfree gas.

<br/>5.&nbsp; Using a 1.0 mL syringe tipped with a 22 gauge needle, withdraw the cell suspension from the vial, transfer it to the broth, and incubate at 70°C.&nbsp; Plate 0.1 mL of the inoculated culture onto a non selective medium and incubate aerobically at 37°C.

<br/>6.&nbsp; Growth should be detected in the broth within 48 to 96 hours.&nbsp; No growth should be detected on the aerobic plate or broth.<br/>&nbsp;<br/>ANAEROBIC CONDITIONS:<br/>a.&nbsp; Balch tube refers to a special type of test tube that is designed to be pressurized and is suited for anaerobic work.&nbsp; Balch test tubes can be purchased form Bellco glass (www.bellcoglass.com stock no. 204800150).

b.&nbsp; Resazurin is a commonly used redox indicator which is pink when the redox potential is above 50 mv, and colorless when the redox potential is below 110 mv., i.e. highly reducing.&nbsp; Most strict anaerobes require this low redox potential for optimal growth.

<br/>c.&nbsp; To obtain a fully reduced medium, it is necessary that the medium be anoxic and that a reducing agent be added.&nbsp; Common reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate.

<br/>d.&nbsp; Syringes can be made anaerobic by one of two methods.

1.&nbsp; Displace the dead space in the syringe with a sterile oxygenfree



Cells appear as single or paired rods surrounded with a sheathlike structure which protrudes balloonlike beyond the ends of the cells.<br/>Mercaptoethane sulfonic acid, also known as Coenzyme M, at a final concentration of 0.05% (Sigma M1511)<br/>can also be used as a reducing agent.&nbsp; It has been found to increase cell recovery. Hydrogen is inhibitory to growth.<br/>Purified genomic DNA of this strain is available as ATCC ®&nbsp; 43589D2TM.



=== Chinese Method ===

''T. maritima'' strain MSB8 (JCM10099), a gift from Institute of Microbiology, Chinese Academy of Sciences, was activated and cultured at 80°C in modified medium containing (per liter of distilled water) 10.0 g starch, 5.0 g yeast extract, 10.0 g tryptone, 27.0 g NaCl, 0.5 g KH<sub>2</sub>PO<sub>4</sub>, 0.5 g K<sub>2</sub>HPO<sub>4</sub>, 0.5 g MgCl<sub>2</sub>·6H<sub>2</sub>O, 0.33 g KCl, 1.0 g NH<sub>4</sub>Cl, 0.08 g CaCl<sub>2</sub>·2H<sub>2</sub>O, 0.5 g L-cysteine, 0.5 g Na<sub>2</sub>S·9H<sub>2</sub>O, 1.0 mg resazurin and 15.0 ml of trace minerals solution containing (per liter distilled water) 1.5 g nitrilotriacetic acid, 3.0 g MgSO<sub>4</sub>·7H<sub>2</sub>O, 0.5 g MnSO<sub>4</sub>, 1.0 g NaCl, 0.1 g FeSO<sub>4</sub>·7H<sub>2</sub>O, 0.1 g CoSO<sub>4</sub>·7H<sub>2</sub>0, 0.1 g ZnSO<sub>4</sub>·7H<sub>2</sub>O, 0.01 g CuSO<sub>4</sub>·5H<sub>2</sub>O, 0.01 g AlK(SO<sub>4</sub>)<sub>2</sub>, 0.01 g H<sub>3</sub>BO<sub>3</sub> and 0.01 g Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O. The medium was boiled under a stream of oxygen-free N<sub>2</sub> gas and cooled to 80°C. Two milliliters of the activated bacteria were inoculated to 100 ml of medium and then cultured at 60, 70, 80 or 90°C. To monitor the growth rates, the cultured bacteria were withdrawn at time intervals, every 2 hours, and their O.D.<sub>600 nm</sub> was measured. The growth curves of the bacteria were generated by fitting the data to the regression equation A = A<sub>0</sub>+a/(1+Exp(T<sub>1/2</sub>−''t'')/b), in which A is the O.D. value at 600 nm, ''t'' is the culture time, A<sub>0</sub> is the O.D.<sub>600 nm</sub> at lag phase and T<sub>1/2</sub> is the time when the cultures reach the middle of log phase. The “a” constant is the maximum change in the O.D.<sub>600 nm</sub> between A<sub>max</sub> and A<sub>0</sub>, while “b” is the time between t<sub>lag end</sub> and t<sub>log end</sub>. When the bacteria reached T<sub>1/2</sub>, the cells were considered at optimal conditions; the bacteria were harvested by centrifuge at 6,000 g for 15 min at 4°C followed by a wash with ultrapure water. The bacterial cultures at each temperature were prepared in triplicate.


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=== Chinese Method 2 ===

''Thermotoga maritima'' (ATCC 43589) was tested for growth on: (i) modified Luria–Bertani (LB) medium containing 2.7% (w/v) NaCl, (ii) a common marine medium based on one-half-strength Turks Island salts (Merk Index no. 9954) with the modification of 2.7% (w/v) NaCl, 0.5% (w/v) soluble starch and 20 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES) instead of 1,4-piperazine-''N, N''′-bis(2-ethanesulfonic acid) (PIPES) buffer, (iii) TBSY-HEPES medium, which is TB medium ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-6 Childers''et al.'',1992]) with the same modification as for the common marine medium, (iv) ''Thermoanaerobacter ethanolicus'' medium (ATCC 1190) supplemented with 2.7% NaCl (''Te''<sup>''+''</sup> medium), and (v) a simplified ''Te''<sup>''+''</sup> medium, in which the vitamin solution was omitted and Wolf's mineral solution was replaced with Balch's trace mineral solution ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-2 Balch''et al.'',1979]).

The ''T. maritima'' basal medium (TMB) contained, per liter, the following: 1.5 g KH<sub>2</sub>PO<sub>4</sub>; 4.2 g Na<sub>2</sub>HPO<sub>4</sub>·12H<sub>2</sub>O (22 mM PO<sub>4</sub><sup>3−</sup>,); 0.5 g NH<sub>4</sub>Cl; 0.2 g MgCl<sub>2</sub>·6H<sub>2</sub>O (1 mM); 27 g NaCl; 4 g yeast extract (Oxoid Ltd, Basingstoke, UK); 1 mL Balch's trace mineral solution; 1 mg resazurin; 40 mL reducing solution. The reducing solution contained 200 mL NaOH (0.2 M); 2.5 g Na<sub>2</sub>S·9H<sub>2</sub>O; 2.5 g cysteine HCl·H<sub>2</sub>O. When required, thiosulfate was added to TMB from sterile stock solutions to a final concentration of 20 mM. The starting pH of the medium was 7.5 after adding the reducing solution. Anaerobic TMB was prepared in Hungate tubes or serum bottles, either inside the anaerobic chamber (Sheldon Manufacturing Inc., Cornelius, USA), or outside, using the Hungate technique ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-5 Braun''et al.'',1979]; [http://femsle.oxfordjournals.org/content/259/2/254.long#ref-8 Ljungdahl & Wiegel, 1986]). The carbohydrate, including soluble starch, xylan (from birch wood), carboxymethyl cellulose (CMC), glucose or xylose was added to the TMB to a final concentration of 0.5% (w/v) (TMB-starch, TMB-xylan, TMB-CMC, TMB-glucose, TMB-xylose). The cultures were incubated at 80°C after a 2% (v/v) typical inoculation. Samples were taken by sterile injector, and immediately cooled in an ice bath for further study.

In a 10-L fermentor, 7 L of non-reduced steam-sterilized TMB (121°C, 0.1 MPa, 40 min) was flushed with either filtrated nitrogen or carbon dioxide at 0.04 MPa, throughout cell growth. A 140 mL portion of the reducing solution was injected into the medium after its temperature had fallen to 75°C. Sterile soluble starch, glucose or xylose stock solution was added to the desired concentrations. Liquid fermentation was done with the temperature set to 75±1°C, the impeller at 100 r.p.m., and the pH to 6.5, with batch feeding of sterile 5 M NaOH. Samples were taken aseptically and immediately cooled in an ice bath for further study.

Xylan, CMC, HEPES and resazurin were purchased from Sigma (St Louis, MO, USA). All other chemicals of reagent grade were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).



The T. maritima basal medium (TMB) contained, per liter, the following:

1.5 g KH2PO4&nbsp;;

4.2 g Na2HPO4 . 12H2O (22 mM PO<sup>3</sup><sub>4</sub> ,);

0.5 g NH4Cl;

0.2 g MgCl2. 6H2O (1 mM);<br/>27 g NaCl;

4 g yeast extract<br/>1 mL Balch’s trace mineral solution;

1 mg resazurin;

40 mL reducing solution.

The reducing solution contained 200 mL NaOH (0.2 M); 2.5 g Na2S. 9H2O; 2.5 g cysteine HCl. H2O.<br/>When required, thiosulfate was added to TMB from sterile stock solutions to a final concentration of 20 mM. The starting pH of the medium was 7.5 after adding the reducing solution. Anaerobic TMB was prepared in Hungate tubes or serum bottles, either inside the anaerobic chamber


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=== Solid&nbsp; Chinese Method ===

To determine the best agar (Beijing Shuangxuan Co., Beijing, China) concentration for plating efficiency, 1.0%, 1.5% or 2.0% (w/v) of cell-imbedded TMB-starch top agar was tried on 2.0% TMB-starch bottom agar. The sterile non-reduced TMB-starch agar was melted by heating before being reduced and dispensed into autoclaved 100-mL tissue culture flasks in the anaerobic chamber. The flasks were then sealed with butyl rubber stoppers that were sealed with open-top closures to allow manipulation with syringes, and were laid on their flat sides. The inoculum was diluted in a syringe to the desired concentration, and a sample was injected into 4 mL melted-top TMB-starch agar in Hungate tubes. The cell-imbedded top agar was then immediately transferred by syringe into flasks, and was spread equally on the bottom layer. Direct plating was performed as described previously ([http://femsle.oxfordjournals.org/content/259/2/254.long#ref-5 Braun''et al.'',1979]). Inoculated flasks were incubated upside-down at 72°C. The gas-tight flasks with bottom layers, and the Hungate tubes with 4 mL top media were stored at 4°C. Single colonies were selected using a sterile 1 mL syringe equipped with a 15 cm stainless needle with 90° bent and flattened tips. Autoclaving was typically performed at 121°C for 40 min.


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=== Method 3 ===

T. maritima strain MSB8 and M. jannaschii strain DSM2661&nbsp; were&nbsp; grown&nbsp; at&nbsp; 80°C&nbsp; on&nbsp; medium&nbsp; (BSMII)&nbsp; containing:&nbsp; 40&nbsp; g/liter&nbsp; sea salts, 5 g/liter tryptone, 3 g/liter yeast extract, 3 g/liter maltose, 3 g/liter piperazine-1,4-bis(2-ethanesulfonic&nbsp; acid)&nbsp; (PIPES),&nbsp; 0.5&nbsp; mg/liter&nbsp; NaSeO3·5H2O,&nbsp; 0.5 mg/liter NiCl2·6H2O, 0.25 g/liter NH4Cl, 10 mg/liter Fe(NH4)2(SO4)2·6H2O, 1.25&nbsp; g/liter&nbsp; sodium&nbsp; acetate&nbsp; anhydrate,&nbsp; 1&nbsp; ml/liter&nbsp; 1%&nbsp; Resazurin,&nbsp; 10&nbsp; ml/liter Wolfe’s trace elements, and 10 ml/liter Wolfe’s trace vitamins solution (all from Sigma&nbsp; Aldrich;&nbsp; components&nbsp; of&nbsp; Wolfe’s&nbsp; vitamin&nbsp; and&nbsp; mineral&nbsp; solutions&nbsp; can&nbsp; be found in ATCC medium no. 1343). The medium was adjusted to pH 7.0, autoclaved for sterility, and stored at 4°C until use. For H2 inhibition experiments, 400&nbsp; ml&nbsp; of&nbsp; medium&nbsp; was&nbsp; dispensed&nbsp; into&nbsp; a&nbsp; 1-liter&nbsp; glass&nbsp; jar&nbsp; sealed&nbsp; with&nbsp; a&nbsp; rubber septum&nbsp; (Corning,&nbsp; Corning,&nbsp; NY)&nbsp; and&nbsp; heated&nbsp; to&nbsp; 80°C.&nbsp; The&nbsp; medium&nbsp; was&nbsp; then sparged&nbsp; through&nbsp; a&nbsp; syringe&nbsp; needle&nbsp; with&nbsp; the&nbsp; appropriate&nbsp; gas&nbsp; (100%&nbsp; N2 or&nbsp; 80%<br/>H2–20% CO2) for 1 minute and reduced with 4 ml of a 10% sodium sulfide–10% L-cysteine HCl solution (pH 8.0) (Sigma-Aldrich). A 1% inoculum of log-phase cells was used. Culture bottles were agitated at 100 rpm in an oil bath at 80°C



=== Method 4 ===

Thermotoga maritima DSM 3109, T. neapolitana DSM 4359, and T. elfii DSM 9442 were grown in a modified version of the ATCC 1977 medium (see van Ooteghem et al. 2002) containing 1 g cysteine.HCl l -1 as reducing agent, and glucose, xylose, arabinose or glycerol as carbon substrate. The medium also contained 2 g yeast extract l -1 and 2 g peptone l -1 , which are needed by these bacteria. Potassium phosphate was increased from 0.6 to 6.3 g l -1 to increase the buffering capacity of the growth medium (Eriksen et al. 2008), while the concentrations of MgCl.H 2 0 and CaCl 2 .H 2 0 were reduced to 0.2 and 0.01 g l -1 , respectively, to prevent formation of precipitates. MgCl, CaCl 2 and the carbon source (glucose, xylose, arabinose, or glycerol) were autoclaved in a separate solution before mixed with a solution containing the remaining medium constituents. A few crystals of resazurin were added as redox indicator.



=== Method 5 ===

Thermotoga medium per liter: 0.96 g PIPES buffer, 2.4 g NaCl, 0.16g MgSO4.7HO, 0.016g NH4Cl, 0.04g CaCl2.2H2O, 0.16 g yeast extract&nbsp; and 10 mM glucose. The of the yeast extract pH medium was adjusted to 6.8