DSMZ Cells
Thermotoga maritima, ATCC 43589, DSMZ 6858
[edit]ATCC Medium 2114: Thermotoga medium
Storage Temp.
Frozen: 80°C or colder
FreezeDried: 2°C to 8°C
Live Culture: See Propagation Section
Biosafety Level 1
Intended Use
This product is intended for research use only. It is not intended for any animal or human therapeutic or diagnostic use.
Citation of Strain
If use of this culture results in a scientific publication, it should be cited in that manuscript in the following manner: Thermotoga maritima (ATCC ® 43589TM)
Growth Conditions
Temperature: 70°C
Atmosphere: Anaerobic
Propagation Procedure
[edit]ATCC Method
[edit]1. Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flaming the top.
2. If needed exchange the gas in the test tube for 100% N 2 .
3. 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. Let the medium sit at room temperature for 10 to 20 minutes until the resazurin becomes colorless before inoculating.
4. When the Balch tube is ready to be inoculated let the frozen vial thaw at room temperature under a
gentle stream of sterile oxygenfree gas.
5. 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. Plate 0.1 mL of the inoculated culture onto a non selective medium and incubate aerobically at 37°C.
6. Growth should be detected in the broth within 48 to 96 hours. No growth should be detected on the aerobic plate or broth.
ANAEROBIC CONDITIONS:
a. Balch tube refers to a special type of test tube that is designed to be pressurized and is suited for anaerobic work. Balch test tubes can be purchased form Bellco glass (www.bellcoglass.com stock no. 204800150).
b. 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. Most strict anaerobes require this low redox potential for optimal growth.
c. To obtain a fully reduced medium, it is necessary that the medium be anoxic and that a reducing agent be added. Common reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate.
d. Syringes can be made anaerobic by one of two methods.
1. 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.
Mercaptoethane sulfonic acid, also known as Coenzyme M, at a final concentration of 0.05% (Sigma M1511)
can also be used as a reducing agent. It has been found to increase cell recovery. Hydrogen is inhibitory to growth.
Purified genomic DNA of this strain is available as ATCC ® 43589D2TM.
Chinese Method
[edit]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 KH2PO4, 0.5 g K2HPO4, 0.5 g MgCl2·6H2O, 0.33 g KCl, 1.0 g NH4Cl, 0.08 g CaCl2·2H2O, 0.5 g L-cysteine, 0.5 g Na2S·9H2O, 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 MgSO4·7H2O, 0.5 g MnSO4, 1.0 g NaCl, 0.1 g FeSO4·7H2O, 0.1 g CoSO4·7H20, 0.1 g ZnSO4·7H2O, 0.01 g CuSO4·5H2O, 0.01 g AlK(SO4)2, 0.01 g H3BO3 and 0.01 g Na2MoO4·2H2O. The medium was boiled under a stream of oxygen-free N2 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.600 nm was measured. The growth curves of the bacteria were generated by fitting the data to the regression equation A = A0+a/(1+Exp(T1/2−t)/b), in which A is the O.D. value at 600 nm, t is the culture time, A0 is the O.D.600 nm at lag phase and T1/2 is the time when the cultures reach the middle of log phase. The “a” constant is the maximum change in the O.D.600 nm between Amax and A0, while “b” is the time between tlag end and tlog end. When the bacteria reached T1/2, 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.
Chinese Method 2
[edit]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 (Childerset al.,1992) with the same modification as for the common marine medium, (iv) Thermoanaerobacter ethanolicus medium (ATCC 1190) supplemented with 2.7% NaCl (Te+ medium), and (v) a simplified Te+ medium, in which the vitamin solution was omitted and Wolf's mineral solution was replaced with Balch's trace mineral solution (Balchet al.,1979).
The T. maritima basal medium (TMB) contained, per liter, the following: 1.5 g KH2PO4; 4.2 g Na2HPO4·12H2O (22 mM PO43−,); 0.5 g NH4Cl; 0.2 g MgCl2·6H2O (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 Na2S·9H2O; 2.5 g cysteine HCl·H2O. 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 (Braunet al.,1979; 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 ;
4.2 g Na2HPO4 . 12H2O (22 mM PO34 ,);
0.5 g NH4Cl;
0.2 g MgCl2. 6H2O (1 mM);
27 g NaCl;
4 g yeast extract
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.
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
Solid Chinese Method
[edit]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 (Braunet 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.
Method 3
[edit]T. maritima strain MSB8 and M. jannaschii strain DSM2661 were grown at 80°C on medium (BSMII) containing: 40 g/liter sea salts, 5 g/liter tryptone, 3 g/liter yeast extract, 3 g/liter maltose, 3 g/liter piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), 0.5 mg/liter NaSeO3·5H2O, 0.5 mg/liter NiCl2·6H2O, 0.25 g/liter NH4Cl, 10 mg/liter Fe(NH4)2(SO4)2·6H2O, 1.25 g/liter sodium acetate anhydrate, 1 ml/liter 1% Resazurin, 10 ml/liter Wolfe’s trace elements, and 10 ml/liter Wolfe’s trace vitamins solution (all from Sigma Aldrich; components of Wolfe’s vitamin and mineral solutions can 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 ml of medium was dispensed into a 1-liter glass jar sealed with a rubber septum (Corning, Corning, NY) and heated to 80°C. The medium was then sparged through a syringe needle with the appropriate gas (100% N2 or 80%
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
[edit]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
[edit]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 and 10 mM glucose. The of the yeast extract pH medium was adjusted to 6.8
T. neapolitana
[edit]The following medium (modified from ATCC 1977 medium recommended for T. elfeii) was used
to prepare the batch reactors for culture of T. neapolitana: NH4Cl, 1.0 gm: K2HPO4 , 0.3 gm:
KH2PO4 , 0.3 gm: MgCl2 x 6H2 O, 0.2 gm: CaCl2 x 2H2O, 0.1 gm: NaCl, 10.0 gm: KCl, 0.1 gm: Cysteine HCl, 1.0 gm: Yeast Extract, 2.0 gm: Trypticase, 2.0 gm: Vitamin Solution (DSMZ medium 141), 10.0 ml: Trace Element Solution (DSMZ medium 141), 10.0 ml: H 2 O, 1.0 L. The initial pH of the medium was adjusted to 8.5 @ 20°C with NaOH. Trizma base (1.0E-04M) can be added to help maintain pH in an optimal range for longer periods of time during incubation. A primary carbon source (0.25 g/50 ml cellulose, cellobiose, starches, or glucose) was also added.
The medium was always prepared aerobically. Fifty ml. of the above medium was aliquoted into
serum bottles, allowing 110 ml. headspace for gas accumulation. Excess oxygen was removed by heating the batch reactors containing medium and substrate to 100°C while sparging its contents (2-10 min) with nitrogen gas. Sparging time was varied to achieve predetermined concentrations of oxygen of between 1 and 12% in the headspace and then the actual oxygen concentration in the bottle was tested using a gas chromatograph. (The effects of different oxygen concentrations are shown below). Serum bottles were then sealed, capped and sterilized.
Culture conditions
[edit]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 MgCl2.H2 0 and CaCl2 .H20 were reduced to 0.2 and 0.01 g l -1 , respectively, to prevent formation of precipitates. MgCl, CaCl2 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
Batch cultures of Thermotoga maritima, T. neapolitana, and T. elfii were grown in 120 ml serum bottles containing 60 ml medium with shaking at 200 rpm and 80 o C. Before the cultures were inoculated, the bottles were sealed by butyl rubber stoppers and flushed with N 2 .
Cultures of T. neapolitana were also grown in a 3 l Applikon bioreactor containing 2.75 l medium. The reactor was equipped with a Pt100 temperature sensor and an autoclavable pH electrode (Mettler Toledo). Temperature and pH were automatically controlled by a heating blanket wrapped around the reactor wall, and by titration with 1 M NaOH. Cultures were stirred by a 4 bladed Rushton turbine and aerated by headspace or by sparger aeration with nitrogen gas. The exit gas was passed through a condenser at 4 o C to reduce evaporation before it was passed through on-line sensors for quantification of gas-flow rate (AWM3000, Honeywell), and hydrogen (EZT3HYE 'Easy Cal' CiTiceL) and CO 2 (IRcel, City Technology, Portsmouth, UK) in the off-gas.
Two hyperthermophilic bacteria, viz. Thermotoga maritima strain DSM 3109 and Thermotoga
neapolitana strain DSM 4359, were analyzed for H 2 production.
Anaerobic batch cultivations were performed in 120- and 240-mL serum bottles with a working
volume of 25 mL or 50 mL, at constant temperature of 80oC and shaking at 200 rpm. Media were inoculated with a 10% (v/v) pre-culture. Growth on glucose (2.5 g/L) and glycerol (5 g/L) by T. maritima was tested on three different types of media, indicated as medium A1 [23], A2 [23], and M3. M3 media was a modified M2 [21] medium consisting of (amounts are in grams per liter of deionized water): 1.5 g KH 2 PO 4 ; 2.4 g Na 2 HPO 4 ·2H 2 O; 0.5 g NH 4 Cl; 0.2 g MgCl 2 ·6H 2 O; 2.0 mg NiCl 2 ·6H 2 O; 11.9 g HEPES (N-2-hydroxyethylpiperazine-Nƍ-2 ethanesulphonic acid); 2 g yeast extract (YE); 15 ml trace element solution (DSM-TES, see DSMZ medium 141, complemented with Na 2 WO 4 3.00 mg/L); 1.0 mL of vitamin solution (Biotin 2 mg, Nicotinamide 20 mg, p-Aminobenzoic acid 10 mg, Thiamine (Vit.B 1 ) 20 mg, Pantothenic acid 10 mg, Pyridoxamine 50 mg, Cyanocobalamin and Riboflavin 10 mg [24]); 1.0 g/L of cysteine hydrochloride as reducing agent and 1 mg resazurin, which was used as a redox indicator.
Anaerobic conditions were achieved by flushing the headspace of the serum bottles with pure N 2 for 5 min. Growth on glucose (2.5 g/L) and glycerol (5g/L) by T. neapolitana was performed on M3 medium.
The NaCl concentration used was 2.7% (w/v) for T. maritima and 2.0% (w/v) for T. neapolitana. The starting pH of the medium was adjusted to pH 6.9 for T. maritima and pH 7.3 for T. neapolitana with 10 mM NaOH.
The optimal growth parameters (pH, YE concentration) for glycerol (2.5 g/L) conversion by T.
neapolitana was investigated on M3 medium for the pH range of 5-8.5 (YE, 2g/L) and YE concentration range 0- 4 g/L (pH7).
Aquifex pyrophilus (ATCC 51433), DSM 6858
[edit]Propagation Procedure
1. Sterilize the top of the serum bottle by spraying it with 70% ethanol and then flame the top.
2. When the bottle is ready to inoculate, open the vial according to enclosed instructions.
3. For inoculation, use a 1.0 ml syringe tipped with 22 gauge needle. Withdraw 0.5 ml of 2186 and use this to rehydrate the freezedried pellet. Transfer the rehydrated cell into the 2186 broth; from this bottle prepare a 2 nd bottle at a 1:10 dilution. Incubate the bottles at 75 85 o C. Plate 0.1 ml of the inoculated culture onto a non selective medium and incubate aerobically at 37 o C. Inoculate a nonselective anaerobic and aerobic broth and incubate at 70 o C
4. Growth should be detected in the #2186 broth within 24- 48 hours. There should be no growth detected on the aerobic plate. There should be no growth in the nonselective aerobic or anaerobic broth.
| NaCl |
30.00 g |
| MgSO4 . 7H2O |
7.00 g |
| MgCl2 x 6 H20 |
5.50 g |
| KCl |
0.65 g |
| Na2S2O3 |
2.00 g |
| NaBr |
0.10 g |
| NaHCO3 |
2.00 g |
| NH4Cl |
0.15 g |
| K2HPO4 |
0.15 g |
| CaCl2 x 2 H2O |
0.50 g |
| Trace element solution |
10.00 ml |
| Distilled water |
1000.00 ml |
Gas with nitrogen gas for about 20 min and then adjust the pH to 6.5 - 6.8 with H2SO4. Fill 10 ml of medium in serum bottles (120
ml) under a protective CO2 atmosphere and tightly stopper. Exchange the gas phase by a mixture of H2 : CO2 : O2 (300 KPa;
each mixture 79.75 : 19.75 : 0.5 by volume) and autoclave at 121°C for 15 min under pressure. For more details see the original
description (Huber et al. System. Appl. Microbiol. 15:340-351 (1992)).
Trace element solution:
[edit]To 1000.0 ml trace element solution in medium 141 add:
(NH4)2Ni(SO4)2 2.00 g
Na2WO4 10.00 mg
Na2SeO4 10.00 mg
Trace element solution:
Nitrilotriacetic acid 1.50g
MgSO4 x 7H2O 3.00g
MnSO4 x H2O 0.50g
NaCl 1.00g
FeSO4 x 7.H2O 0.10g
CoSO4 x 7.H2O 0.18g
CaCl2 x 2.H2O 0.10g
ZnSO4 x 7.H2O 0.18g
CuSO4 x 5.H2O 0.01g
KAl(SO4)2 x 12.H2O 0.02g
H3BO3 0.01g
Na2MoO4 x 2.H2O 0.01 g
NiCl2 x 6.H2O 0.03 g
Na2SeO3 x 5H2O 0.30 mg
Na2WO4 x 2H2O 0.40 mg
Distilled water 1000.00 ml
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH).
Vitamin solution:
Biotin 2.00 mg
Folic acid 2.00 mg
Pyridoxine-HCl 10.00 mg
Thiamine-HCl x 2H2O 5.00 mg
Riboflavin 5.00 mg
Nicotinic acid 5.00 mg
D-Ca-pantothenate 5.00 mg
Vitamin B 12 0.10 mg
p-Aminobenzoic acid 5.00 mg
Lipoic acid 5.00 mg
Distilled water 1000.00 ml
Medium. The growth medium was the modified SME medium in which yeast and peptone were replaced by
1 g/L yeast extract;
0.5 g/L casaminoacids;
0.4 g/L glucose;
0.4 g/L dextrin (from corn);
0.2 g/L D(+)galactose;
0.2 g/L dextran
0.1 g/L glycogen;
0.2 g/L pyruvate,
0.1 g/L acetate
3 g/Lcolloidal sulphur.
Culture conditions. The medium was sterilized by filtration (Sartroban, 0.22 μm) in 20 l Nalgene bottle containing the colloidal sulphur, previously sterilized by heating twice at 100°C for 30 min on two successive days. The culture was performed at 90°C and pH 6.5. The temperature was regulated and the pH was controlled by addition of either 1 N
NaOH or 1 N HCl solutions. Moreover, the culture was sparged with N 2 (0.1 v.v -1 .min -1 ) to maintain anaerobic conditions, and to eliminate possible volatile fermentation end products that might inhibit the growth of fermentative microorganisms.
Bacto peptone 5 g/L
Yeast extract 1 g/L
Sodium chloride 28 g/L
Magnesium salts 10 ml/L
Solution A 1 ml/L
Solution B 1 ml/L
Solution C 1 ml/L
Resazurin (1 mg/mL) 1 ml/L
Vitamin Solution 0.5 ml/L
Cysteine HCL 0.5 g/L
Magnesium salts
Magnesium sulphate 7H2 O 180g/L
Magnesium chloride 6H2 O 140g/L
Solution A g/L
Trisodium citrate 4
Manganese (II) Sulphate 4H2O 9
Iron (II) Ammonium Sulphate 6H2O 10
Zinc Sulphate 7H2 O 2.5
Copper (II) Sulphate 5H2 O 0.15
Potassium Aluminium Sulphate 12H2O 0.3
Cobalt (II) Chloride 6H2 O 0.3
Nickel (II) Chloride 2.5
Solution B g/L
Calcium Chloride 2H2O 56
Potassium Chloride 16
Strontium Chloride 4
Sodium Bromide 25
Potassium Iodide 10
Solution C g/L
Dipotassium Hydrogen Phosphate 500
Sodium Tungstate 2H2O 3.3
Boric Acid 7.5
Sodium Molybdate 2H2O 0.15
Sodium Selenite 5H2O 0.005
Vitamin Solution (modified from Balch et al) mg/L
Biotin 40
Folic Acid 40
Pyridoxine-HCL 200
Thiamine-HCL 100
Riboflavine 100
Nicotinic Acid 100
DL-Calcium Pantothenate 100
Cyanocobalamin 2
Lipoic Acid 100
The stock solutions A B and C were prepared as 1000× concentrated solutions and the vitamin solution was prepared at 2000× concentration to be used. The vitamin solution was prepared as a 50% v/v ethanol/water mix. The ethanol soluble components were first dissolved in ethanol and the water soluble components dissolved in water and the solutions mixed. In the event of precipitation, the solution was shaken before use.
The components of the medium described above were mixed in the following order when the medium was prepared:
(1) Sodium Chloride
(2) Magnesium Salts
(3) Solution A
(4) Solution B
(5) Solution C
(6) Resazurin
(7) Vitamin Solution
(8) Bacto Peptone
(9) Yeast Extract
(10) Cysteine-HCL
(11) Sodium Hydroxide
When a sulphur-containing medium was required, 30 g/L elemental sulphur was added in addition to the above.