Editing DSMZ Cells (section)

== Aquifex pyrophilus&nbsp;&nbsp; (ATCC 51433), DSM 6858 ==

Propagation Procedure<br/>1. Sterilize the top of the serum bottle by spraying it with 70% ethanol and then flame the top.<br/>2. When the bottle is ready to inoculate, open the vial according to enclosed instructions.<br/>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.&nbsp; Transfer the rehydrated cell into the 2186 broth; from this bottle prepare a 2 nd&nbsp; bottle at a 1:10 dilution. Incubate the bottles at 75  85 o C.&nbsp; 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<br/>4. Growth should be detected in the #2186 broth within 24- 48 hours.&nbsp; There should be no growth detected on the aerobic plate.&nbsp; There should be no growth in the nonselective aerobic or anaerobic broth.

{| style="width: 500px" border="1" cellpadding="1" cellspacing="1"
|-
| NaCl<br/>
| 30.00 g<br/>
|-
| MgSO4 . 7H2O<br/>
| 7.00 g<br/>
|-
| MgCl2 x 6 H20<br/>
| 5.50 g<br/>
|-
| KCl<br/>
| 0.65 g<br/>
|-
| Na2S2O3<br/>
| 2.00 g<br/>
|-
| NaBr<br/>
| 0.10 g<br/>
|-
| NaHCO3<br/>
| 2.00 g<br/>
|-
| NH4Cl<br/>
| 0.15 g<br/>
|-
| K2HPO4<br/>
| 0.15 g<br/>
|-
| CaCl2 x 2 H2O<br/>
| 0.50 g<br/>
|-
| Trace element solution<br/>
| 10.00 ml<br/>
|-
| Distilled water<br/>
| 1000.00 ml<br/>
|}

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<br/>ml) under a protective CO2 atmosphere and tightly stopper. Exchange the gas phase by a mixture of H2&nbsp;: CO2&nbsp;: O2 (300 KPa;<br/>each mixture 79.75&nbsp;: 19.75&nbsp;: 0.5 by volume) and autoclave at 121°C for 15 min under pressure. For more details see the original<br/>description (Huber et al. System. Appl. Microbiol. 15:340-351 (1992)).

=== Trace element solution: ===

To 1000.0 ml trace element solution in medium 141 add:<br/>(NH4)2Ni(SO4)2 2.00 g<br/>Na2WO4 10.00 mg<br/>Na2SeO4 10.00 mg

'''Trace element solution''':<br/>Nitrilotriacetic acid 1.50g<br/>MgSO4 x 7H2O 3.00g<br/>MnSO4 x H2O 0.50g<br/>NaCl 1.00g<br/>FeSO4 x 7.H2O 0.10g<br/>CoSO4 x 7.H2O 0.18g<br/>CaCl2 x 2.H2O 0.10g<br/>ZnSO4 x 7.H2O 0.18g<br/>CuSO4 x 5.H2O 0.01g<br/>KAl(SO4)2 x 12.H2O 0.02g<br/>H3BO3 0.01g<br/>Na2MoO4 x 2.H2O 0.01 g<br/>NiCl2 x 6.H2O 0.03 g<br/>Na2SeO3 x 5H2O 0.30 mg<br/>Na2WO4 x 2H2O 0.40 mg<br/>Distilled water 1000.00 ml<br/>First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH).

'''Vitamin solution''':<br/>Biotin 2.00 mg<br/>Folic acid 2.00 mg<br/>Pyridoxine-HCl 10.00 mg<br/>Thiamine-HCl x 2H2O 5.00 mg<br/>Riboflavin 5.00 mg<br/>Nicotinic acid 5.00 mg<br/>D-Ca-pantothenate 5.00 mg<br/>Vitamin B 12 0.10 mg<br/>p-Aminobenzoic acid 5.00 mg<br/>Lipoic acid 5.00 mg<br/>Distilled water 1000.00 ml



Medium. The growth medium was the modified SME medium&nbsp; in which yeast and peptone were replaced by<br/>1 g/L yeast extract;<br/>0.5 g/L casaminoacids;<br/>0.4 g/L glucose;<br/>0.4 g/L dextrin (from corn);<br/>0.2 g/L D(+)galactose;<br/>0.2 g/L dextran<br/>0.1 g/L glycogen;<br/>0.2 g/L pyruvate,<br/>0.1 g/L acetate<br/>3 g/Lcolloidal sulphur.<br/>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<br/>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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5 g/L<br/>Yeast extract&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 g/L<br/>Sodium chloride&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 28 g/L<br/>Magnesium salts&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10 ml/L<br/>Solution A&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 ml/L<br/>Solution B&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 ml/L<br/>Solution C&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 ml/L<br/>Resazurin (1 mg/mL)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 ml/L<br/>Vitamin Solution&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.5 ml/L<br/>Cysteine HCL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.5 g/L

Magnesium salts<br/>Magnesium sulphate 7H2 O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 180g/L<br/>Magnesium chloride 6H2 O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 140g/L

<br/>Solution A&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; g/L<br/>Trisodium citrate&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4<br/>Manganese (II) Sulphate 4H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9<br/>Iron (II) Ammonium Sulphate 6H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10<br/>Zinc Sulphate 7H2 O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.5<br/>Copper (II) Sulphate 5H2 O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.15<br/>Potassium Aluminium Sulphate 12H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.3<br/>Cobalt (II) Chloride 6H2 O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.3<br/>Nickel (II) Chloride&nbsp;&nbsp;&nbsp; 2.5

Solution B&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; g/L<br/>Calcium Chloride 2H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 56<br/>Potassium Chloride 16<br/>Strontium Chloride&nbsp; 4<br/>Sodium Bromide&nbsp;&nbsp;&nbsp;&nbsp; 25<br/>Potassium Iodide&nbsp;&nbsp; 10



Solution C&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; g/L<br/>Dipotassium Hydrogen Phosphate&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 500<br/>Sodium Tungstate 2H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.3<br/>Boric Acid&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 7.5<br/>Sodium Molybdate 2H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.15<br/>Sodium Selenite 5H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.005

Vitamin Solution (modified from Balch et al)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; mg/L<br/>Biotin&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 40<br/>Folic Acid&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 40<br/>Pyridoxine-HCL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 200<br/>Thiamine-HCL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100<br/>Riboflavine&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100<br/>Nicotinic Acid&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100<br/>DL-Calcium Pantothenate&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100<br/>Cyanocobalamin&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2<br/>Lipoic Acid&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 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.
[[Category:Cell Lines]]