Editing DSMZ Cells (section)

=== 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.