TITLE: Anaerobic	Media	Preparation	Protocol
AUTHORS: Orkun Soyer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a generic protocol for the preparation of </span><span style = "font-style:italic;">any</span><span> strictly anaerobic media. The exact media composition is assumed to be known by the user of this protocol (for example, we routinely use this protocol together with the media sheet; OSM01_01, available from OSS Lab website).</span></div></div>

[STEPS]
?. [Prep. and mixing stage]
In  dissolve the salt mix of the medium, while stirring the liquid on a stirrer plate.
[milliq-H2O]
This dissolved salt mix can be stored without any problem (e.g. contamination would not be expected as it does not contain any carbon source, most salts should be stable, etc.)
?. [Prep. and mixing stage]
Add the buffer solution (e.g. NaHCO3, Na2CO3).
?. [Prep. and mixing stage]
Add the trace metal solution.
?. [Prep. and mixing stage]
Add the carbon source (as defined by your media sheet and/or microorganism).
Depending on the nature of the C-source, you might want to add it after the degassing stage. For example, methanol, that can be used for M.barkeri, could have high volatility and be lost during degassing.
?. [Prep. and mixing stage]
Add , an indicator of redox level, if it was not already contained in the salt mix or buffer solution.
[Resazurin]
?. [Prep. and mixing stage]
Fill up to  with milliq-H2O. Make sure you stop stirring, while topping up to , as the stirring can cause you to misjudge the volume.
1 L
1 L
?. [Prep. and mixing stage]
Place media flask on a pH-meter and adjust the pH with HCl / NaOH (or KOH in case of sodium-free media) to the level required by the specific media.
Once the pH is adjusted, we are ready for the degassing stage. At this point, the 1L media can be split into batches of smaller volume as desired. For example, degassing stage can be quicker for a smaller volume. Also, a 200ml batch can be used to fill 40 Hungate tubes with 5ml each.
?. [Heating and degassing stage]
Heat media close to boiling point but try to avoid boiling (as this would increase the loss of solvents). The heating step can entail;- Placing the flask into a water bath that has been heated to  (works well for Duran flasks). - Round neck flasks might be easier to heat on a hot-stirrer plate that has reached the  heat-marking.
80 °C
100 °C
Option: Place the	media in	the microwave with a stopper and bring	it just below boiling temperature	prior to placing it	in water	bath or hot stirrer	plate. This can save time. Experience shows	that	there	are no detrimental effects	of microwave treatment, but note that this might	change with specifics	of the media	composition. If using	microwave,	a guide is to use	1 minute per	100ml of media.	IMPORTANT: It must be noted that heating in the	microwave can generate a	vacuum	in the flask, pulling in the	stopper.	To avoid	the stopper being	fully pulled in, make sure you  loosen it well before putting the flask in the microwave and also pull the stopper out immediately	when you get the	flask out of the microwave– note that the flask will be hot!
?. [Heating and degassing stage]
Bring the heated flask next to the gassing manifold and set it on a heating/stirring plate.
?. [Heating and degassing stage]
Insert a redox and temperature probe into the media and the gas-line into the headspace. Secure a stopper in place, to minimize gas exchange between flask and atmosphere.
Option: The use of redox and temperature probes should be optional, however note that the use of Resazurin as redox indicator provides only an estimate of the success of degassing. Resazurin goes colourless at a redox value of -51mV and can interact with certain organics. Having a redox probe would provide an exact measure of redox and hence more information. In our experience, Rezasurin remaining pink correlates well with a media that will not support anaerobic growth but this might change with specifics of media and species. In general, we recommend use of redox probe where possible.
?. [Heating and degassing stage]
Turn on the gas cylinder and start gas flow at 1 liters per minute (LPM) using the appropriate gas mixture with your gassing manifold. As a rule of thumb, when the medium contains bicarbonate or carbonate, use a N2/CO2 (80/20) mixture, otherwise use 100% N2. A gas flow of 0.5 LPM will be sufficient for 200 mL in round-neck flasks. The gassing setup is shown in Picture 1.
?. [Heating and degassing stage]
When the medium has reached 80° C, push the gas-line down into the liquid, so it bubbles through the medium. If in step 10, no temperature probe was used, use the water bath or heating plate temperature and the time that the media spent on the heating device as a proxy to judge media temperature. Note that pushing the gas-line into the liquid would not be possible for 200 mL in round-neck flasks, unless your gas-line/canula is long enough.
?. [Heating and degassing stage]
After  -  degassing at , turn off the heating system and add the vitamin solution, and Cysteine-HCl (it is assumed here that there are no additional compositional elements but this might change with the specifics of your media).
80 °C
?. [Heating and degassing stage]
Continue gas flow, while the medium is cooling and until a redox value of - 300 mV or less is reached AND the media turns colourless, i.e. the Resazurin dye shifts from pink to colorless.
?. [Heating and degassing stage]
Remove any probes that are still in the flask. This might cause the media to turn pink. In that case, continue degassing a bit more.
?. [Heating and degassing stage]
Remove the gassing line (while gas is still flowing), by pressing the stopper in place and carefully pulling the gas line (while minimizing any chance of air flow into the flask). Immediately push the stopper fully in place and tape it secure (this is important as moving into the anaerobic chamber in the next step might cause the stopper be displaced due to pressure changes).
?. [Dispense and finalise stage]
Move the flask into the anaerobic chamber air lock and start the gassing sequence without any delay.
?. [Dispense and finalise stage]
Once the airlock cycle is finished, move the flask into the anaerobic chamber. Ideally, you should let the flask in the chamber and wait a bit ( - ) before opening it. This would allow the anaerobic chamber to recover/equilibrate from the airlock sequence and reach low O2 levels again.
?. [Dispense and finalise stage]
If you have a hand-held probe in the chamber, open the flask and measure the pH and redox of the media and record it in your lab book.
?. [Dispense and finalise stage]
Dispense medium into Serum flasks or hungate tubes as needed. If media shows any precipitation, place it on a stirrer during dispension, so to ensure homogenous media.
?. [Dispense and finalise stage]
Seal tubes with butyl-rubber septa.
?. [Dispense and finalise stage]
Place any remaining medium in a 1L Duran flask sealed with rubber butyl septa. Label the flask with your initials, date, and the pH and redox measured in step 19.
?. [Dispense and finalise stage]
Take media out of the anaerobic chamber and place aluminium crimp seals on the tubes you used.
?. [Dispense and finalise stage]
Autoclave media placed in flasks/tubes in step 20, as well as media to be stored (from step 22).
?. [Dispense and finalise stage]
Store in the dark and at .
80 Room temperature
IMPORTANT NOTE: Before using media for inoculation, the final component; Na2S should be added. This component is used for additional reducing of redox potential and also as Sulfur source. Its addition at this late stage of inoculation is because it is quite unstable and not ideal for storage. When adding Na2S, make sure you use the right amount for your culture volume (and given your stock solution concentration).
?. [Dispense and finalise stage]
Any media that is older than 8 months should be discarded. Any stored media younger than 8 months can be used provided it has maintained its pH and redox in the required range.
?. [Dispense and finalise stage]
If possible, measure pH and redox in the anaerobic chamber before using any stored media. If the measurements are not within range (redox, below -300mV and pH, as defined by specific media) discard media and prepare new one. Do not attempt adjusting these values with media additions.