TITLE: CCMA Coculture Media (Red Cap)
AUTHORS: Catherine Gohar, Ada de la Cruz

[STEPS]
?. [Equipment for making media]
For making media you will need: 2 --> 500 mL graduated cylinders1 --> 100 mL graduated cylinder1 --> 10 mL graduated cylinder2 --> small funnels2 --> weigh boats1 --> lab spatula 1 --> pipette + tip1 --> 1000 mL or 2000 mL flask (depending on if you're making 1 L or 2 L of media)
?. [Making Media (Before autoclave) for 1 L]
Pour 500 mL of nanopure water into beaker
?. [Making Media (Before autoclave) for 1 L]
Add 2.28 g of NaCl
?. [Making Media (Before autoclave) for 1 L]
Add 100 mL of 10x general salts sol'n
?. [Making Media (Before autoclave) for 1 L]
Add 5.6 mL of 60% Na DL-lactate syrup
?. [Making Media (Before autoclave) for 1 L]
Add 1 mL of resazurin
?. [Making Media (Before autoclave) for 1 L]
The remaining 375 mL are nanopure water
?. [Making Media (Before autoclave) for 1 L]
For 2 L of media, you will add:1,000 mL of nanopure water4.6 gof NaCl200 mL of 10x general salts11.2 mL of Na DL-Lactate Syrup2 mL of resazurin750 mL of nanopure water
?. [Making Media (Before autoclave) for 1 L]
If not immediately autoclaving, place aluminum foil (and a rubber stopper if wanted) on the top to prevent evaporation, label, and store in fridge.
?. [Making media (autoclaving process and initial hooking up to gas) for 1 + 2 L]
When autoclaving:Pour media that was mixed prior into the Widdel flaskensure that the red caps are loosened put aluminum foil on both ends of the black rubber tubesplace autoclave tape of the flask select the "P12 30 min liquid" program
?. [Making media (autoclaving process and initial hooking up to gas) for 1 + 2 L]
After autoclaving, connect the Widdel flask to N2CO2:Turn on the gas tank Add new plastic filter and needle tip on the cannula tip that will be going through the rubber tube with the cotton filter Place the needle with the flowing gas into the rubber tube w/ the cotton filter and ensure that gas is running into widdel flask (red caps will rattle or you can check by turning on the air flow to the other cannula tip)
?. [Making media (autoclaving process and initial hooking up to gas) for 1 + 2 L]
Once connected to N2CO2, bring the media to room temperature by:Placing flask into red ice bucket on top of stir plate and putting ice around it + turning on stir platepour a bit of cold water + more ice to speed up the cooling process
?. [Making media (autoclaving process and initial hooking up to gas) for 1 + 2 L]
If you haven't done so beforehand, while the media cools, it is a perfect opportunity to autoclave a sufficient amount of clean balch tubes and stoppers
?. [After media has been hooked up to gas and has cooled]
Remove ice bucket from below the widdle flask and readjust the apparatus holding it upright.
?. [After media has been hooked up to gas and has cooled]
For 1 L of media you will need: 1.1 mL of 1 M K2HPO425 mL of 1 M NaHCO31 mL of trace minerals1 mL of Thauer's vitamins1 mL of 1 M cysteine1 mL of 7.8% w/v Na2S
?. [After media has been hooked up to gas and has cooled]
For 2 L of media you will need: 2.2 mL of 1 M K2HPO450 mL of 1 M NaHCO32 mL of trace minerals2 mL of Thauer's vitamins2 mL of 1 M cysteine2 mL of 7.8% w/v Na2S
?. [After media has been hooked up to gas and has cooled]
Pour 70% ethanol sol'n on top of the containers of the aforementioned ingredients and use a flame to sterilize them
?. [After media has been hooked up to gas and has cooled]
Using a new needle and tip each time to maintain sterilization (1 mL, 3 mL, 5 mL, and 30 mL syringes + 23 gauge needles will be used) add the aforementioned chemicals into the waddel flask through the rubber membrane in the red cap
?. [After media has been hooked up to gas and has cooled]
Once everything has been added, allow the media to reduce (it will go from blue --> purple --> pink --> clear) before dispensing into tubes
?. [To dispense reduce media into tubes:]
Sterilize the cunnula that will be dispensing the gas into the balch tubes as well as thin tweezers using the ethanol and flame process
?. [To dispense reduce media into tubes:]
Remove the cotton filter from the top of a 5 mL pipette with the sterile tweezers
?. [To dispense reduce media into tubes:]
Attach the pipette to the end of the black rubber tube with the dispensing clamp (ensure this is done in a sterile manner)
?. [To dispense reduce media into tubes:]
Ensure that the red caps on the Waddel flasks are tightened and tape down the black stopper to avoid it being popped out from built up pressure
?. [To dispense reduce media into tubes:]
Ensure that all clamps are loosened and squeeze the dispensing clamp to test the flow. Ensure that there is a waste bucket below to catch any dripping media.
?. [To dispense reduce media into tubes:]
Dispense 20 mL of  media into each balch tube (This will be achieved by using the 3 mL mark on the 5 mL pippette as a guide)
?. [To dispense reduce media into tubes:]
Stopper the tube while pulling out the gassing cannula
?. [To dispense reduce media into tubes:]
After all media has been transferred to the tubes, crimp an aluminum seal onto the cap (red)
?. [To dispense reduce media into tubes:]
Place the sterile cannula inside the balch tube and let it dispense N2CO2 for about ten seconds