McClean:Basic Chemostat Guide

Overview

This is a simple introduction into how to set up a chemostat experiment using the Botstein Lab Chemostat Room. Adapted from the see Botstein Lab Chemostat Guide and Lab Chemostat Protocols. This will go through how to clean and set up a vessel, make glucose limited media, and how to start the apparatus. Additional Protocols are available for how to induce metabolic cycling and how to sample cells.

Sign-Up

There is a sign-up whiteboard outside the chemostat room, make sure to sign up well in advance to make sure that there is enough space and time for you to run your experiment.

Autoclaving

Before you can start anything you have to make sure to autoclave the 10 L media carboys and the chemostat vessels. This should be a day in advance of when you want to start the experiment.

Carboys

A Carboy usually consists of a large glass vessel a cork and several tubes: An air intake tube with a sterile luer filter and a media output tube. To prepare for autoclaving:

1. Ideally the Carboy should already be sterile from its last user, but to be safe rinse out the carboy and the tubing with DI water.
2. Leave al the tubing unclipped, but make sure there are clips on each of the valves.
3. Put aluminum foil on the ends of all the output tubes. It is important to put them on in such a way where it is easy to take them off. One easy way to do this is to first fort horizontally and then fold a bit of the top down. Tape is in the chemostat room.
4. Tape down the cork with electrical tape. Use two perpendicular pieces over the top and then one long long piece to secure the ends of the other 2 pieces to the vessel neck.
5. Add a clamp to the media output valve but leave it open. Once you remove the carboy from the autoclave make sure to clamp the output valve immediately to ensure the media tube remains sterile !
6. Autoclave on the liquid cycle with paper towels underneath the vessel.
7. Once the carboy is finished autolaving make sure to clamp the media output vessel immediately with the clamp placed earlier.

Vessels

Each individual vessel should have a culture vessel, O-ring, stirrer, port assembly (essentially the lid),effluent drop tube, probes (temp and O2), air inflow, air release, and effluent tubing, and clamp Before you can autoclave the vessel you have to make sure to assemble it properly:

1. Rinse out the vessels and tubing with DI water. Replace any old tubing that looks too distorted or gross. (NEVER USE SOAP TO CLEAN)
2. Make sure the stirrer is snug by adjusting the small o-ring to be closer to the joint and place the entire top port in the vessel
3. Put the big O-ring on the vessel rim and make sure it isn't too flat or it won't seal.
4. Look at how the stirrer is sitting in the vessel, you want it to be fairly centered.
5. Clamp the vessel shut slowly, you don't want to shut the clamp too fast and crack it.
6. Depending on which side of the chemostat room you are using there are either inserted temp probes or a probe that connect directly to the port apparatus. If using a dO₂ probe first remove the cap and place in the appropriate bucket. Get some O₂ electrolyte solutions (LOCATION?!) to replace in the probe. First take off the bottom of the probe. Push out the mesh membrane. Dump the old electrolyte solution and refill about half way and put the probe back together (some of the solution will spill over so be ready to wipe it up).
7. Make sure to adjust the affluent vessel such that it is pulled all the way out, we will adjust it after we autoclave it.
8. All ports that are not in use either need to be closed off with a tube that is clamped or filled with silicon.
9. Any tubing that is supposed to be left open/unclamped (ie air intake, media input, effluent tube) should be covered in foil as described above.
10. Autoclave on the liquid cycle for 15 minutes.

Mixing Media

Depending on what you want to Limit, Media has to be mixed differently, but for glucose limitation its fairly straight forward.

1. Take out the 1000x Vitamins and 1000x Metals from the stocks. Makes sure to put the metals on a stirrer to mix them up.
2. Fill a 1000 mL graduated cylinder and fill it to ~700 mL of milliQ water and place it on a magnetic stirrer with a stirbar.
3. Rinse out a measuring carboy with MilliQ water thoroughly
4. Fill a measuring Carboy to about 7L using the autofill function.
5. In the meantime add 10 mL of the vitamins and the metals to the graduated cylinder.
6. Measure out the appropriate amount of glucose. Ex: for .8% glucose you would want .8 g/L or 8 g total. Add to the cylinder. Make sure the solution mixes thoroughly.
7. Add the graduated cylinder volume to the measuring carboy.
8. Add 1000 mL of 10x salts to the measuring carboy.
9. Using the graduated cylinder slowly add MilliQ water to the fill line.

Filling the Carboy

This step sterile filters the media and fills the carboys at the same time. Everything should be done as quickly as possible to avoid contamination.

1. Connect the air filter to to the vacuum unit.
2. Take 500 mL sterile filter and cut off the bottom of the package.
3. Take the top of a 100 mL bottle and quickly screw it into the bottom of the sterile filter.
4. Remove the sterile filter + bottle form the package and tighten it between a clamp on a ring stand.
5. Using forceps sterilized in ethanol, open the output valve and remove the cotton filter, this will allow the media to easily flow through the tube into the carboy.
6. Replace the output valve and connect it to the media output tube of the carboy by removing the foil
7. Take off the clamp that was closed after autoclaving.
8. Open the top of the sterile filter and slowly pour 500 mL of the media from the measuring carboy into the the chamber. It is easier to first our from the measuring carboy to the large pitcher and then use the pitcher to slowly add the media to the sterile filter.
9. Turn on the vacuum and allow media to flow into the carboy,
10. Do NOT allow the media to go above the air input line, as this would cause it to travel into the vacuum system.
11. Once the carboy is full turn off the vacuum and clamp the media output valve. Remove the vacuum line and the sterile filter form the carboy tubes. The carboy is now ready for use
12. Unscrew the bottle and cover it with its original cap. This will act as a negative control for sterility for the media. Watch it over several days at room temp to see if any growth/contamination happens.