πŸ“–
eVOLVER
  • eVOLVER Documentation Wiki
  • General
    • About Us
    • eVOLVER Community
      • Code of Conduct
  • Getting Started
    • Buying eVOLVER
    • Part Sourcing
    • Unboxing and Setting Up
    • Software Installation
      • DPU Installation
      • Electron App (GUI) Installation
    • Configuring Computer and Networking
      • Router Setup
    • Calibrations
      • Temperature Calibration
      • Optical Density Calibration
      • Pump Calibration
      • Manual Calibration - calibrate.py
  • Upgrade Base eVOLVER Hardware
  • Experiments
    • Starting an Experiment
      • Carboy Media Prep
      • Preparing Vials
      • Setup Waste Carboy
      • Sterilizing Lines
      • Loading Vials and Setting Initial Conditions
      • OD Blank
      • GUI Start Guide
      • Command Line Start Guide
      • Cleaning Up After Experiment
    • Growth Curve
    • Chemostat
    • Turbidostat
    • FAQs
    • Tips and Tricks
  • Guides
    • Use the GUI to Control Parameters
    • Building a Smart Sleeve
    • Making media bottles and splitters
    • Emergency Efflux
    • View the Server Log and Restart Server
    • Updating the eVOLVER Server
    • Change Your conf.yml File
    • Arduino Software Installation
    • Raspberry Pi Configuration
    • Command Line Usage
    • Millifluidics Guides
      • Designing Millifluidics Using Eagle
      • Constructing Laser Cut Millifluidics
      • Constructing Millifluidics via SLA Printing
      • Calibrating IPPs
      • Operating Millifluidics
  • Troubleshooting
    • Troubleshooting Overview
    • Experiment Troubleshooting
      • Contamination
      • Vial Overflow, Pump Failure, and Spills
      • Tubing and Connector Blockage and Bursting
    • Optical Density (OD) Readings
    • Vial Troubleshooting
      • Replacing Photodiodes and LEDs
      • Heating Element
      • Stirring
    • Server Troubleshooting
    • Vial Platform Troubleshooting
      • Motherboard Troubleshooting/Replacement
      • 12V Power Supply Troubleshooting/Replacement
    • GUI Troubleshooting
    • eVOLVER Maintenance
  • Hardware
    • Overview of Hardware Architecture
    • Overview of Fluidics
      • Tubing and connectors
      • Peristaltic Pumps
      • Fluidics box
    • Overview of Millifluidics
      • IPPs (Integrated Peristaltic Pumps)
      • Pressure Regulator
    • Vial Caps
      • Universal Vial Cap Construction Guide (Luer Connectors Only)
      • 5 and 7 Port Nylon Tubing Caps Construction Protocol
    • Smart Sleeve
      • Vial Board
      • πŸŒͺ️Stirring
      • Temperature
      • Optical Density
        • OD90 vs OD135
    • Motherboard Layout and Circuitry
      • 🌑️Arduino
      • Sensor/Actuator Board Slots
      • Pulse Width Modulation (PWM) Boards
      • Analog-to-Digital Converter (ADC) Boards
    • Raspberry Pi
    • Chassis
    • Light Blocker / Splash Guard
    • Known Issues
  • Software
    • Overview of Software Architecture
    • DPU
      • Calibration
      • custom_script.py
      • Experiment Data Files
      • eVOLVER.py
    • Arduino
    • Server (Raspberry Pi)
      • Calibration Files
      • Configuration Files (conf.yml)
    • Known Issues
  • Extensions
    • Adding A New Experimental Parameter
      • Power Supply
      • Specific Applications
      • Custom Calibration Code
    • Custom Experiments
      • ePACE
        • ATTiny1634 Writing
        • LUX Board Troubleshooting
      • Morbidostat
      • Extractor Column
        • Extractor Volume Maintenance
        • Experiment Start
          • Sterilizing Extractor Fluidics
          • Setting up your experiment
          • Using the extractor script
        • Extractor Analysis
        • Troubleshooting
        • Example protocols
      • Phototroph Growth
        • Setup Phototroph eVOLVER
        • Photo-eVOLVER Smart Sleeves
          • Photo-eVOLVER Smart Sleeve Construction Guide
        • Experiment Guide
    • Custom Fluidics
      • Adding a Third Pump Rack
      • Bubblers / In-Vial Aeration
        • Bubbler Construction Protocol
        • Bubbler Cleaning Protocol
      • Running the slow pumps
    • min-eVOLVER
      • About
      • min-eVOLVER Construction
        • Parts
        • Construction Protocol
      • Fluidics Setup
      • Software Installation and Startup
      • send_command.py
      • Calibrations
      • Starting an Experiment
      • ePACE with min-eVOLVER
        • [v1.1] ePACE with min-eVOLVER
      • Troubleshooting
    • Interfacing with Other Systems
  • Contributing
    • Guidelines for Contribution
    • Reporting a Bug / Hardware Failure
    • Documentation
      • Making a Forum Post
      • How to Edit the Wiki
    • Software Development
    • Hardware Development
Powered by GitBook
On this page
  • Materials
  • Media Requirements
  • Protocol
  • Relevant Forum Posts

Was this helpful?

Edit on GitHub
Export as PDF
  1. Experiments
  2. Starting an Experiment

Carboy Media Prep

Considerations while preparing large volumes of media for continuous culture in 20L carboys.

PreviousStarting an ExperimentNextPreparing Vials

Last updated 1 year ago

Was this helpful?

Materials

Media Requirements

A primary consideration when running eVOLVER experiments is predicting the media consumption rate, which is typically orders of magnitude higher than in typical batch experiments. The required media for continuous culture can be estimated using the following equation: π‘€π‘’π‘‘π‘–π‘Ž π‘…π‘’π‘žπ‘’π‘–π‘Ÿπ‘’π‘‘ [π‘šπΏ] = πΆπ‘’π‘™π‘‘π‘’π‘Ÿπ‘’ π‘‰π‘œπ‘™π‘’π‘šπ‘’ [π‘šπΏ] / 𝐴𝑣𝑔. π·π‘œπ‘’π‘π‘™π‘–π‘›π‘” π‘‡π‘–π‘šπ‘’ [β„Ž] βˆ— π‘π‘’π‘šπ‘π‘’π‘Ÿ π‘œπ‘“πΆπ‘’π‘™π‘‘π‘’π‘Ÿπ‘’π‘  βˆ— π·π‘’π‘Ÿπ‘Žπ‘‘π‘–π‘œπ‘› [β„Ž] (1) It should be noted that additional media (~20mL) is needed during device setup to flush media input lines.

Protocol

20L of media will take a significant amount of time to cool. It's best to autoclave your media the day before you want to use it for an experiment.

  1. Before preparing media, steam clean the carboy by filling with ~2L of water and autoclaving on the liquids cycle for at least 45 minutes. Be sure to keep the lid loose to prevent the carboy from collapsing. Pour out the water.

If the carboy does collapse, you can take the lid off and place it back into the autoclave for 30m-1h. The residual heat should allow it to pop back.

2. If you are able to prepare a concentrate of your media, you can do so as a 10x 2L solution (or whatever makes sense for your media) in a beaker with a stir bar. It is also possible to mix everything directly in the carboy. Add your media components in small batches and try to mix as you go.

3. Fill the carboy up to 20L - volume of media components that will be supplied after the autoclave step. For example, if you need your media to be 2% glucose, you should fill the carboy to 18L and then add 1L of filter sterilized 40% glucose after autoclaving.

4. Place a male Luer cap on the outlet of the carboy lid. Place a filter on the inlet, and cover it with foil. Loosen the entire lid.

You do not necessarily need a filter on the inlet - this depends on the context of your experiment. For most applications, capping the inlet and keeping the lid loose for the duration of the experiment is sufficient - the media should not become contaminated.

5. Place the carboy into an autoclave bin, then place into an autoclave. Autoclave on the liquids cycle for 2 hours.

6. Take the carboy out of the autoclave.

WARNING: The carboy and media will be extremely hot, and there will be ~1L of hot media in the autoclave bin. Ask someone for help removing from the autoclave!

Media will take a significant amount of time to cool (overnight) - be sure to take this into account before running an experiment with the media.

7. If media components need to be added after autoclave (glucose, antibiotics, supplements, etc.) place the carboy on a cart and move next to a bench flame. Sterilize the area thoroughly with ethanol before quickly unscrewing the lid to make the additions. Mix by placing the carboy on a cart and moving the cart in a circular motion, or by placing on the floor, tilting ~30 degrees, and moving the top in a circular motion.

8. You can place the autoclave on the floor next to your experiment. Be sure to keep the lid loose to prevent negative pressure buildup in the carboy which could prevent media from going into the culture vessels.

9. After the carboy is empty, rinse thoroughly with water, being sure to run water through all of the tubing. Steam clean as described in step 1, then pour out the remaining liquid.

Relevant Forum Posts

Nalgene 20L carboy
Nalgene fitting/venting closure
About the volume of required media
Making Media Bottles and Media Splitters