---
description: >-
  Considerations while preparing large volumes of media for continuous culture
  in 20L carboys.
---

# Carboy Media Prep

## Materials

[Nalgene 20L carboy](https://www.fishersci.com/shop/products/nalgene-polypropylene-carboy-17/0296020A)

[Nalgene fitting/venting closure](https://www.fishersci.com/shop/products/thermo-scientific-nalgene-filling-venting-closures-pp-3-port-83b-closure-3-x-1-4-6-35mm/0292313Z)

## 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: 𝑀𝑒𝑑𝑖𝑎 𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑑 \[𝑚𝐿] = 𝐶𝑢𝑙𝑡𝑢𝑟𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 \[𝑚𝐿] / 𝐴𝑣𝑔. 𝐷𝑜𝑢𝑏𝑙𝑖𝑛𝑔 𝑇𝑖𝑚𝑒 \[ℎ] ∗ 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓𝐶𝑢𝑙𝑡𝑢𝑟𝑒𝑠 ∗ 𝐷𝑢𝑟𝑎𝑡𝑖𝑜𝑛 \[ℎ] (1) It should be noted that additional media (\~20mL) is needed during device setup to flush media input lines.

## Protocol

{% hint style="warning" %}
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.
{% endhint %}

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 <mark style="color:red;">**keep the lid loose**</mark> to prevent the carboy from collapsing. Pour out the water.

{% hint style="info" %}
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.
{% endhint %}

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.&#x20;

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.

{% hint style="info" %}
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.
{% endhint %}

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.

{% hint style="danger" %}
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!
{% endhint %}

{% hint style="warning" %}
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.
{% endhint %}

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

[About the volume of required media](https://www.evolver.bio/t/about-the-volume-of-the-required-media/330)

[Making Media Bottles and Media Splitters](https://www.evolver.bio/t/making-bottles-and-media-splitters/63)
