Cleaning Protocols

Simple Protocol

1. Soak the lids in 10% bleach (without any purging or vacuum)

2. Blow out the resultant bleach/media mixture with an air supply or syringe

3. Rinse with DI water

Salty Media Removal and Thorough Cleaning

Although autoclaving the lids with bubblers removes the risk of contaminating the next experiment, you'll ideally want to remove media residue and any biofilm that may have accumulated on the bubbler. This is best achieved in the following process, which utilizes 3 valves and Y connectors to control the pressure in the tubing connected to the bubbler (pictured below): a valve to pressurize the bubbler, a valve to apply vacuum to the bubbler, and a valve to "neutralize" the bubbler to ambient pressure. This can also be achieved manually with a syringe.

1. Remove all lids from their vials

2. In a 1L beaker full of 10% bleach, pressurize the bubbler to purge any residual media/bacteria into the bleach

3. Apply vacuum to the bubbler to pull bleach into it

1. Pour out the bleach, rinse the lids with water a few times, and refill the beaker with water

2. Pressurize each bubbler to purge the bleach inside each bubbler into the water

3. Apply vacuum to pull in water (or 70% ethanol), and then apply pressure to purge

Materials

• 3D printed (SLS, nylon PA-12) vial cap with ports for nylon tubing

• 3D printed (SLS, nylon PA-12)

• (1/8" OD)

• - 5 micron filter, 1/4" Diameter, 1/16" Thick

• (Recommended)

• (Recommended)

• (Recommended) for testing bubblers

Construction Protocol

1. Cut Nylon Tubing to Height in Vial

The length of the tubing connecting the lid to the bubbler will be dependent on the culture volume of your experiments, which is determined by the length of your efflux straw. As a good starting point, a 2 inch efflux straw will correspond to a 20mL culture volume, and a 2.5 inch length of tubing connecting to the bubbler will be ideal for that volume. If your culture volume is different than this, the best way to find an ideal tubing length is by experiment:

1. Cut a length of nylon tubing and fit it in the cap and 3D printed bubbler holder. Make sure that you cut so that your bubbler is below your efflux straw!

2. HOWEVER, use caution with how low you set the bubbler; if the bottom of the bubbler body is below the 15mL line in the vial, it will begin to interfere with OD readings

3. Using the length of tubing you found as a template, cut equal lengths of tubing for the remainder of bubblers you will be making.

2. Assemble Bubbler

Materials

• Mixing tray (here a small weighboat)

• Disposable applicator

• Foreceps/tweezers

• Frit

Steps

1. Use gloves to avoid getting epoxy on your hands

2. Follow the epoxy directions to mix up a small amount of epoxy in a disposable dish or a piece of cardboard. Use even pressure to get even amounts of resin and hardener.

3. Use a pipette tip or other similarly sized applicator to put the minimum amount of epoxy around the rim of the 3D printed bubbler body where the frit will be placed

3. Test Bubblers

1. Passivate the frits by submerging in liquid

   1. Option 1: Soak in water overnight

   2. Option 2: Soak in LB-Miller media (or any rich salty media) or 5% citric acid for 1 hour. Lemon juice will do in a pinch.

4. Attach Bubbler to eVOLVER Cap

Options

1. Epoxy directly into cap

   1. Advantages:

      1. Pre-defined and rigid position in the cap

Epoxy Directly Into Cap

1. Similar to the assembly of the nylon tube into the bubbler body, apply expoy to the other end of the tube attached to your bubbler, insert into one of the ports on the bottom of the vial cap (whatever one corresponds to your preferred layout; my preference is for media in on the right, bubbler in the back, efflux straw on the left).

2. Make sure bubbler is pushed all the way in and steel frit is facing sampling port

   1. Being inconsistent about this will mean different caps will have different scattering properties that will change OD

Attach via Silicone Tubing

See protocol .

The slow pumps are currently operated independently of the main eVOLVER GUI and pump liquid at a rate of X mL/min.

The eVOLVER slow pumps currently operate exclusively through the . The navigate to the run_slow_pumps.py file similarly to starting an experiment via command line.

In the run_slow_pumps.py script you only need to modify one of two lines for time settings, one for modifying specific pumps and the other for modifying all pumps

Traditionally it takes about 200-300 seconds to fill the lines with liquid using the slow pumps, however this is dependent on the length of tubing.

Please cite Daniel Hart's paper when it comes out!

Overview

An in-vial aerator, which can be hooked up to a gas stream of your choice. Normal stir bar-based mixing in the eVOLVER does not provide enough gas exchange in many situations. These bubblers produce small bubbles that greatly increase gas exchange.

Requirements

Cap with Nylon Tubing

Allows the nylon tube of the bubbler to slot into cap. See for options.

Long Stir Bars

The normal eVOLVER stir bars allow large bubbles to adhere if you are bubbling. This creates lots of noise in OD readings. It is therefore necessary to use thinner stir bars that don't allow such large bubbles to stick to them. We have found these to be the only ones that work with bubbling: .

Long Stir Bar Jittering and Low Stir Stability

These stir bars are unfortunately prone to jitter and not stir. This is a function of at least the following:

1. Distance to magnets on the fan used for stirring

   1. The newest eVOLVERs have a bigger distance to the fan because of the 3D printed magnet holder and 1/4" acrylic spacers

   2. Try ordering thinner acrylic spacers

Please let us know on the forum if you find other stir bars that work with bubbling. However, several people have looked and not found any.

Stop Stir During OD Reads

While bubbling, you must stop stirring while taking OD readings to allow bubbles to float to the surface. Otherwise, bubbles will create too much variability in OD readings and make your OD calibration useless.

During OD Calibration

While calibrating OD, we mimic experimental OD readings as much as possible. To do this:

1. Turn off stirring via the GUI

2. Calibrate with long stir bars (see above) and caps with bubblers

3. Swirl OD standards at least every other time you move them to avoid cell sedimentation

During Experiments

Load a conf.yml onto the server that pauses stir whenever an OD measurement is taken

1. Guide | example stir pause conf.yml file

2. This decreases data acquisition rate (20 seconds to 60 seconds), but greatly increases OD accuracy

Considerations

Variability Between Bubblers

The assumption is that there will be variability between the bubblers that you make -- even though you will screen them before putting them in a vial cap! This is compensated for by bubbling much more than we need for a given microbe's gas consumption needs. Worse bubblers will not be a problem if the worst bubbler you have provides more than enough gas exchange.

Biofilming

Aerators are great substrates for biofilming. If your strain biofilms, make sure you swap in new bubblers when bubbles noticeably diminish. See .

Media Type Affects Bubble Size

Bubblers will produce different sized bubbles depending on media type. This is related to the amount of salts, proteins, carbohydrates, etc that are in the media. These can act as surfactants to create smaller bubbles. Therefore, largest bubbles will be in water and smallest bubbles will be in rich media, as can be seen below:

<Add picture of bubbler in water vs algae media vs LB>

Overview

This apparatus is designed to save you time when you are cleaning bubblers.

Construction

Parts

Infrastructure (You may already have this in place)

1. 1X - Gas regulator with pressure gauge capable of regulating to 10 psi

   1. For controlling air pressure to bubblers (we don't want 150 psi going through our bubblers!)

   2. 1X -

Cleaning Apparatus

1. - sealant for leaky threaded fittings (tightly wrap it around and break it off)

2. At least 6ft - (1/8" OD)

3. 5X -

Assembly

1. Assemble apparatus

1. Assemble the 3 push-button connectors using directions that come with them

2. Connect nylon tubing in the above configuration

3. Connect 3/32" tubing in the above configuration

2. Assemble a vacuum trap to prevent liquids from entering building vacuum line

3. [Bubbler testing only] Assemble the push-to-connect cap

Used for easily attaching / detaching bubblers for testing

Testing Protocol

1. Attach the bubbler to the black 2-way connector connected to the green falcon tube lid as pictured below. To attach, push the bubbler in; to detach, push the black ledge into the connector and then pull the bubbler out.

1. Fill a vial with ~15mL of rich media like LB, YPD, or BHI. BG11 media will also work. As a rule of thumb, the less salt and peptides your media has dissolved in it, the larger your bubbles will be.

2. Place the green lid with the bubbler attached into the vial with media. Ensure the bubbler is submerged in the liquid.

1. Ensure the air supply is turned on and set to 5-10psi. Check that the bubbler cleaning apparatus tubing is attached to the air supply. If air is flowing through the apparatus, you should feel a rush of air coming out of the red tubing.

Parts

1. Buy another pump rack from Fynch

2. 2X - 3D printed pump extension mount (Front)

3. 2X -

4. 2X -

   1. Final length 5.7 inches (144.78mm)

5. 8X -

7. 2X -

Protocol

Machine 40-20 Aluminum Tubing

Do for both of the aluminum rails. Diagram can be found .

1. Cut to final length 5.7 inches (144.78mm)

2. If necessary, drill out the hole on each side so that your M6 x 1mm tap can work

3. Tap each side with M6 x 1mm tap

Assembly

1. Unscrew 4 screws from top of original pump mount

2. Remove 20-20 aluminum tube

3. Replace with 40-20 tube. Screw in

4. Add 3D printed extenders