TITLE: Assembly and sample collection in the MetaFlowTrain
AUTHORS: Guillaume Chesneau, Johannes Herpell, Sarah Wolf, Silvina Perin, Stéphane Hacquard

[DESCRIPTION]
The following protocol is for the preparation of a MetaFlowTrain experiment. It provides the required materials, and the procedure. We also have a video protocol to assist you in handling the MetaFlowTrain
>>Video Protocol<<

Nothing is set in stone with the MetaFlowTrain. We welcome all input to improve the system and are eager to craft new adapters/microchambers tailored to your scientific needs.

[STEPS]
SECTION: Procedure
1. DAY 1 : PREPARE MATERIAL AND BUILD MICROCHAMBER TRAINS
SECTION: Procedure
1.2. 1.2. BUILDING MICROCHAMBER TRAINS (Video: 00:21:12) Video Protocol:

1.2.1 Surface sterilize all equipment with Bacillol and expose it to UV light for 15-30 minutes. Items to be surface sterilized include boxes with autoclaved microchambers and connectors, silicon rings, box for opened filters, and box for the final trains. 
Comment: This process ensures the sterility of your working environment.

1.2.2 Unpack the filters and place them in a sterile box.
Comment: (Video : 0:25-0:30).

1.2.3 Put a silicon ring in the connector and screw it to a syringe filter, ensuring a tight fit!!
Comment: You should see pressure on the silicone ring (Video : 0:51-0:55).

1.2.4 Attach the microchamber, put a silicon ring in each female connector of the microchamber and connect it to another syringe filter (Figure 4)
Comment: (Video : 0:57-1:04).

1.2.5 Place the completed trains in a sterile box 
Comment: (Video : 1:04).

1.2.6 You can repeat this step if you need a train of more than 1 microchamber.
Comment: (Video : 1:05-1:26).

 

 



SECTION: Procedure
2. DAY 2 : METAFLOWTRAIN EXPERIMENT
SECTION: Procedure
1.1. 1.1  PREPARE MATERIAL :

1.1.1 Cut 1cm-wide strips of biocompatible tape (Polyolefin Diagnostic Tape, Nr 9793R, 3M) with precuts every 2cm (Figure 1) and store it in a container (e.g. glass beaker). 
Comment: This tape cannot be autoclaved, which is not a problem since the sticky side is sterile. however, we will still UV sterilize the surfaces later to maintain a sterile environment while handling the MetaFlowTrain system.

 

1.1.2 Prepare and sterilize your media. 
Tip: ensure your media is already in the container you will use for your MetaFlowTrain experiment.

1.1.3 Sterilize the necessary number of microchambers and connectors.
Comment: All 3D-printed pieces can be autoclaved for sterilization, as the resin is autoclavable. 
Tip: Place the microchamber end connectors in separate boxes to help keep the bench organized during building. Boxes on the video protocol: 0:28.

1.1.4 Sterilize one box to store the opened sterile filters during the building of microchamber trains. 
Comment: Box on the video protocol: 0:25-0:31.

1.1.5 Sterilize boxes to store the final built microchamber trains 
Comment: Box on the video protocol: 0:28.

1.1.6 Assemble the 15 ml Falcon tube adapters by connecting the tubing (Tygon E-LFL Pump Tubing: SA-AVX42007 from Darwin Microfluidics). Place the assembled 15 ml Falcon tube adapters (Figure 2) in a box (Box on the video protocol: 4:07) and sterilize them by autoclaving.
Comment : The length of the tubing can vary depending on your specific setup but should be consistent (e.g., 12 cm for 15mL Falcon tube adapters)

 

1.1.7 Prepare the Duran bottle adapters by connecting the tubing (Platinum-cured Silicone Tubing: SE-TUB-SIL-1*1, Darwin Microfluidics). Place the assembled Duran bottle adapters (Figure 3) in a box (Box on the video protocol: 2:24) and sterilize (autoclave) them.
Comment : The length of the tubing should be 22 cm for a 500 mL Duran bottle, but adjust it based on the type of bottle/Flacon you’re using to ensure it touches the bottom of the bottle. 

 

1.1.8 Place silicon rings in a container and sterilize (autoclave).
Comment: Beaker on the video protocol: 0:40.


SECTION: Procedure
2.1. 2.1 PREPARE YOUR MICROBIAL INOCULUM
This step depends on your protocol. 
We inoculate with 40uL of bacteria OD600, or 40uL of fungi, 2mg.mL.
Comment : The final volume in the microchamber is 400uL. Inoculating 40uL in the microchamber leads to a dilution to the tenth of your inoculum.

SECTION: Procedure
2.2.  2.2 PUMP TUBE CONNECTION AND STERILIZATION (Video: 01:38:00) Video Protocol:

2.2.1 Expose the pre-cut biocompatible tape (3M‱, Figure 1) to UV light for at least 5 minutes on each side, then return it to sterile container.

2.2.2 Pump tubes sterilization:
        a. Place the peristaltic pump on a clean bench.

        b. Install the tubing (3-stop Platinum-cured Silicone Tubing: SE-TUB-SIL-SSS-1*1 (Darwin microfluidics) and tighten them to 7 notches. 
Tip: can be done the day before (Video : 1:42-1:48).

        c. Run 70% ethanol through the pump for 15minutes at speed 500uL.min. Then discard the Ethanol.
Comment : (Video : 1:48-1:59).

        d.  Run 500mL of water to clean the Ethanol, and empty the system.
Comment : (Video : 2:02-2:17).
Tip : Always keep the tips of your tubing in a sterile container (e.g., a sterile beaker) to prevent contamination from touching your bench.

        e. Carefully place the Duran bottle adapter (Figure 3) on your bottle filled with media and wrap it with Parafim or Micropore tape.
Comment : (Video : 2:20-2:42).
 
       
       f. Connect all the pump’s tubing to the Duran bottle adapter. 
Comment : (Video : 2:45-2:58).
Tip : If needed you can use a flat tweezers to help you adapting the tubing to the Duran cap adapter.


SECTION: Cleaning microchambers
3. 3.1 After finishing your experiment, collect the microchambers and connectors in separate glass containers.  

3.2 Remove all the tape from the top of the microchambers.  

3.3 Fill the glass containers with water until all the parts are fully submerged.  

3.4 Place the glass containers in a sonicator for 15 minutes.  

3.5 Thoroughly wash the parts using a wash bottle with water, for microchambers, ensure the inside is thoroughly cleaned from the top hole.

3.6 Let them dry completely.  

3.7 Store them dry in a box, in the dark, at room temperature until the next use. 


 




SECTION: Procedure
2.4.   2.4. CONNECT THE METAFLOWTRAIN (Video: 03:50:00) Video Protocol:

2.4.1 Connect the trains to the pump’s tubes (3-stop Platinum-cured Silicone Tubing: SE-TUB-SIL-SSS-1*1 (Darwin microfluidics))
Comment : (Video : 3:54-4:06).

2.4.2 Connect the tubes with the 15mL Falcon adapter to the end of the train
Comment : (Video : 4:07-4:24).

2.4.3 Repeat these steps for the 24 channels of your pump.

2.4.4 Screw 15mL Falcon tubes to the Falcon tube adapters, one per train (24 per pump).
Comment : (Video : 4:07-4:24).
 

2.4.5 You're now ready to remove your pump from the clean bench and start your experiment !
Comment: We are using a 90 cm x 60 cm plate with two handles to move the entire system from the clean bench to our growth chamber.


SECTION: Procedure
2.5. 2.5. START THE PUMP

2.5.1 Once all your pumps are prepared and settled in your growth chamber, fill the media at a high speed (e.g., 200 µL/min) until it reaches the first filters in the trains.

2.5.2 Next, adjust the flow rate to a maximum of 20-50 µL/min to fill the microchambers.  
Comment: It’s crucial to fill the microchambers slowly to avoid flushing your microbial inoculum into the filters.

2.5.3 When the liquid begins to exit the final filter in your train, set the pumps to your experimental flow rate (e.g., minimum flow rate = 7.349 µL/min).  
Tip: You can visually confirm that the microchambers are filling. Similarly, observe the filters; they will change color from dry (whiteish) to wet (grayish) as they become saturated. If a train fails to fill, abandon it and continue with your experiment.

2.5.4 Wait for the desired duration of the experiment.

 


SECTION: Procedure
2.3. 2.3 MICROCHAMBER INOCULATION (Video: 03:00:00) Video Protocol:

2.3.1 Position your trains on racks (Figure 5)
Comment : (Video : 3:03-3:10).
 

2.3.2 Add 40ul of the strain into your microchambers (your inoculum will be diluted 10 times in the microchamber).
Comment : (Video : 3:11-3:23).

2.3.3 Seal the microchamber with biocompatible tape (3M‱, Figure 1). 
Comment : (Video : 3:26-3:48).
Tip: Securely seal the tape with a PCR plate sealer to prevent leaks.


SECTION: TROUBLESHOOTING
4. 4.1 Leaks on top of the microchambers:  
This is often caused by poor print quality of the microchambers, likely due to scratches or imperfections on their surface. Potential causes include:  
1. Errors during the 3D printing process.  
2. Insufficient drying of 3D prints before curing or autoclaving, which can cause cracks during heating and damage the prints.  
3. Damage during removal of the prints from the support or platform.  
Cracks on the microchambers can compromise their permeability. Refer to your printer's instructions and best practices to ensure high-quality prints.

4.2 Leaks between microchambers:
This is typically due to a loose connection. Ensure you see pressure on the silicone rings when screwing the microchambers or connectors with the syringe filters. Screwing should not require excessive force, but you should notice the silicone ring slightly compressing.

4.3 No media flowing into the hoses from the bottle:
This may be caused by a loose connection to the bottle adapter. Check that no air is entering the connection, and ensure the hoses are fully plugged into the adapter. Also, inspect the adapter for any leftover 3D printing supports that could create air gaps and compromise the connection. In general, this issue often arises from poor print quality or improper cleaning/removal of supports from the print.

4.4 Air in the top of the microchambers:
This is a common occurrence, as some air naturally passes through the MetaFlowTrain system. It’s important to remove this air to ensure the microchambers are filled with liquid, especially before harvesting. To remove the air, tilt the microchambers to guide the air bubble toward the chamber’s outlet. You can simply tilt the entire rack, and you’ll notice the bubble moving toward the outlet. After a few minutes, the flow rate should push the air bubble out on its own.