TITLE: Autoclave Instructions for the BD FACS Aria Fusion Sheath Fluid Tank
AUTHORS: Jamie C Tijerina

[DESCRIPTION]
It is good practice to clean sheath tanks regularly to prevent biofilm formation, rust, and contamination of cytometers or sorters. The sheath tank is a common point of cytometer and cell sorter contamination because opening and closing the lid requires a high amount of contact, exposing the interior of the tank and its contents. Additionally, some experiments require strict decontamination of the system, including the sheath tank.

There are rare instances of contamination in which chemical-resistant biofilms can form on the interior of the metal sheath tubing within the tank. In these instances, autoclaving the tank is required. If autoclaving cannot remove the biofilm, then complete replacement of the tanks and parts would be necessary. 

This protocol can be adapted for sheath tanks found on other sorters and cytometers. Generally, the differences would be the presence of different probes and connectors that need to be removed if the material cannot withstand autoclaving. The BD FACS Aria Fusion tank is one of the more complex tanks to clean due to the presence of the probe and the number of connectors that need to be considered.

[BEFORE_START]
Ensure that you have access to a clean MilliQ or similar DI water dispenser. Do not use tap water. Ensure that you have access to a working biosafety cabinet. Ensure all consumable supplies listed in the materials section are stocked before starting the cleaning procedure.

[STEPS]
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1. Wipe down the surface of a biosafety cabinet with 70% Ethanol or CaviWipes. Switch on the air flow.

*Make sure that your biosafety cabinet is cleaned regularly, including underneath the grates and surface. You may need to lift these components out in order to do this. This should be done at least once a month.

*Perform as many steps as possible within the biosafety cabinet. It may be challenging to soak or rinse the tank inside the hood, but many other components can be placed to dry or soak inside the hood throughout the process as needed.
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2. Line the flat surface inside the biosafety cabinet with either CaviWipes or Paper Towels sprayed with 70% ethanol.

*Lining the surface with CaviWipes or ethanol-sprayed paper towels increases the contact time between the surface and the ethanol and/or disinfecting agents, as opposed to only wiping down the surface and placing the items to dry.
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4. Carefully remove the sheath probe/sensor from stainless steel sheath tank using a wrench. THE PROBE CANNOT BE AUTOCLAVED. Rest the probe on the lined surface. Remove all teflon tape residue, using q-tips and razor as needed to remove pieces. 
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5. Prepare 1000mL (1L) of 70% ethanol in a 1000mL graduated cylinder. Set aside 300mL in a separate container for other uses. Leave the cylinder filled with 700mL of 70% ethanol and place it inside the biosafety cabinet.

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6. This step is optional, but useful if contamination is suspected, or if rust is observed: carefully remove the two connector fittings using a wrench. Remove all remaining pieces of teflon tape from the fittings. 

If no rust is detected on the connectors: Soak them in 70% ethanol for 10 minutes in a small container using the 300mL of 70% ethanol prepared in the previous step. Once the 10 minutes have passed, rinse them with DI and place them to dry on a lined surface in the biosafety cabinet. These connections can also be autoclaved in a separate autoclave safe container; such as the one where the lid and its components will be placed.

If rust is detected on the connectors: Soak the fittings in distilled white vinegar overnight in a small container. The next day, use a stainless steel brush to remove the rust easily from the connectors. Rinse and dry well.

If rust is detected on the surface of the tank: use Mr. Clean Magic Eraser to remove the rust. These are milder than steel wool and will remove most rust spots from the tank surface without damaging the tank finish.
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7. Using a long Q-tip, razor, or similar instrument, remove any remaining pieces of teflon tape from the thread where the probe was seated and where the connector fittings were seated (if the connector fittings are removed).
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8. Remove the pressure gauge using a wrench. 

*This is optional as the pressure gauge can be autoclaved. However, this step is included as there is a very small risk that it could stop working.
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9. Spray tank interior with 70% ethanol and allow to sit for 1-2 minutes. 

*10 minutes is the ideal contact time for cleaning with 70% ethanol, but it is possible that the ethanol may dry out if left to sit for that long. The main purpose of this step in this context is to remove any organic matter from the tank. 1-2 minutes has been sufficient for this purpose.

Autoclaving does not remove organic matter, dirt, teflon tape remnants, or dried out sheath fluid. This is why it is necessary to take these steps to prepare the tank for the autoclave.
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11. Rinse tank with filtered DI water twice. You do not have to fill the tank with DI, but you need to ensure that all ethanol, organic matter, dirt, teflon tape remnants, or sheath fluid residue is removed.

Make sure to dry the tank well using a sturdy paper towel that is resistant to disintegrating. DO NOT USE KIMWIPES as they are not strong enough for this task and will leave remnants behind.
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12. Once the tank has been cleaned, cover all openings with aluminum foil. Use autoclave tape to secure the aluminum foil.
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3. Set aside the tank lid and rubber o-ring.
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13. Begin working on the tank lid and o-ring. Remove the plastic fittings from the tank lid. Separate the o-ring from the lid. 
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14. Using either a CaviWipe or 70% ethanol spray, wipe the stainless steel tank lid and the plastic fittings. The purpose of this step is to remove any organic matter, dirt, or dried out sheath fluid. Also inspect the o-ring for cracks and fissures. If the o-ring appears to be compromised, discard it and use a new one.

Dry the tank lid with a paper towel.
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15. Dry the tank lid and o-ring with a paper towel.
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16. Place the tank lid and the o-ring in an autoclave-safe container. Then cover all openings of the container with aluminum foil. Secure the foil with autoclave tape.
 

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17. Autoclave the tank for 1 cycle: 121°C, 15 psi, 20 minutes.

*Every autoclave is different. On the autoclave that is regularly used for this protocol, the dry exhaust cycle includes the 20 minutes plus additional necessary time, totaling 49 minutes.
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18. At the end of the autoclave cycle, carefully remove the tank and the container with the lid from the autoclave. Make sure you are wearing your autoclave gloves or welding gloves to protect your hands and arms from the heat as the tank and container will be very hot.
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19. Allow the tank to cool to room temperature before proceeding with relevant steps that require handling the tank. It is not safe to work on the tank while it is hot. Additionally, you should not refill the tank with cold or room temperature sheath fluid until it has cooled completely as it could damage the tank.

*Time management suggestion: allow at least 2 hours for the tank to cool. Or, wait until the end of the day to place the tank in the autoclave. Remove the tank from the autoclave once the cycle is done and allow it to cool overnight.
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20. Before replacing the probe, take the probe to the graduated cylinder prepared with 700mL of 70% ethanol. Then place the probe in carefully in the cylinder, taking care to make sure that the connector and wire do not come in contact with the liquid.
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21. Set a timer for 10 minutes. Soak the probe for 10 minutes.


 


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22. Remove the old paper towels from the biosafety cabinet surface. Then, once again line the surface in the biosafety cabinet with either CaviWipes or Paper Towels sprayed with 70% ethanol
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23. After 10 minutes have passed, remove the probe from the cylinder and carefully rinse the probe with DI water. Then place it on the newly lined flat surface.
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24. Re-insert the probe: ensure a proper seal by first wrapping the thread neatly with teflon tape and tighten with wrench. 

If the connections were also removed, wrap the thread neatly for each with teflon tape and tighten with a wrench. 

Add the pressure gauge back to the tank. Use fresh teflon tape and a wrench.

If the tank will be filled with sheath for use on the sorter right away, fill it immediately in a biosafety cabinet with fresh filtered sheath fluid. Close the lid carefully.

 

If the tank will be placed in storage, place the lid with the o-ring back on the tank lid and close the tank. Alternatively, you can remove the tank immediately from the autoclave, skip removing the foil, skip taking the lid out of the autoclave-safe container, and wait to re-install the probe. You can store as-is until you are ready to re-fill the tank. If you do store it in this manner, you may wait to perform the 10 minute chemical decontamination of the probe until immediately before you place the tank in use, and re-install the probe at that time.
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25. Before fluidics startup: When the tank is connected to the air-line of the instrument, listen for the sound of air escaping. It is common after autoclaving of the tank that there may have been some shifting. Or if parts have been removed and replaced, they may need to be tightened further. Most commonly, there are issues with the seating of the lid and the o-ring as they may very slightly warp. This is easy to correct, but it may require that the lid be tightened further by hand.

Do not machine tighten any parts. Always tighten by hand using a wrench. 
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10.