Sad CO2 Incubator...

[Yvonne Couch]

Hi all,
So I am in the process of moving into a new lab. As everyone does I come in with pre-conceived ideas about how to do things and obviously everyone does things differently but I am genuinely concerned about their CO2 incubator. It is almost ENTIRELY rust on the inside. Its very smooth so there's no flaking but I'm concerned that it's so rusty. My questions are as follows:
a) is rust bad in a cell culture incubator? I assume yes but I've genuinely never seen it before so I couldn't really say...
b) how would you get rid of it without making the incubator permanently toxic? There is no second incubator running with the same gases at the moment so the rust would have to be cleaned a shelf at a time so anything that lingered on the shelves might affect the cells.
Any thoughts?
Y.

[WS]

Hi Yvonne,

sure it's rusted iron and not oxidized copper? Actually, if steel is used, then it should be stainless steel.

Anyway, like DK, I would not be upset too much, as iron oxide is not volatile and not likely to support growth of bacteria and fungi (maybe some extremophiles will like it, however :).

[Cathal Garvey]

-----BEGIN PGP SIGNED MESSAGE-----
Hash: SHA256

Hi Yvonne,
I don't know what to suggest about removing the rust without further
compromising the surface, unless you wanted to re-enamel over the
surface after rust removal? There will be commercial products for this
sort of thing, most likely targeted towards oven repair.

I can imagine how Rust would increase the chances of contamination,
both by providing a ready source of Iron (often limiting to biofilms)
and by providing a rough surface to protect contaminants from exposure
to sprays etc..

I can also imagine this being a common problem; I used to work in a
lab that sprayed everything with strong bleaching agents, which (I am
lead to understand) can lead to pitting of stainless steel over time,
making rust more and more likely. They also used copper sulphate as a
biocide in the water tray of the incubators, which was a good idea,
but the tray was also steel. I'm not sure why it didn't ruin the tray,
but copper sulphate will usually react very quickly with
iron-containing surfaces, which would pit them even faster. If someone
had the "bright idea" of using copper biocides to spray the inside of
your incubator, that would help explain the extreme corrosion.

Best,
Cathal

> _______________________________________________ Methods mailing
> list Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>

- --
Please note my new email: cathal...@cathalgarvey.me
PGP Key: 988B9099
Bitmessage: BM-opSmZfNZHSzGDwdD5KzTnuKbzevSEDNXL
Twitter: @onetruecathal
Code: https://gitorious.org/~cathalgarvey
Blog: http://www.indiebiotech.com
-----BEGIN PGP SIGNATURE-----
Version: GnuPG v1.4.11 (GNU/Linux)
Comment: Using GnuPG with Thunderbird - http://www.enigmail.net/
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=AC/Z
-----END PGP SIGNATURE-----

[WS]

[Deitiker, Philip R]

Questions I would ask are:

How was the incubator being cleaned previously, does the manufacturer have a specific recommendation for cleaning?
If the rust is on the outside I would ask whether the incubator had been kept in a cold room or taken back and forth from a cold room, whether it was stored in an area where strong acids were used?
Is it possible that the deposits were transported from outside to inside the incubator?
Who made the incubator, do they have a problem with rusting?
The reason why I would ask further questions is that rust is not a typical problem inside a stainless steel incubator.

For typical rust problems (lime buildup etc) phosphoric acid can, with adequate input of 'elbow grease' get the corrosion off. To compare the two acids, hydrochloric acid (see end of reply) reacts with metal oxides and metal carbonates on the surface forming soluble metal chlorides and releasing or absorbing carbon dioxide or hydroxide, respectively. The metal phosphates are not as soluble as the chloride salts, but they change the crystal structure adequately to loosen minerals and allow them to be scrubbed away. Phosphoric acid typically does not produce deadly gases and the phosphate stabilizes the surface of the metal. Of course the surface needs to be effectively washed and neutralize when completed. You can use a dilute sodium bicarbonate to neutralize followed by rinsing to remove sodium ions.

Special notes on cleaning.
1. Certain components in the incubator may not be of the plate-metal component. For example you might have silver/mercury welds holding plates together, there may be brass, copper or aluminum components. There are specific cleaners designed for copper and brass that are safer to use. Hydrochloric acid should never be used on aluminum. Physical scrubbing methods are best, but mild phosphoric acid can be used and cleaned off quickly.
Welds that contain mercury may release mercury on treatment with acid, so wear gloves and use ventilation.

For the most part, I leave the patina on the brass components as long as it is not flaky, I have never had a problem with this type of oxidation in an incubator. With the sensors the most I have done to clean is used light spraying of 70% EtOH to sterilize to wipe and allow to air dry. If you sufficient modify the environment of the sensor, you may inadvertently change CO2 calibration.

2. Certain components (e.g. internal ventilation ducts, shelves and shelf holders) can be removed and cleaned outside the incubator, mechanical cleaning is a good way of getting much of the rust off. Cleaning with green pads or brillo pads will get bad spots, what's left - use the phosphoric acid to scrub off. The good thing about these removable components is that after scrubbing they can be autoclaved. Autoclaving helps to remove residual acids and also sterilize the components. Always unplug the incubator to remove ductwork as these can damage the circulation fan.

3. Check your ventilation. Certain older incubation have a fan that sets in the chamber. If the fan is slow, rattling, or stopped then it may need to be disassembled and the bearings cleaned and lubricated. It is better to remove or wrap the sensor/fan assembly if possible for deep cleaning of the incubator. Tape off the hole where the instruments set as to prevent corrosive substances from entering the plenum space. Since you got the fan out, see if you can get some light machine oil and lubricate the bearings, inspect the fan blade, if it vibrates with running replace the fan blade. Since this is an electrical component you need to unplug the incubator, and you will have to disconnect the fan motor and sensor relays. In some cases the fan motor can be removed from the sensor/fan assembly, in these case you can wrap the sensor assembly in a plastic bag and proceed with cleaning. The removed motor can be inspected and lubricated as needed.

4. Check the door seals. One reason for rust (or where it generally occurs) is around the door seals. This is a place where harmful bacteria tend to grow and they produce acids that can over time rust equipment. The CO2 in the incubator already lowers the pH to around pH5.0, so it does not take much to lower the pH into the range where stainless steel starts to rust. These seals need to be occasionally cleaned (with a mild soap like SDS) and lightly lubricated and carefully wiped dry. Avoid strong cleansers on rubber components and plastic, certain plastics are easily damaged by organic solvents. In general SDS, followed by water rinse. I have learned through experiences (mainly bad) that many soaps sold as antimicrobial or powerful cleansers (such as glassware soap) are damaging to rubbers and plastic. Alcohol, for instance, should never be used with many forms of plastic (e.g. plexiglass). Other plastics should never be autoclaved with certain soaps and some plastics will get damaged if autoclaved with soap residues that are not pH neutral.

5. Preventing rust. Our folks have found that for wet incubators you can use a large pyrex cooking tray for a water reservoir so that water does not need to cover the bottom. The benefit of using pyrex is that media spills and drips with condensate into the water, this eventually causes bacteria to grow. By having a pyrex glass water reservoir you can take it out frequently, clean, autoclave and add fresh sterile water. The bottom can be sprayed and wiped with 70% EtOH with minimal incubator downtime. Microbial growth in the bottom of an incubator is likely the source of rust and discoloration.

6. When you reassemble your wet incubator, always remember that the incubator CO2 concentration needs to be checked with the proper device. The incubator needs to run for 24 hours with a filled water reservoir and then CO2 should be checked with a Fyrite Test Kit. In most cases (in fact in all cases where deep cleaning of the incubator was required) the CO2 recalibration needs to be done. Also a good idea to check the thermostat/thermometer, there have been cases when this instrument is no longer accurate or has failed completely.

7. If your incubator has a water jacket, check your water jacket, rusting around the stopper hole can cause evaporation and loss of water. Loss of water in the jacket can cause uneven or unreliable heating or ultimately incubator failure. If the stopper hole is rusted then it should be cleaned and repaired and water level should be set according to manufacturer specification.

As per the question, does rust cause contamination. The answer is no. But rust creates more substrate areas for contamination to grow. And it makes it difficult to remove contamination. In addition it is a sign that the equipment has not been treated properly in the past, and this may also indicate ongoing abuse and other problems (such as in the sensor/fan assembly). Rust on stainless steel occurs because the pH has dropped below pH4 for an extended period of time.


Corrosion and rust can be completely removed with strong monovalent acids such as hydrochloric etc, assuming this is stainless steel and badly rusted.
Hydrochloric acid is very strong; however, and the fumes are difficult to work with. Even 0.1N or 0.01N hydrochloric acid can be difficult to work with, and it may damage electronic sensors in the machine. The less ventilated the work area the lower the concentration that would be safe to work with, and at some point the acid will simply not be effective at removing thicker deposits of metal oxides. With certain metals HCl also causes gas formation with can quickly degrade the metal and produce hydrogen gas, which is explosive (I have a good story about how explosive this reaction can be, so ...). Once a surface is treated with hydrochloric acid to remove the rust the surface needs to be rinsed with water and the washed with sodium bicarbonate, quickly and then rinsed with water. Metal surfaces that are treated this way are extremely reactive and remain so until they react with rare atmospheric gases, to prevent further corrosion lightly coat the metal with machine oil (e.g. sewing machine oil) and clean completely the next day. Note: failure to neutralize a stainless steel surface treated with strong acid will result in that surface rusting again within 24 hours, so once you start cleaning with strong acids you are committed to neutralize and shield the surface. Ergo this would be a technique reserved for spot treatment of really badly rusted stainless steel. HCL will pit all metals to some degree, that rust may be the only thing between the inside of the incubator and the water jacket, so any degree of pitting may cause complete failure.


Have 6 incubators in operation that were built in 1984, seen my fair share of problems.

-----Original Message-----
From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio.indiana.edu] On Behalf Of Yvonne Couch
Sent: Monday, March 11, 2013 3:07 PM
To: met...@magpie.bio.indiana.edu
Subject: Sad CO2 Incubator...

Hi all,
So I am in the process of moving into a new lab. As everyone does I come in with pre-conceived ideas about how to do things and obviously everyone does things differently but I am genuinely concerned about their CO2 incubator. It is almost ENTIRELY rust on the inside. Its very smooth so there's no flaking but I'm concerned that it's so rusty. My questions are as follows:
a) is rust bad in a cell culture incubator? I assume yes but I've genuinely never seen it before so I couldn't really say...
b) how would you get rid of it without making the incubator permanently toxic? There is no second incubator running with the same gases at the moment so the rust would have to be cleaned a shelf at a time so anything that lingered on the shelves might affect the cells.
Any thoughts?
Y.

_______________________________________________
Methods mailing list

http://www.bio.net/biomail/listinfo/methods