Usually I add some copper sulphate to my (filtered) autoclaved distilled water in the water reservoir pan, but something orange-white (fungus?) keeps appearing in the water. What else can i add to it to prevent growth in it? SDS?
Dear , pls clean the pan thoroughly, sterilize it in Oven (100 degree for 20 minutes), add sterilized distilled with with increased concentration of copper sulphate. TQ and good luck for you cell culturing work. TQ
Hi, We also use CuSO4. I think you just need higher conc. Also take out the racks once in few weeks, clean them with alcohol and may be autoclave as suggested already. Nothing should grow then. Please see if your racks have good quality body, else it might be that due to moisture the coating is coming off and floats on the water you add to your incubator!
Hello,
To avoid the contamination of CO2 incubator water , several commercial products exists such as :
* Aquagard (from Promokine company) : http://www.promokine.info/products/microbial-detection-elimination/disinfectants/aquaguard/
* Aqua EZ Clean (from T-Pro Biotechnology company) : http://www.tprobio.com/?aqua-ez-clean,38
These products are used as additive in the water, and leads to a protection against several potential contamination sources of the incubator water.
Sincerly,
In our lab, we use Spor-Klenz to wipe down the inside of the incubator. The water in the pan at the bottom is ddH20 that has been autoclaved. We also add Clear Bath to the water, 250ul/L of H20.
If you change the water once a week you do not have to add any chemicals. 30 years ago manufacturers sold copper lined CO2 Incubators stating that the copper acts as an antibacterial/fungal agent. WE STILL GOT CONTAMINATION even putting copper sulphate solution into the water tray.
Modern CO2 Incubators are lined with 304 grade stainless steel and in one piece i.e. it is easier to clean. They also have have temperature decontamination cycles to aid in combating microbial contamination.
Good lab practice should prevent all types of contamination in cell culture incubators
@ Sawsan Elhalawani : I have read that sodium azide when it reacts with water forms highly toxic volatile fumes. Have you experienced any sudden cell death or morphological distortions in your cultures?
Jhoti,
We’ve had some luck adding chemical solutions to the humidity pan Though, often times, the mysterious contaminant will return. If you haven’t already, I would suggest autoclaving your pan or even ordering a replacement. Certain distilled waters or chemicals can cause pitting in the metals of the pan, which creates a microenvironment for microbial growth. Have you eliminated other sources of contamination? Microbes often reside in or on the water bath, on lab coats, in the flow hood, and on the benchtop and can easily (and inadvertently) carried into the incubator where they’ll thrive.
Does your incubator allow any sort of contamination control or decontamination? The latest incubators provide constant or post-door-opening UV irradiation during day-to-day culturing. In these incubators, the UV light is focused in such a way (using a back plenum) that it irradiates and decontaminates circulating air and your humidity pan, but does not irradiate your cultures. It’s a great way to prevent recurrent microbial growth.
Recurrent contamination can also be facilitated by higher than necessary humidity levels, perhaps caused by an overfilled humidity pan. Some incubators have Peltier-cooled dew sticks that help reduce contamination problems from overhumidification (or condensation). The Peltier-controlled dew stick is hooked up to a probe that monitors ambient temperature and cools the dew stick slightly (without affecting the incubator temp) to draw in condensed water (similar to a cold glass of water on a humid day). The stick itself has an antimicrobial coating that resists contaminants and dips the condensate back into the humidity pan. These types of sticks function best when the incubator allows continuous, shielded UV irradiation of the dew stick and humidity pan to prevent spread of microbial growth through condensation.
Copper has been identified as harmful to the environment, and you should try to phase it out of your lab. Stanford even has a prohibition on copper-based algaecides. Your local city regulations might be similar, so check with them.
https://lbre.stanford.edu/sites/all/lbre-shared/files/docs_public/water_bath_algicides.pdf
The notice suggests the following copper-free alternatives
Chloramine-T
Thermo Revco Cleaning Agent
Spectrum Clear Bath
I use a a foot of 18 gauge bare copper wire balled/twisted up and stuffed into a 5 or 15 mL conical tube with several small holes drilled in it...this allows for plenty contact with the water, while preventing direct metal-to-metal contact between the copper and stainless steel, which could cause galvanic corrosion.
I've found that this copper metal method prevents microbial growth in water baths of incubators and rotary evaporators without the need for copper sulfate or proprietary commercial products. Its best to use DI water in the water bath, as its a slightly more "aggressive" solute than plain old tap water...in theory this helps ensure a higher level of dissolved copper ions in solution, but who knows.
For a more aggressive water-bath biocide, I would recommend 0.05-0.1% benzalkonium chloride...its a (relatively) cheap, stable and non-volatile broad-spectrum biocide of the quaternary ammonium ("quats") family...AKA the stuff in Lysol other than ethanol...downside is that its toxic and possibly a persistent environmental pollutant, but don't quote me on that last bit.
The best thing to do is remove your water pan once a week and autoclave it. While the chemicals work sometimes, they can also be harmful to your cells and the environment. I suggest stripping your incubator and thoroughly cleaning it. Get in a good rotation of autoclaving the pan and shelves once a week. This should keep your incubator clean without having to worry about chemical additives.
Additionally, do not add more water than is necessary to your pan. 1-1.5L for a 37 degree C incubator should be all that you need to last the week.
Hi,
I recommend neoLab BAD-Stabil (R) , never failed us. However, I agree that you should remove the pan once a month, clean it (with Bleach or Ethanol) and refill it with fresh water + BAD-Stabil (Or similar).
Hi
I usually add 0.2 gr copper sulphate per 100 ml autoclaved water distillation twice.
Leilasadat, many jurisdictions (at least in California, US) are prohibiting the use of copper solutions as they can cause damage to aquatic life.
Our lab uses Aquaguard-1 (Biological Industries), specifically made for humidified CO2 incubators. Wide spectrum product, and pretty safe chemical profile.
Go to Home Depot cut about 2in piece of copper pipe from a plumbing department throw it in the autoclave, and stick it in the incubator pan after sterilized.
I use benzalkonium chloride 0.02% final concentration in distilled water at the water tray and change it ever 15 to 20 days and works well.
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