"What are your tips and tricks for preventing contamination in an animal cell culture lab? Any hard-learned lessons or overlooked practices you'd recommend?"
I use a decent amout of antibiotics in medium and dpbs. Also always make sure the tips are sterile and if I accidently touch anything outside the bottles with the tip, I discard it and take a new one. However sometimes I can't notice that and disaster happenes. If I suspect something might have gone wrong I filter all the liquids with 0.22 syringe filter or take a new bottle. However, if contamination happenes, I learned how to fix it with almost lethal doses of antibiotics exposure for a short time. When cells are about to die, bacteria are already dead, lol. Then I wash it for a few times and add a new meduim, so for next passages my cells are free of contamination.
Should i autoclave the distilled water in the CO2 incubator, can it be a source of contamination ? Lina M. Yanygina
Can you tell me what is the protocol to fix the contamination with lethal doses of antibiotics? Lina M. Yanygina
Can you please help me to define what is this in my cell culture? Philippe Paget-Bailly
Here's what I'm currently doing: Before every experiment—or even just checking cells under the microscope—I disinfect all surfaces and devices in the lab. I disinfect every small part inside the biosafety cabinet, all disposables, tip boxes, micropipettes, and basically anything my hands will come into contact with. It uses a lot of alcohol.
After that, I run UV light for 40 minutes in both the lab and inside the safety cabinet. I also make sure the air curtain is running. Despite all of this, I’m still experiencing a high rate of contamination. It's driving me crazy.
What else can I do? Should I consider fumigation? Philippe Paget-Bailly
Yasmine Mohsen I used penstrep but this year contamination has been happening quite often so I switched to gentamicyne. I must mention that I work with MSC from adipose tissue and ARPE19 - they both feel great on that antibiotic. I don't have lab grade gentamycine, so I took in pharmacy ampules with 40 mg/ml solution (2 ml in 1 ampule). I washed celles in flasks thoroughly with DPBS to remove as much bacteria as possible and added a mix of DBPS and gentamycine for incubation for 1-2 minutes. (50 ml DBPS and 1 ml gentamycine, or you can try your volumes). I shook the flask to wash over the walls. I watched in microscope how my cells go round and ready to detatch and die and before that removed the liquid quickly and added fresh medium. They attached again, spreaded and continued to grow normally. In medium bottle of 500ml I add 70 microliters of gentamycine as a penstrep substitute now. I have the same flasks for several months and the same cells (MSC) and don't have any sign of bacteria. The method is risky though, I guess.
If you have contamination often and you use a lot of ethanol and disinfect everything including your hands\gloves in sterile hood with laminar flow, you may consider washing the hood with antiseptics, changing UV lamp in the hood and filter in laminar flow. They can be expired. Check your liquids as well, they can be contminated. If you move anything inside the hood bath it in ethanol again including your gloves. Your coat sleeves and hair also can be the sourse of contamination, keep it in mind. Wash Co2 incubator once a week with h2o2.
Thank you so much Philippe, i will take it into considerations Philippe Paget-Bailly
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