This is my first post on Research Gate, so bare with me...
I am a new grad student who recently picked a dissertation lab and will be doing A LOT of cell cultures (specifically these are primary neuronal cultures of hippocampal tissue from P1-3 mice, supplemented at DIV 10 with microglia). The room we are using for our own in house cultures was not originally designed to be a culture room but works fairly well according to previous students. Recently, I started a new culture only to find that my incubator has become contaminated!!! I talked with the previous grad student and he says this happens occasionally because the room has moderate traffic from other students, and isn't the most clean to begin with. Does anyone have any tips for maintaining a clean culture room in a 'not-so-clean' environment?
Thanks!
Dear Joe
I think it will be possible to work cell culture in your lab - it is mainly organization. Where we are working was an office before! And the environmental conditions are not the best here, too.
What we did was separating processes:
- One specific working area *exclusively* for cell culture with *restricted access*.
- Only personnel who needs to work there can enter. Only those are doing the cleaning.
- We use shoes and coats exclusively for working in cell culture to reduce possible contaminations.
- When students want/need to work cell culture, they have to do a training with the professor/responsible of the area to guarantee good working procedures. No self-teaching or "Chinese whispers".
- We do NOT use antibiotics in cell culture for not covering bad working habits.
- Material/glass ware used in cell culture are marked or different shaped (e.g. square vs round bottles) for exclusive use in cell culture. Those have not to be mixed with other laboratory areas/processes.
- Our incubator has a Cu-alloy and for humidifying we use water with CuSO4 to reduce bacterial growth.
- The clean bench is not a storeroom - only material used in the experiment should enter. We use 10 min UV irradiation to sterilize surfaces. Only 1 person can work at the clean bench at the time.
- Use gloves and spray with sufficient EtOH (70%) material, which enters the clean bench (including hands).
- If the incubator got contaminated, it has to be decontaminated immediately: 180 °C and/or EtOH. If you are lucky you have 2 incubators and can clean one regularly w/o interfering in your experiments.
With this we have contaminations very seldom and usually we get them by the samples to be tested (e.g. natural extracts).
I hope these ideas help. Not "good luck" but "success"!
Kind regards
Peter
The personnel flow control definitely will help. I assume biosafety cabinet is being used for manipulation of the cells. To ensure the cabinet work efficiently, minimal equipment (mainly micro pipettors) should be kept in the cabinet. Thirty minutes UV irradiation can be used prior and after the work. Keep the lab coats clean (regular washing and autoclaving).
The incubator requires maintenance every six months. The metal racks and shelves can be taken off and autoclaved, and the HEPA filter (which is very important for the air exchange) needs to replaced. The primary cells can be a source of contamination. The sterilization of tissue surface can be considered if the mouse has been exposed to non-SPF environment. Chronic mycoplasma infections can be a potential issue that is not easily found but would severely alter the cell metabolism, a microbiology assay is required for excluding such contamination.
you may use all of the above suggestion, however I am adding if you can use an isolated cell culture chamber in your incubator to prevent the contamination of your long term cultures
https://www.nippongenetics.eu/en/cat/cell-biology/storage-4/cell-culture-chamber-storage-4/