Dear Rania,

I have only cultured macroglia, and I have never had to re-filter my media. I buy high quality FBS that is triple-filtered to remove as much contamination as possible, and the DME is tested for endotoxin, etc. I use gentamicin rather than Pen/Strep however. I find it works better with primary neuronal cells, particularly when they are derived from model organisms where it can be difficult to perform a sterile dissection (e.g. fish have a slime coat). In our lab we have usually had each investigator make their own small bottles of supplemented media in order to prevent cross-contamination, particularly when someone is new to the lab.

I have only used vacuum filtration for sterilization when I make my own media from powder. When I have made my own supplements (N2, etc.) I used syringe filters for sterilization before they are aliquotted for storage. With vacuum filtration the pH can change, particularly if there is a lot of sodium bicarbonate in the media (DME has lots).

You can always perform a sterility test first of your media by incubating some in plates without cells, but handled in the same hoods in the same facility where you will work. It could be there is something in the air or room of the other lab that is causing a problem (or even in the incubator itself).

Good Luck!

Jill

Hi Sebastian and Jill,

Thank you so much-This was great feedback and also great advice! As of now I am not supplementing my media with differentiation factors, but only with Heat-inactivated Low-endotoxin FBS and Pen/Strep (& all our solutions and media are purchased, we don't really make anything).

One issue I have realised is that the tissue culture hood we have access to does not have a working UV, and to keep the hood clean the blower is always turned on and the hood is constantly cleaned with a combination of virox/clidox and ethanol. Interestingly, this hood belongs to the lab that recommended we use vacuum filters for preparing our media... I wonder now though if the UV not working and the method of cleaning the hood is really enough to maintain the sterility of the hood. As I mentioned earlier, I am working to set-up primary mixed glial cultures and then will work to isolate primary microglia cultures. Should I be worried about the effects this may have on the baseline activation of microglia in culture?

Thank you again for your constructive advice!

Dear Rania,

Trying to determine sterility of the airflow in a hood can be a problem, especially in common use facilities. I never used the UV lamps in our hoods because they lose power over time, and so you have to keep cleaning the area anyway. What we would have to do is have our hood certified annually (especially the hoods where we handle biohazards). There is usually a company that has a contract with your institution that does this on a regularly scheduled basis.

However, if the HEPA filters are functioning and the air flow is always on, you have a pretty good chance of maintaining a sterile field. I suggest you talk to the lab that owns the hood to see if they have done a sterility test lately, or if it is scheduled for re-certification.

Having said all of this, the cost of the animals from which you will derive your primary cultures is not insignificant. Syringe filters are relatively inexpensive. If you are worried, filter small batches of media if you need to get started on your project (e.g. you have timed pregnant animals) before you have determined the source of the problem.

I will also note that sometimes contamination can come from interesting sources: lab coats, sloppy co-workers, dust on plastic ware or supplies stored on open shelves, cardboard in the culture room, poorly cleaned instruments, Pipetmen (use filter tips!), even glove powder (I only use unpowdered gloves). And, as Sebastian mentioned, prep the brains in a different area. Animals are just a natural source of contaminants, and it is one of the challenges of primary cell culture.

I also use several sets of dissection tools: One to open the skin, one to open the cranium, a third to remove the brain, another set to remove meninges. And I transfer to a new dissection plate with new dissection media to remove the meninges, and then put the brain in a separate plate to dissect my area of interest. I then collect my dissected brain areas in a new plate in order to be chopped into smaller pieces before dissociation. Dissection media is cheap, and serial washes help dilute possible contaminants.

Good Luck!

Jill

Jill & Sebastian, I appreciate your guidance and your thoughtfully detailed responses. Thank you so very much for your help.

Best wishes on your endeavors,

Rania