Primary Neuron Contamination - (Apr/24/2012 )

I've been running into an issue recently and I was hoping someone here might have some insight. I've been culturing primary neuronal cell cultures from P0/1 animals for about a year now with little to no problem. However, in the past week or two I seem to be getting contamination in my cultures right from the beginning. I get a noticable smell after 3-4 days in the incubator when I go to change out the media, and I can see little black spots in the cultures, mostly associated with the neurons themselves but also between neurons.

Just yesterday I changed out all my media/B27 opened up new aliquots of L-glut and poly-d-lysine for coating and a new bottle of sterile H2O. However, after I plated the cells I looked at them about an hour later, and there already appeared to be quite a bit of contamination that was almost entirely associated with the neurons themselves with very little between neurons. They are black spots that show a low level of movement. I can't imagine such a rapid infection is coming from one of my newly opened up bottles of reagents...

The only other things I can think of is:

1) my dissection tools are contaminated (I soak them in 70% EtOH for 10-20 minutes prior to use, but perhaps I'll start autoclaving them)

2) the animals are infected

Not sure how to handle the second one if it is the case, has anyone had any experience with these sorts of issues and come to a resolution?

Thanks so much.

MM

Ah, the delights of primary cell culture.

To check your medium for contamination, set up a well of medium with a coverslip but no cells and leave it in the incubator for a couple of days. If that stays clean, then you know your reagents are OK.

It sounds more likely that the contamination is coming from your tissue harvest.

1. Autoclaving instruments is a good start, or using a glass bead sterilizer to sterilize your instruments just before harvest, if you have one.

2. An infection within the animals is unlikely, but it could be worth asking the vivarium staff if they have had any recent issues with the sentinel animals in your room. It's more likely that the contamination is from the animals' skin during tissue harvest. Do you disinfect the animals' skin before you start the operation? One method I know people use that would be simple with neonates is to just immerse the animals in 70% EtOH and dry them off before starting (after euthanizing of course).

Also, do you use any enzymes to dissociate the cells before plating? If so, that could be the source of contamination. If it persists, it may be worth it to buy new batches of enzyme.

Thanks for your thoughtful response gfischer...I have incubated all of the components of my media (individually and separately) to see if any of that is contaminated. So far, there doesn't appear to be anything but it's only been about 24hrs.

I may try autoclaving my instruments, although I'd be surprised if all of a sudden this bit is an issue, but who knows?!

I have not been sterilizing animals prior to harvest, perhaps I will try spraying them down with EtOH after I sacrafice them... at the moment I'm testing all the hoods we use for contamination and am going to try growing the contaminant in LB to see if it is indeed bacteria.

As for the enzyme, I do use papain, but I filter sterilize it after I dilute it, and these buggers are quite large so I don't think that is a likely source.

You are right, it is probably not the animals (just trying to consider all my options) as I just heard back from some other folks in another building that also do neuronal cell culture but are not having this problem. However, someone else in the same TC suite as me but using all different media/tools etc. is having the same problem...the thing that just baffles me though is that I see the infection immediately after plating the cells (within about an hour). They really do seem to be there from the start, that is what is so bizarre...

Any further thoughts would be more than welcome.

Cheers,

MM

Hi Mighty Mouse,

It is quite inusual to have a contamination just from the begining, so I suppose that you are getting a bit paranoic with the contamination problem. If you changed your mediums you did almost all. I do not autoclave my material, only EtOH spray them before use. I recomend you two things, first add Pen/strep to all your mediums, and second autoclave the incunbators and clean the hood with Deconex and EtOH amd change all the material (Pasteur Pipettes and tips) from the hood to avoid any bacteria source.
All the best.

Hi MM,

Do you use serum? If yes, do you filter it? Sometimes there's a lot of debris in serum. I thought I had contamination once (also primary neuronal cultures), but it was just a lot of gunk that came with unfiltered serum.

HogtheHedge

MM -

Were you able to discover the problem? I'm seeing exactly what you have described in your cultures - I UV all my tools prior to use and spray each head with 70% ETOH before making the cultures....

Just wondering what solved your issue.


Thanks.