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Rat Oligodendrocyte Precursor Isolation Issues - (Jun/13/2011 )

Hey,

I am following an established protocol for isolating rat OPCs: http://www.nature.com/nprot/journal/v2/n5/full/nprot.2007.149.html

I unfortunately have a couple of problems I'm running into. First, I've had some sort of contamination in 4 out of 5 litters. The first I simply bleached and dumped, second I created a slide with the culture medium and stained using crystal violet (I did not have gram's-iodine for gram's staining), third had a couple of flasks that looked possibly infected so I bleached and dumped to be safe, fourth was clear of visible infection and the fifth again shows infection.

I've created slides from the latest infection and will perform a gram's stain on these ones to see what's there. The previous slide with crystal violet had abundant cocci.

Steps I've taken so far to eliminate the source(s) of infection:
-Started using disposable plastic sleeve protectors over my lab coat
-After the ice anesthetizing, dip the pup in 95% ethanol before cutting the head off
-After removing the pups head, soak in 95% ethanol for 1 minute before rinsing in HBSS
-Adding pen/strep the HBSS the cortices are held in
-Rinsing the removed brain with HBSS before isolating cortices
-Cleaned incubators with detergent, sanitizer then ethanol
-Reautoclaved all equipment
-Dissecting equipment goes in 95% ethanol between pups
-For primary glial cultures I change the Pasteur after each flask


So the infection is the first problem, the second is to do with isolating OPCs. The flasks that weren't infected and did make it to the shaking isolation of OPCs seemed fine. I counted and resuspended my OPCs at 1 10^4 per cm^2, unfortunately the first set did not seem to grow great and yields were too low for western analysis. The second isolation worked better but I am still having trouble with 1)low OPC yield from each flask (~200,000 OPCs per T75 mixed glial culture) and 2)there seems to be a number of astrocytes contaminating the OPC isolates.

Sorry for the long post. Anyone with some experience with primary cultures have any ideas?

Thanks.

-Bob

-rjs625-

So I performed another dissection and have contamination again! Anyone have any suggestions? please and thanks.

-Bob

-rjs625-

Uploaded a picture of the adherent contamination...
Attached File

-rjs625-

Hi all,

Still having contamination in our primary cultures. I have tried culture media containing gentamicin (20ug/mL), pen/strep (50units/50ug/mL & 100units/100ug/mL). The latest attempt was adding 100ug/mL gentamicin, 1ug/mL amphotericin B, 400 units pen, 400 ug/mL strep to the dissection media (HBSS); this HBSS was used to rinse isolated cortices 3x before proceeding with tissue disassociation.

In addition to my original post:
-We have went back to 70% ethanol for the dipping/soaking steps
-Purchased new sporicidal disinfectant and cleaned all incubators/hoods/benches with it.
-All media used is supposed to be sterile filtered by our media department, we have tried resterile filtering it (0.2micron corning filter unit) before use.

All but 3 out of 13 separate dissections have produced grossly contaminated primary cultures. Even the 3 that were visibly clear may have had some low level contamination that went undetected. I am racking my brain to figure out the source! Considering all the steps taken to eliminate contamination, it seems like the animal is the only explanation; but even that seems like it should be addressed with the 1 minute soak in alcohol and the rinses of the cortices.

We are sending our cultures to a bacteriology lab. Hopefully they can culture the bacteria and provide susceptibilities. However, I have 2 more dissections this week and will not have results from the bacteriology lab; does anyone have any suggestions as to what I could try?

Thank you for any advice you can provide!
-Out of Ideas.

-rjs625-