Getting crazy because of cell contamination - (Sep/29/2005 )

Hello,

As a starting PhD-student, I am working now with cell cultures (human tumour cell lines like A549, CAL-27, H292,...) for about 1 year. I seldom had contaminations (we don't use antibiotics), but since last month, almost every test I do is contaminated after several days!!

Description of the phenomenon:
- the contamination is only apparent after several (sometimes more than 4) days
- the colour of the medium doesn't change that much, but it becomes quite trouble and "cloudy"

I already cleaned the whole laboratory, the incubator,... and I already made new glutamine, trypsine, sodium pyruvate, serum,... I even have already unfrozen new cells, but after a while they get contaminated too (and no, it is not the stock which is contaminated, unless the whole N2-stock is contaminated suddenly, which seems me not very realistic).
The contamination just keeps appearing and I am really getting desperate...

Can anyone suggest what the cause could be?
Or can anyone give me some advise about how I can find (and eliminate!) the cause?

Thanks!!
An

How are you sure your freeze down stock is not contaminated? The time-line you see the "bloom" in contamination sounds like something that came from the freezedown stock, plus it sounds like you have taken many of the other obvious measures to avoid future contamination.

If the prob is not the freeze stock then... try to check if something ahas been recently changed in your materials, solutions or lab instruments that can be involved with your cells contamination. It seems your a taking all the required attention to avoid it but ther must be a cause for this contaminations,

I dont think something can survive N2 condition... but I think bacteria which can be already in your cell colture before freezing can survive freezing procedure cause they receive the same treatment as the others cells... I am not expert on microbiology but I heard theyr resistant.

Hope to be someway helpful

Well let's try to give you some help !

How does the culture look under the microscope ? Do you tiny points dancing happily ? Or do you see kind of rods merging together like this picture ?

Even if you clean thouroughly the incubator it can still be in the ventilation system specially for fungi contamination cause they are very hard to get rid of (spore forms)

Did you try to incubate a flask with only medium ? If it get's contaminated the chance is pretty high that it comes either form media components or from the incubator !

Let's see !

pesji

Guys


bacteria are way happy in N2....actually they froze beatifully...and when you thaw your cell, the sleeping beautys will rise and shine...

Hello!

First of all: thanks a lot for all your tips!!

To respond at your suggestions:

- The contaminations look like little (black) dots, but I can't detect any rods or other forms. In addition, I think the dots are not adherent to the bottom of the flask. And the morphology cells themselves doesn't seem to change that much: they don't lose their adhesion to the bottom and no death cells are visible.

- Yes, I already incubated a flask with medium alone. And sometimes this medium is contaminated too. However, sometimes I used the medium already, so I am not really sure if the medium isn't contaminated because it came in contact with a contaminated culture (if you can follow me). Therefore, I will now make a flask of medium alone immediately after I made the medium but before I use it (i.e. after the addition of serum, glutamine and sodium pyruvate).

- My intuition tells me the frozen stock is not the problem. I rather think one of the components I use, or the incubator are the cause of my troubles.
Does anyone knows a procedure to detect or figure out what the contaminating organism is? Is there maybe a colouring test or something else I could use?

have you attempted to streak a loop onto blood agar? if it is yeast (very common) or bacteria from skin (also very common) you would likely see growth in a day or two. noting the growth rate, in combination with a simple gram stain will give you some clues as to what it is, at least generally. you could also do a quick gram stain of some juice from the culture itself.

it sounds like bacteria, if it is tiny black spots. probably some staph or something.

as an aside:
we once thought we were getting some very strange new contiminant in our epithelial cultures. right after changing the media, we were seeing little black dots that we were just sure were some form of yeast. after testing all sorts of theories over a couple of days, we realized that we were looking at air bubbles. we had changed pipet vendors; the bore of the pipet was a little different and more air bubbles were induced during pipetting. we sure felt dumb..but it was nice not to have contamination. when two air bubbles were immediately next to each other, it even looked like the yeast were budding...

Hello!

First of all: thanks a lot for all your tips!!

To respond at your suggestions:

- The contaminations look like little (black) dots, but I can't detect any rods or other forms. In addition, I think the dots are not adherent to the bottom of the flask. And the morphology cells themselves doesn't seem to change that much: they don't lose their adhesion to the bottom and no death cells are visible.

- Yes, I already incubated a flask with medium alone. And sometimes this medium is contaminated too. However, sometimes I used the medium already, so I am not really sure if the medium isn't contaminated because it came in contact with a contaminated culture (if you can follow me). Therefore, I will now make a flask of medium alone immediately after I made the medium but before I use it (i.e. after the addition of serum, glutamine and sodium pyruvate).

- My intuition tells me the frozen stock is not the problem. I rather think one of the components I use, or the incubator are the cause of my troubles.
Does anyone knows a procedure to detect or figure out what the contaminating organism is? Is there maybe a colouring test or something else I could use?