9 replies to this topic

[maverick3006]

Hi All,

I am trying to grow primary fibroblasts from skin of sclreoderma patients. Since the very beginning, I have been observing tiny oval structures in bunches (like grapes) contaminating my cultures. This has also resulted in increased TNF levels.

I suspect this to be yeast contamination, I am not sure.

Please tell me:

• How to ensure what kind of contamination is this?
• Assuming that it is yeast contamination, how to get rid of it?

I am aware that I have given very limited information regarding my predicament, but I was unsure of what more to add. Please help me out, I'll be obliged

Regards
Maverick

[Kamran]

Staphylococcus spp. show grapes like arrangements which could be easily be confirmed by inoculating 50-100μL culture supernatant on LB agar or any other agar medium you have at your disposal.

Yeast would also grow on these agar media however, colonial morphology and size would be significant different and subsequent gram-staining would confirm what kind of contamination was that.

To get rid of these miscreants you need to adopt good aseptic conditions in addition to adding antibiotics/antimycotic solution.

[maverick3006]

Thnx Kamran, will get back to you regarding what happens next.

[leelee]

Do they grow more abundant over time?
It surely sounds like contamination.

The only reasonable option is to throw them out and begin again.

(after thoroughly cleaning your equipment, incubator and hoods, and discarding all of your open media and additives, PBS and trypsin)

It is probably a waste of your time to find out what they are, as they will hopefully be a one off occurrence and you will never really know where the contamination occurred anyway.

If they do appear again either:
- your frozen stocks are contaminated
- your aseptic technique needs work

Its probably a good idea to have someone with a great deal of tissue culture experience watch you work a few times anyway- just to see if there are any mistakes in aseptic technique that you might be making.

Good luck

[leelee]

Also, best practice is to culture cells without the addition of antibiotics or anti fungals- as these can have an impact on the behaviour of your cell line, and may mask low level contaminations.

[maverick3006]

Thanks Leelee.......Unfortunately, this problem repeated itself even after repeated sterilisation procedures like you have outlined. I will try culturing the cells without antibiotics, though and hope for the best!

[rhombus]

Hi All,

I am trying to grow primary fibroblasts from skin of sclreoderma patients. Since the very beginning, I have been observing tiny oval structures in bunches (like grapes) contaminating my cultures. This has also resulted in increased TNF levels.

I suspect this to be yeast contamination, I am not sure.

Please tell me:

• How to ensure what kind of contamination is this?
• Assuming that it is yeast contamination, how to get rid of it?

I am aware that I have given very limited information regarding my predicament, but I was unsure of what more to add. Please help me out, I'll be obliged


Regards
Maverick

Dear Maverick,

Have you thought that the contamination is as a result of the underlying sclreoderma ?????
It may be that these patients are highly susesptable to skin yeast infections as they are regularly prescribed steriods to reduce their inflammatory respones....but which as a side effect makes it difficult to fight infections?????

Is it possible for you to include a nice phase contrast image of your infected cells?

Yeast (under the microscope) have an unmistakable morphology and would therefore be easily recognisable.

I look forward to your response

Kindest regards

Uncle Rhombus

[maverick3006]

Hi all.......here are some pix of these intruders. These have been taken 12 hours after changing the media in a culture maintained for 4 days. They look horrible, don't they??

45 and 46 are at 10X mag and 50 at 40X mag.

Uncle Rhombus- your suggestion about this being something to do without scleroderma itself makes sense to me. So, I am going to culture fibroblasts from healthy volunteers in a day or two and hope that they donot show any contamination.

In the meantime, based on these pic can anyone help me?? Can these cultures be salvaged or should I discard them?

Thanks all.

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[rhombus]

Hi all.......here are some pix of these intruders. These have been taken 12 hours after changing the media in a culture maintained for 4 days. They look horrible, don't they??

45 and 46 are at 10X mag and 50 at 40X mag.

Uncle Rhombus- your suggestion about this being something to do without scleroderma itself makes sense to me. So, I am going to culture fibroblasts from healthy volunteers in a day or two and hope that they donot show any contamination.

In the meantime, based on these pic can anyone help me?? Can these cultures be salvaged or should I discard them?

Thanks all.

Dear Maverick 3006

They are definetely contaminated and need to be chucked away. It is hard to tell if it is a yeast but I could not make out one contaminant that had a "budding" morphology, which is indicative of yeast infections.

The problem with "infections" in general is that the bug will release bacterial wall contents into the culture and this WILL affect the skin fibroblasts. Just the presence of bugs may affect the cells directly. If you try and use an antibiotic to kill the infection ...then this WILL definetely affect the fibroblasts.

Your idea of preparing fibroblasts from so called "healthy" patients is a good one.

Generally speaking fungal contaminations come from the local environment i.e. the CO2 incubator

Yeast and other bacterial infections can come from poor aseptic technique as leelee quite correctly states.

Good luck

Kindest regards

Uncle Rhombus

[maverick3006]

Hi All,

Thanx a bunch for all the advice. Now, I have discarded my contaminated cultures and ordered new HEPA filters for the CO₂ incubator. Next step will be to culture samples from healthy controls, and then to recruit fresh patients. Lets hope this works.