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Yeast contamination in cell culture - hood to blame?


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6 replies to this topic

#1 Josthashi

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Posted 14 December 2009 - 12:13 PM

Hello,
I have worked for years on various cell cultures without any major contamination problems. Last week, however, I was plating my cells under the hood (actually a class 2 type A1 biosafety cabinet), when I noticed the cabinet blower was really starting to struggle. It seemed like it was coming dangerously close to shutting off entirely before then returning back to normal flow. This happened a few times and I noticed after a few days that all my plates had the same yeast in them (the incubator had just been cleaned, so that is likely not the infection source). The reason I'm posting here is that I was told a malfunctioning blower is not a likely route of contamination and I would like a second opinion. Thoughts?

#2 aimikins

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Posted 14 December 2009 - 12:56 PM

of course it could. TC contamination can come from anywhere. have you done everything you can to test all other parameters?
"it is a miracle that curiosity survives formal education" -A.E.

#3 Josthashi

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Posted 14 December 2009 - 01:09 PM

Yes, and I've been driving myself insane doing so. One does not simply become terrible at cell culture techniques and, like I said, I've never had any problems with contamination before, never mind an issue of this size. I think part of the reason that I was told what I was told is that no one wants to accept that the hood is broken/contaminated (then accepting all the time and work that has to go into fixing it) - but we've got another hood that I'll be using from now on and hopefully my contamination-free cells will be convincing enough.

#4 rhombus

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Posted 15 December 2009 - 03:17 AM

Yes, and I've been driving myself insane doing so. One does not simply become terrible at cell culture techniques and, like I said, I've never had any problems with contamination before, never mind an issue of this size. I think part of the reason that I was told what I was told is that no one wants to accept that the hood is broken/contaminated (then accepting all the time and work that has to go into fixing it) - but we've got another hood that I'll be using from now on and hopefully my contamination-free cells will be convincing enough.



Biological safety cabinets these days have very sensitive monitoring devices that usual monitor:
Inflow rates
Downflow rates
Seals within the cabinet front

If any of these are below acceptable levels, then the alarms should be going off. These are all under Britsih standards and apply to all cabinet manufacturers. If your cabinets are that old that they do not have these devices....THEN DO NOT USE THEM.

What is not covered is the integrity of the HEPA filter...this is the filter that gives you the sterile laminar flow. If this has been compromised then it is possible that product protection has been compromised as well.

I do have to say that most yeast infections....come from the operator themselves. It would be useful to pour "settle plates" and test the cabinet and lab environment. The standard practice is to:-

Open 6 plates inside the cabinet for upto 4 hours and then incubate at 37oC for 3 days.
Open up plates (upto 4hrs) all around the TC lab... i.e. next to the waterbath, on top of incubators etc and then incubate as above.
Open up plates INSIDE the Incubators (upto 4hrs).
Open up 1 plate and touch the agar with your fingers..this acts as a positive control.
Have 1 unopened plate...this acts as your negative control (to make sure that your plates are sterile in the first place)... and then incubate at 37 like the others.


I have done this on many occassions and have always found the hood and TC Enviroment clear.....the only positives I normally get are the finger test and the Incubators (inside)

#5 rhombus

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Posted 15 December 2009 - 03:18 AM

Yes, and I've been driving myself insane doing so. One does not simply become terrible at cell culture techniques and, like I said, I've never had any problems with contamination before, never mind an issue of this size. I think part of the reason that I was told what I was told is that no one wants to accept that the hood is broken/contaminated (then accepting all the time and work that has to go into fixing it) - but we've got another hood that I'll be using from now on and hopefully my contamination-free cells will be convincing enough.



Biological safety cabinets these days have very sensitive monitoring devices that usual monitor:
Inflow rates
Downflow rates
Seals within the cabinet front

If any of these are below acceptable levels, then the alarms should be going off. These are all under Britsih standards and apply to all cabinet manufacturers. If your cabinets are that old that they do not have these devices....THEN DO NOT USE THEM.

What is not covered is the integrity of the HEPA filter...this is the filter that gives you the sterile laminar flow. If this has been compromised then it is possible that product protection has been compromised as well.

I do have to say that most yeast infections....come from the operator themselves. It would be useful to pour "settle plates" and test the cabinet and lab environment. The standard practice is to:-

Open 6 plates inside the cabinet for upto 4 hours and then incubate at 37oC for 3 days.
Open up plates (upto 4hrs) all around the TC lab... i.e. next to the waterbath, on top of incubators etc and then incubate as above.
Open up plates INSIDE the Incubators (upto 4hrs).
Open up 1 plate and touch the agar with your fingers..this acts as a positive control.
Have 1 unopened plate...this acts as your negative control (to make sure that your plates are sterile in the first place)... and then incubate at 37 like the others.


I have done this on many occassions and have always found the hood and TC Enviroment clear.....the only positives I normally get are the finger test and the Incubators (inside)




I do hope this is useful and gives you some ideas.......



Kindest regards

Uncle Rhombus

#6 Josthashi

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Posted 15 December 2009 - 05:31 AM

Thanks so much, I'm sure it's a combination of human error and equipment malfunction, I'll just be so relieved when I figure it all out.

#7 Josthashi

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Posted 22 December 2009 - 10:47 AM

Just an update:

Yes, our hood blower was broken and we have to assume the old hood is contaminated until it's fixed/cleaned, and yes, some of the contamination may be traced to that. However: the main source of the contamination was found to be the collagenase!

So remember: always filter-sterilize! Nasty yeast may be living in your collagenase! Don't let a seemingly tiny mistake ruin all your hard work!




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