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Autoclaving is my problem

autoclave decontamination

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32 replies to this topic

#1 madelingirly

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Posted 26 December 2011 - 10:12 PM

Dear new friends,
I need an urgent advice, I am a student in a material science lab on which they recently establishing cell culture unite, the responsible senior post doctor bought all the devices and left the lab after that, so I am suffering from his choices in devices no one in my lab can help me, as this senior was only the one with previous experience \, even my professor is new in this area.

My Autoclave problem: After autoclaving the items are coming wet from, I don`t know what I can do, I dry them in an oven after that,
is this enough???
besides how I can maintain the sterility if I take them wet from autoclave, and walk aroun 6 meters till oven, ut them in 60-80C wait around 30-60 minute till they are dry, then take the items, as a result of my uncertainty of sterilization I suspect my work all the items, especially when spherical shaped objects appeared on my cultured plates

Please Does any one has a similar experience with no drying option autoclave machine.
and what are the proper steps for using this kind of autoclave.

This is my autoclave
http://www.alpco.co....eries/auto.html
Model is KT-2346(20L)

Best Regards

#2 science noob

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Posted 27 December 2011 - 12:05 AM

Dear new friends,
I need an urgent advice, I am a student in a material science lab on which they recently establishing cell culture unite, the responsible senior post doctor bought all the devices and left the lab after that, so I am suffering from his choices in devices no one in my lab can help me, as this senior was only the one with previous experience \, even my professor is new in this area.

My Autoclave problem: After autoclaving the items are coming wet from, I don`t know what I can do, I dry them in an oven after that,
is this enough???
besides how I can maintain the sterility if I take them wet from autoclave, and walk aroun 6 meters till oven, ut them in 60-80C wait around 30-60 minute till they are dry, then take the items, as a result of my uncertainty of sterilization I suspect my work all the items, especially when spherical shaped objects appeared on my cultured plates

Please Does any one has a similar experience with no drying option autoclave machine.
and what are the proper steps for using this kind of autoclave.

This is my autoclave
http://www.alpco.co....eries/auto.html
Model is KT-2346(20L)

Best Regards


You would dry the items in oven (60-80C). Sometimes we leave it in there for 24 - 48 h (at 60C) to completely dry it plus rid of any potential contaminants. So, it is normal for items to come out of the autoclave with water vapour. There are also a few functions - solids or solutions. Try not to autoclave solutions (e.g. water or LB) together with lab equipments.

I presume that you asking this questions under 'Tissue & Cell Culture" would be using the items for culture? It's advisable to use commercial-grade tissue culture materials which come in sterile packs. What we do is seal it if there are still flasks left in the pack.

#3 afRNA

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Posted 27 December 2011 - 08:30 AM

ya, me too get this problem when i start lab works early days. Therefore, I usually dry the plastics before autoclave it. then I pack them in a bag then only autoclave it. after it finished again i dry it until needed.

U make sure before autoclave that whether it dried or not..
while autoclave the falken tubes do not close them tightly or do not allow them while open..

Thanks.

#4 madelingirly

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Posted 27 December 2011 - 08:41 PM

Thanks guys for your help,
now I have a fungal infection problem in my cultured plate, I think it may be yeast, I dont know.
So I want to make sure that my sterilization is complete and perfect.

So according to ur previous experience
science noob, afRNA,

Drying in an oven is enough to overcome the problem and maintain sterility, I hope so.
So could one suggest what might be the source of yeast infection,

#5 Biouday

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Posted 28 December 2011 - 01:24 AM

Why not you call the company itself to solve it.

#6 madelingirly

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Posted 28 December 2011 - 05:58 AM

Enthusiast, Thanks for the advice, I `ve already done it, and they told me it is normal for items to be wet, but they are just sales, I am asking about real experience on dealing with no-drying function autoclave as I have no similar experience on this machine



#7 leelee

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Posted 28 December 2011 - 06:54 PM

Autoclaves sterilise using steam heat under high pressure, so it is natural to expect items to come out wet. Drying as others have described is perfectly acceptable and is the norm.

Your contamination issues are not caused by this wetness.

In almost all cases of tissue culture contamination, it is the poor aseptic technique of the person doing the culturing that is the problem. Given how new you are to tissue culture, and the lack of experienced staff to train you, I'd say this is the case for you.

Is there anyone in a lab nearby that could demonstrate technique to you, and then watch you do your work to check for any possible errors?

Failing that, go to the tissue culture section on this forum, and read through the posts of others who have had contamination issues, as there are many good suggestions for correct technique etc (especially from Uncle Rhombus, bob and the like...).

Also, find yourself some tutorials or resources online, where you can read about the principles of autoclaves, sterility and aseptic technique (in relation to tissue culture), as you need to fully understand what you are doing to be able to find any mistakes you are making.

Hope that helps.

#8 madelingirly

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Posted 29 December 2011 - 07:25 PM

Dear leelee,

I am very grateful for your helpful advice, I have watched several tutorial online, and read several books regarding cell culturing.
I can`t really determine where is my leak in my aspectic techniques and devices.
In out lab we have only one refrigerator, so I am putting my reagents in it, besides some other lab colleagues putting some juice in it, I know it is wrong, but only one fridge, what I can do is only rinsing my bottles thoroughly with 70% alcohol.
Another suspected reason, is the oven which I share with my colleagues too, on 60 C only.
Another suspected reason is my water bath, which I share with others, and I clean every weak with soap and 70% alcohol.
The Incubator may be the problem, from couple of days ago, I said I will clean my incubators, so I putted my 2 plates in the hood, remove all the trays and wiped inside incubator with 70% alcohol and each tray was wiped too, then I putted every thing back, and put my plates in place, the next day one of my plates was infected with typical fungi, which can be seen by naked eye, so I throw it, however, the other plate has nothing in it, except these small spherical shapes, which I suspect to be yeast, but no turbidity, or precipitation on my plate is observed yet, I dont know.

Another suspected reason, is me,
As I am very new in this, some times my gloves touch outside the clean bench, then I rinse it with 70% ethanol.
some times a drop of medium is wetting the inside wall of my plate which may be the reason for contamination.

I wish if any one with similar position could share his experience with me

Finally, I am deeply indebted for your nice comments

Another

#9 science noob

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Posted 30 December 2011 - 12:55 AM

Dear leelee,

I am very grateful for your helpful advice, I have watched several tutorial online, and read several books regarding cell culturing.
I can`t really determine where is my leak in my aspectic techniques and devices.
In out lab we have only one refrigerator, so I am putting my reagents in it, besides some other lab colleagues putting some juice in it, I know it is wrong, but only one fridge, what I can do is only rinsing my bottles thoroughly with 70% alcohol.
Another suspected reason, is the oven which I share with my colleagues too, on 60 C only.
Another suspected reason is my water bath, which I share with others, and I clean every weak with soap and 70% alcohol.
The Incubator may be the problem, from couple of days ago, I said I will clean my incubators, so I putted my 2 plates in the hood, remove all the trays and wiped inside incubator with 70% alcohol and each tray was wiped too, then I putted every thing back, and put my plates in place, the next day one of my plates was infected with typical fungi, which can be seen by naked eye, so I throw it, however, the other plate has nothing in it, except these small spherical shapes, which I suspect to be yeast, but no turbidity, or precipitation on my plate is observed yet, I dont know.

Another suspected reason, is me,
As I am very new in this, some times my gloves touch outside the clean bench, then I rinse it with 70% ethanol.
some times a drop of medium is wetting the inside wall of my plate which may be the reason for contamination.

I wish if any one with similar position could share his experience with me

Finally, I am deeply indebted for your nice comments

Another


So what cells exactly are you culturing (human cell line?)? And what medium are you using?
The sources of these things are crucial because if the stocks are already contaminated, no matter how sterile you work, the fungus/yeast/bacteria (which is most likely by the sounds of it) is already there.

If there is a persistent contamination, you will clearly see fungus or yeast growing inside the incubator. All incubators have a tray which you need to fill with sterilised, distilled water - that needs to be monitored as well.

What I would recommend is place a petri dish of media (the one you're using) into the exact same 'problematic' incubator and allow it to be in there for a week or so. Take a look at it under the microscope to see if there are any suspicious-looking microbes floating around.

I'm still puzzled as to what exactly are you autoclaving? Is it the cell culture plastics/glass? What are you growing your cells in?

#10 madelingirly

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Posted 30 December 2011 - 01:59 AM

Dear
science noob

At first I am very grateful for your helpful comment
I am culturing human cancer cell line, the medium and cell lines are from reliable sources (Japan cell bank, Nacalia Tesque)
I am autoclaving pastuer pippettes, and plastic bottles on which I put my medium instead of using the whole stock medium bottle,
when I autoclave my pasteur pippettes I want to wrap with with aluminium foil, but I dont know which is better, wrap it or sterile it free, but it in a container and close it with aluminium foil.
all other things are disposables that includes serolgical pippettes and cultured plates.

#11 science noob

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Posted 30 December 2011 - 03:36 AM

It would be good if there was a post-doc or senior member of the lab which you can physically show him/her the issue(s). Do you have to report to your supervisor or lab manager if there is a contamination to the cell culture facility?

Try to aliquot your media into sterile commercial tubes (you can buy from BD etc). This will rule out any faults in the autoclave step.
Examples of the products I recommend:
http://www.bdbioscie...rue&from=dTable
http://www.bdbioscie...rue&from=dTable

These containers/tubes are guaranteed sterile if you remove it from its packaging properly, seal the packaging and store it properly.

Pasteur pipettes too can be purchased and comes in sterile packages (remember to reseal the packaging if you're done and there's still some left in it)

For your cell line stocks, do you have a few in storage? Did you expand them yourself or someone else did it for you?

Is there antibiotic in your medium?

#12 madelingirly

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Posted 30 December 2011 - 03:46 AM

Dear
science noob

it is purchase from cell bank, I am the first one who cultured them, of course no stock so I need to save them, I used 80 U pencillin per mililiter medium.
Of course if this problem continue to appears I have to report as my data will not be reliable data.
could you tell me in steps what is the proper unpacking and packing steps.
as I opened my cultured petridishes pack from the sign, then I putted in my clean bench with UV lights on all the time.
my serological pippettes are in their boxes and I store them near my clean bench so I take one by one while I am working.

#13 science noob

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Posted 30 December 2011 - 04:15 AM

Dear
science noob

it is purchase from cell bank, I am the first one who cultured them, of course no stock so I need to save them, I used 80 U pencillin per mililiter medium.
Of course if this problem continue to appears I have to report as my data will not be reliable data.
could you tell me in steps what is the proper unpacking and packing steps.
as I opened my cultured petridishes pack from the sign, then I putted in my clean bench with UV lights on all the time.
my serological pippettes are in their boxes and I store them near my clean bench so I take one by one while I am working.


With the packaged sterile stuff, reseal it with tape after using it and store it in the cell culture area. Open the package, take out the items required and store the rest. Before you start cell culture, UV the biohazard hood for at least 15 min. After that, sterilise the inside with 70% alcohol and wipe it dry. You can also allow the alcohol to evaporate by itself. Golden rule is sterilise (spray) everything that goes into the hood with alcohol. The cell culture plates can be exempted if you follow the packaging steps.

And most importantly, wear gloves when handling anything under the biohazard hood (sterilise the gloves with alcohol). Try not to touch any surfaces with the tip of your pasteur and serological pipettes once you remove it from it's plastic pack.

My current advice to you is place the medium you're using into a culture plate and place it into the incubator. You don't have to have cells in these plates. Just see if any contamination occurs.

2nd advice would be, do not use the autoclaved glass bottles to store your media for the time being since no one in your lab can confirm the sterility of the materials. Use a sterile commercial plastic container.

Cell culture techniques is the most crucial bit of any research involving cell biology because it is the first step in producing the cells which you will later on use in your experiments. It is important to have healthy, uninfected cells at the end when you start your experiments as you want to keep a consistency. Very important for an expert to show you how to do it in your lab. An actual demonstration is the best way to learn, not reading into books.

#14 madelingirly

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Posted 02 January 2012 - 02:20 AM

Dear
science noob,

I have a simple question regarding taking wet object out of autoclave into oven , shall I wear gloves while doing so, or I can carry them with my bare hands?????

#15 Biouday

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Posted 02 January 2012 - 04:04 AM

Dear science noob, I have a simple question regarding taking wet object out of autoclave into oven , shall I wear gloves while doing so, or I can carry them with my bare hands?????


What ever it is autoclaved or not, and autoclaving only kills cells not chemicals. It is good practice to use Always Gloves

Edited by Biouday, 02 January 2012 - 04:04 AM.






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