5 replies to this topic

[madelingirly]

Hi every one,
this was a reply on my previous topic, but I think it deserves to be a separate topic.
I am a PhD student who works in a lab of chemists, our professor is establishing cell culture unite for application of his material.
so he recruit a post doc with good experience and he bought all the devices and left our lab.
then I am stuck with no one to train me on any technique or any thing, only searching online, or in books for answers
I cultured my first plate from 2 weeks ago, however, I have  spherical shapes which I suspect to be yeast, I can`t really determine where is my leak in my aspectic techniques and devices.
I have watched several tutorial online, and read several books regarding cell culturing.

In our 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 completely 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,In advance, I am deeply indebted for your nice comments, thoughts and experience

[lamaksha77]

Briefly taking your hands outside of the BSC, or touching something outside the BSC is fine as long as your spray your gloves before resuming work inside. We don't clean the oven or the water bath or incubator in close to 6 months, and haven't had problems with contamination yet....You might want to prepare fresh media and PBS solutions. Secondly, UV irradiate the inside of your BSC (with pipettes, tubes etc inside) before starting work, in case on of these equipments is contaminated....Good luck!

[bob1]

I suggest having a good read of books on cell culture, especially R.I. Freshney's Culture of Animal Cells: a laboratory manual.  There should be plenty of videos of aseptic technique and cell culture on youtube, which would be a good place to start too.

It would be best if you could find someone else in your institute who is doing cell culture and ask them if you can spend a couple of days observing what they do, and even get some cells so that you can copy them and get them to critique your technique.

[Biouday]

Hi you can get a

Cell Culture Laboratory Handbook from Sigma as free (Hard Copy).

I subscribe it through link given here in protocol online and got it. Follow the link given below


http://www.protocol-...lture-handbook/

Edited by Biouday, 01 January 2012 - 08:58 AM.

[madelingirly]

Thanks Guys for your helpful advices,
I have searched for the Freshney's Culture of Animal Cells: a laboratory manual. online and found it, thanks God, I am downloading it right now and I have subscribed for the sigma hand book, hopefully, it may help me.
Thanks Guys again., Hopefully, if any one can help me with other thoughts, experience, or advices.

[casandra]

madelingirly, on 01 January 2012 - 05:14 PM, said:

Thanks Guys for your helpful advices,
I have searched for the Freshney's Culture of Animal Cells: a laboratory manual. online and found it, thanks God, I am downloading it right now and I have subscribed for the sigma hand book, hopefully, it may help me.
Thanks Guys again., Hopefully, if any one can help me with other thoughts, experience, or advices.

you can also check this out: http://www.level.com...ination-COR.pdf (Corning's tech bulletin on Understanding and Managing Cell Culture Contamination)

if you're working at the uni, perhaps you can inquire if there are places where you can get cell culture training ie from the biology or biomedical depts etc and of course, given by experienced personnel...

Edited by casandra, 02 January 2012 - 08:22 PM.