Autoclaving is my problem - (Dec/27/2011 )

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.bdbiosciences.com/ptProduct.jsp?prodId=364249&key=tube&param=search&mterms=true&from=dTable
http://www.bdbiosciences.com/ptProduct.jsp?prodId=364183&key=container&param=search&mterms=true&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?

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.

madelingirly on Fri Dec 30 11:46:45 2011 said:

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.

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?????

madelingirly on Mon Jan 2 10:20:52 2012 said:

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

Biouday on Mon Jan 2 12:04:09 2012 said:

madelingirly on Mon Jan 2 10:20:52 2012 said:

Most of the time, we use the inside of autoclaved bottles to fill up with media for cell culture. So, technically you can do whatever you want to the outside JUST follow the golden rule - sterilise everything that goes into the cell culture hood with ethanol.

but similar to Biouday's point, I would automatically put on a pair of gloves whenever I step foot into a lab regardless if I'm doing cell culture or not. It's your hands and you don't know what chemicals have been around it. Gloves are cheap but you only have 2x hands and can't afford to stuff up your experiments.

When taking objects out of the autoclave, you should absolutely be wearing gloves- thick, heat resistant ones! The inside walls of the autoclave remain hot for some time and you really don't want to burn yourself on that. All it takes is a slight touch, and I've seen the results. Not pretty.

Dear
science noob,

I have followed ur advice in putting an empty petridish with medium only in my incubator to check if my incubator is contaminated or not, I can see the circular shapes in it, from 3 days of incubation, it is much less than the plates which contains the cells but it is there, I have added a photo with my 10X lens,
however, this result may suggest too that my medium may be the source of contamination, because it is the same one which I use in my plates, I dont know, but it is much less compared to original plates with cells

Hi Guys,
I have upload some pictures of my plate, I will be deeply grateful if any one can give me more opinions and comments about these pictures and spherical shapes.
Best Regards

madelingirly on Tue Jan 3 03:28:26 2012 said:

It is very common to find precipitates in FBS. The photo you have provided is not clear enough to be able to tell if these are precipitates or some other contamination.

madelingirly on Wed Jan 4 06:31:45 2012 said:

Plate 1 shows a clump of cells that either hasn't attached when you last split the cells, or has detached from the plate.
Plate 8 shows what looks like a very confluent plate with the cells looking a little worse for wear (either need to be split or medium changed or they are just getting old). The objects you have put arrows to are all either cell debris (red arrows) or intracellular structures such as vacuoles (black arrows).
Plate 7 the arrows point to floating (and probably dead) cells. It is very normal to see dead/floating cells in any population of cells you are growing, indeed some cell types such as SH-5YSY have a proportion of their normal living population that floats as suspension cells.