8 replies to this topic

[petenl2004]

Hi all,

I'm growing some yeast cultures to test certain enzyme activity. Recently I always got contamination in my yeast cultures, seen as big white clumps in the liquid cultures

<a href="http://news.webshots.com/photo/2529854620103365543DDRSuf"><img src="http://inlinethumb26.webshots.com/49177/2529854620103365543S425x425Q85.jpg" alt="2012.06.01 Yeast C"></a>

,
under microscope

<a href="http://news.webshots.com/photo/2856770470103365543AZDQhl"><img src="http://inlinethumb16.webshots.com/8271/2856770470103365543S425x425Q85.jpg" alt="2012.05.14 Yeast D 1"></a>

.

I tried to make the cultures from the new streak plates, autoclaved carefully all my medium, glassware, toothpicks....but the contamination was still there. Has anybody here ever encountered this problem before? Could you give me some tips to identify the contamination?
Thanks a bunch!

Cheers,

Edited by petenl2004, 01 June 2012 - 01:31 AM.

[petenl2004]

Photo taken with pocket camera

Attached Thumbnails

• 

[petenl2004]

Contamination seen under 100x oil lens

Attached Thumbnails

• 

[Arun Kumaran]

If your yeast strain is resistant to any antibiotic, use it by increasing concentration. Clean your incubator with fungicide.

[petenl2004]

Thank you Arun. Indeed my yeast is resistant to Amp. However, I am growing the yeast in SD-DO medium without Tryptophan and Uracil, plus on sucrose as a carbon source instead of glucose (because I transformed the invertase-deficient yeast with the plasmid carrying sucrose synthase gene that helps it to break down sucrose). Due to that, this yeast grows really slow (at least 4 days to reach OD600 of 0.8). I already thought about adding Amp into the medium, but did not do it yet because I was afraid of causing the growth even more retarded. But since this contamination is occurring so often recently, I think I will follow your advice, putting the antibiotic into the growth medium, and also clean my incubator with fungicide!!

I really hope this will help me prevent the contamination Will update when the result is out

Thanks again

[Arun Kumaran]

You are welcome. I think you can try carbon source as 25% Glucose + 75% Sucrose. So, that yeast will start growing fast by consuming glucose initially and then produce more invertase to breakdown sucrose later.

I never tried this. This is just an idea!

All the Best

[Phil Geis]

Pictures are nbot convincing that the apparent contaminant is biological - suggest you answer that question 1st.  If you're confident the contaminant is indeed biological and your yeast master culture is not contaminated,  advise you focus on aseptic technique rather than adding an antibiotic to gloss over faulty execution.

[petenl2004]

@ Arun: Thank you. Indeed, I'm also adding very little bit of glucose (0.02%) into the growth medium, which really boosts the yeast growth.
@ Phil: Thank you for your advice. I will also try to focus on aseptic technique. Before, I used the autoclaved toothpicks to pick the yeast colonies for inoculation, maybe they were not really sterilized??. This time I'll use the metal inoculating loop instead of the toothpicks to see if the contamination is still there.

[petenl2004]

Hi Arun and Phil,

My new yeast cultures were contamination free now. I just started with every new: new colony plates, new media which I prepared in MQ water. In addition, I used the metal inoculating loop to inoculate my liquid cultures instead of the toothpicks, and using the Airpore tape sheet (from Qiagen, actually this is made for the 96 well plate) as the lid for the Erlenmeyer flasks instead of the aluminum foil (which is more expensive, but it works fine for me).

Well, so, now I know what to do and hope that the contamination will not show up again.
Once again, thank you for your advises

Cheers