Well, you re-order the strain but I think you will need to check if your glycerol stock conserve the mitochondrial DNA. Usually when once take a colony of a culture in YPD plates could pick a petite colony. To check it you can do 2 things:

1. Growth your wild type strain in Synthetic Complete with Glycerol (non-fermentable sugar). If the strain doesn't grow is because it is petite and for this reason you can observe a slower grow.

2. Sequence a mitochondrial gene as COX2. You can find the primers in this paper: Belloch C, Querol A, García MD, Barrio E. 2000. Phylogeny of the

genus Kluyveromyces inferred from mitochondrial cytochrome

c oxidase II gene. Int J Syst Evol Microbiol 50: 405–416.

http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCkQFjAA&url=http%3A%2F%2Fijs.sgmjournals.org%2Fcontent%2F50%2F1%2F405.full.pdf&ei=O03UUayUFcjUyQHr4YDADQ&usg=AFQjCNFkj06vnAWdygVkC0unh0jzNMkLlQ&bvm=bv.48705608,d.aWc

I hope it helps!

Good luck!

Have you measured the growth rate during exponential phase of your cultures? If the exponential growth rate is reduced then you know your cells are sick cells. Otherwise, you might be seeing an extra long lag phase. What's the OD of the preculture after 24 h? If the cultures are saturated after at the end of the preculture you will see a significant lag phase once you innoculate the new culture. Either way, I recommend plotting a growth curve. This should help understand the problem better.

One thing which comes to my mind is a recent change in the media kitchen (water purification system and/or dish washer).

A colleague of mine has had severe problems with growing yeast for several weeks. In the end it turned out that the dishwasher in the media kitchen has been replaced (due to age) and this caused the bad growth of the cultures. Probably some chemicals left from the manufacturing process which were not properly removed before delivery and got onto the glas flasks used for incubation. I suggest you use water and new sterile glass flasks from another media kitchen to check if this is really the source of the problem.

In that particular case this solved the problems, I hope this is also the case for you.

Hi!

I also working with various S.cerevisiae strains created by myself and purchased from Euroscarf. I noticed strange behaviour for my strains that grown after a large number of passages and that can be explained by spontaneous mutations. But if you have strange growth of cultures obtained from Euroscarf or from frozen stock, I think in your case the matter is not in the number of passages. I would suggest to pay attention to WATER that you use to prepare culture medium. In our lab even mQ can be contaminated with some microorganisms. Their presence can be detected only on plates, when they form colonies after about 3-4 days of cultivation at 30C. Before that time they are undetectable, but yeast cannot normally grow in the media prepared with contaminated water. I can suggest that the water that you use is contaminated with some kind of toxins from some microbes. May be you need to check the water purifying system, microbes like to grow on old cartridges.

I think that you selected a mutant. Take your mutant that grows as expected and replace the mutant gene with its wt counterpart thereby reconstructing the desired wt.

I agree with David Peris, this sounds like classic petite behavior. Many yeast strains lose segments of their mitochondrial DNA spontaneously at a high frequency, and this makes the respiration-incompetent. It sounds as if the EuroSCARF isolate may already be petite. As growth on SC-glycerol can be painfully slow, we check for grande/petite on YP-ethanol/glycerol/succinate:

YEPEGS

Additions gm/liter

succinic acid 10

dissolve in 500 mI H2O

then add

yeast extract 10

bacto peptone 20

glycerol 26 (approx. 20.8 ml)

8 m1 0.5% Adenine (in 0.05M HCI)

adjust pH to 5.5 with 1 M NaOH adjust volume to 970 ml, then add

bacto agar 22

adjust to pH 5.5 (5.5m1 1 M HOI)

after autoclaving and cooling, add 27.5 ml of 95% ETOH

Petites will not grow on this medium.

The other possibility is that the EuroSCARF "wild-type" is not really; see if it can grow on SD minimal supplemented for all of the advertised auxotrophies.

Good luck!

1) Do you have any strains that are behaving properly?

2) If not, have you changed anything about your glassware processing? residual detergent can have stochastic effects due to traces left on the culture flasks or in the bottles used for media. We once had a sporadic poor-growth problem, which we eventually traced to the foam plugs--the glasswashing facility had started buying fungicide-impregnanted plugs for the mammalian cell people and it leached into our flasks. The effect was maddeningly inconsistent as reuse of the plugs led to multiple autoclaving and reduced the toxicity.

Good luck

Wow, so many useful answers, thanks so much!

So, I will address a few points that have been made:

1) glycerol stocks, mitochondrial DNA and petites

As I understand the petite phenotype, my colonies on YPD plates should then be smaller. This is not the case in a) freshly thawed yeast from a somewhat "old" -80ºC stock and b) 2-4x passaged yeast obtained directly from Euroscarf (on agar). For passaging, I always pick more than 1 colony for restreaking, to avoid picking a random mutant. And the problem persists on different days with different colonies (or more material) from the same YPD plate.

2) Media kitchen/contamination of water/MilliQ

Indeed, that was my second idea, as well. Our dishes and glass tubes are processed without detergent, and nothing was changed compared to last fall, where everything worked.

The problem concerns only the BY4741 wildtype, no other wild type (e.g. BY4742, which is genetically very close). Wouldn't one expect that a general issue with dish processing would equally affect not only one particular wild type, but also the mutants?

Also nobody in my lab seems to have the problem (but they are using other wild types). Indeed, we have had a water purification problem a few years back, which caused a failure to purify organelles - but usually such a thing concerns everybody in the lab.

Same thing for microbial water contamination:

I often keep plates at RT or 30ºC for more than 2 days, since I have some cold-sensitive mutants or do drug resistance tests, and never see strange colonies pop up. Also, all solutions including MilliQ water are filtered at 0.2 µm. Any smaller toxic molecules would, in my opinion, also affect other strains.

3) Testing the Euroscarf for petite isolate, auxotrophy, growth curve

I will do that.

4) Do I have other strains that behave properly?

Yes, as I said BY4742 wildtype behaves normally, and the mutants in BY4741 background that previously grew just like wt in YPD, HC and SC continue to grow very well.

5) OD and morphology of the preculture

The OD after 24h is between 4.0-6.0/ml and indistinguishable between mutants and wild type. This is one reason why it is so strange that if I inoculate the same volumes into ON cultures, I get 10x less growth.

But: I did a phloxine stain for dead cells today on a fresh preculture of BY4741 and 4742. And it looks as if the 4741 contains more phloxine-positive and slightly smaller cells, which are probably beginning to die. That means that the initial preculture contains less healthy cells, i.e. my inoculum in the ON culture is already "less alive".

Thanks to all for your help, I really appreciate it. Please keep more suggestions coming, I am running out of explanations...

First, I will test in complete media, for example YPD,

second, I will check for the auxotrophic requirements of your strain

and finally, check your drop out solution to include the auxotrophic requirment in the solution...

The similar observation was there with me when I was working on Diploid yeast Candida albicans. The colony after 72 hours in presence of AZT upto 144 hours i found some dead cells. Obviously i did not observed petite colony but there is switching in colony morphology. The idea after your observation of auxotrophic needs I can think now there are rearragements in mitochondrial DNA as well as Nuclear DNA. Theoretically there was a Paper on differenciation of colony after formation by Z. Palov in 2004 EMBO as review. By your observation and differenciation observation by Palov, I feel now theoretical IDEA that there is switching state, autrophic state, differentiation state and finally mutation state of a microbial cell. In a nut shell, the rearragements of nuclear material is existing for the opportunity of a microbial cell for a particaular state.

Get new stock of that strain. Avoid repeated subculturing.

So I tested again most of what was suggested.

1) The auxotrophies of the strain are ok, everything is as supposed to be.

2) Growth rates of the strain are identical to BY4742 in both YPD and SC complete media, between 82 and 90 min.

3) Plating on YEPEGS plates revealed that my suspicious BY4741 can very well grow on this media, so petite phenotype can be excluded. Also, since colonies on YPD look normally sized, I didn't think it was this.

4) I noticed that when I make precultures (1d) in YPD and inoculate this into SC complete overnight, growth is just fine. So apparently the strain doesn't support well preculturing in synthetic media, but YPD is ok. Still strange...

I will now re-order the strain from a different supplier.

Thanks everybody for the help, I for sure learned a lot of new things.

Dear Maria,

Did re-ordering the strain help resolve the problem?

Thank you.

Hello,

re-ordering from a different supplier didn't really help - but in fact I assume that this supplier ordered it from Euroscarf, and then sent it to us.

I have not been using this strain for some time now, will do some experiments next week and we'll see. Sometimes those things miraculously appear and disappear...

Maria,

I happened upon this paper by chance. It seems to describe a problem similar to what you've described. I hope it may be of help. You could also try to obtain BY4741 directly from Prof. Jef Boeke at Johns Hopkins University.

http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2007.00798.x/full