Are you sporulating on plates or in liquid? If you are sporulating in liquid, make sure that your cultures are getting very good aeration, as these cultures are oxygen-limited. We shake at about 350 RPM.

Sporulating in liquid seems to give the best efficiency. If you are sporulating in liquid, I would follow the suggestion from Dr. Lichten. I would add that when you sporulate diploid strains, it may work best if they have been grown fresh for each experiment, and grown to the same mid-late log cell density each time.

Thank you so much for valuable suggestions @ Dr.Lichten and Dr. Robinson.

Actually, I am sporulating in liquid. I used to shift overnight primary culture (in YPD) to Pre-sporulation media (0.02 OD) and let it to grow up to an OD of 1.2-1.6. Then I used to replace the media with Sporulation media (2% pot. acetate). I used to grow for next 24hr. During the change of media from pre- to sporulation media, I started to wash cells with distilled water recently. I got less efficiency of sporulation.

But I had seen about 80% sporulation without any wash with water during media change.

So, now I am little confused whether the wash with water is causing this problem or some thing else..?

(or)

May I directly transfer my primary culture from YPD to sporulation media..?

what else can I do to get good consistent sporulation..?

recommend any thing more to improve this protocol..

water wash sounds harsh--I would wash with sporulation medium. Also, I would try for a narrower range of OD for the presporulation growth--1.4 to 1.5.

I also would wash with sporulation medium rather than water. I would use the presporulation medium between YPD and spor, especially since you had been having good luck that way. Otherwise, your protocol sounds fine (and the yeast seemed to think so too, before you added the dH2O wash). For reproducibility, the other suggestion from Dr. Lichten is a very good one- maybe you could compare portions of the same culture at OD 1.2, 1.4 and 1.6 to see which works best (if any works better than another) and then use your cultures at that OD each time.

I think these suggestions should work much better for reproducibility, to solve my problem. I will start experiment with above suggestions and let you know the results in soon.

Thank you so much for spending your valuable time.

I am adjusting the pH of pre-sporulation media to 5.5. is this pH variation will affect the sporulation efficiency?

Also, I have a small doubt about pot.acetate solution. can I autoclave sporulation media (2% pot. acetate) or dissolve pot. acetate in autoclaved water..?

which will work better..?