Hi,
would anyone have a protocol or be willing to share their settings for a yeast (S. cerevisiae) proliferation assay in 96-well plates?
We have a Tecan Spark 10M platereader, but I`d imagine different Tecan models should behave similarly. I`ve tried various settings (linear vs orbital vs double orbital; shaking continuously vs in intervals; flat vs round bottom flasks), but I didn`t find any where cells wouldn`t settle or at least where they would settle uniformly and the manufacturer also wouldn`t have a solution.
So I`d be especially interested in the shaking parameters and type of plate. The starting OD and any special tricks used would also be interesting.
I`m interested in testing the effect of mutations on proliferation upon various treatments.
Thanks a lot,
Michael
Hi Michael,
we have succesfully measured the growth rate via OD600 of S. cerevisiae in our Tecan M200 readers using the following settings:
Temperature: 30C
Shaking (Orbital) Duration: 240 s
Shaking (Orbital) Amplitude: 4 mm Absorbance
Kinetic Cycles: 720
Interval Time 00:06:00
Mode Absorbance
Multiple Reads per Well (X-Line) 2 x 2
Multiple Reads per Well (Border) 2500 µm
Wavelength 600 nm
Bandwidth 9 nm
Number of Flashes 18
Settle Time 0 ms
We use standard clear flat bottom 96well plates (Thermo Fisher Scientific-Nunclon 96 Flat Bottom Transparent Polystyrene Catalog No.: 269620/269787/439454/442404/475094). We start with our cells at an OD600 of 0.04 and culture in synthetic minimal or yeast peptone media with glucose for two days.
At the end of the measurement (after two days), one can see that cells are accumulating in the middle of the wells. At that point the yeast have probably flocculated and it is difficult to keep them in solution. But by then they are anyway in stationary phase.
Hope this helps and good luck with your experiments!
Best,
Greg
Hi Greg,
that`s amazing. Thanks a lot for the parameters, I`ll give it a try.
How much volume do you put into each well? You don`t have any problems with different behavior of the margin rows/columns to the rest, do you?
Best,
Michael
Hi Michael,
I forgot - we use 200ul in total and usually run each cultivation in triplicate. We did not see significant edge effects, but if you are worried about this you can always exclude the data from the edges afterwards.
Best,
Greg
Hi Michael,
the thing is, high-precision microplate readers are not actually developed for continuous shaking. The available read modes (fluorescence, luminescence, etc.) all require highly precise positioning of the plate carrier below the measurement head. Prolonged shaking may lead to misalignment of the plate carrier and the need for technical readjustment by a service engineer. However, before this becomes even noticeable there may potentially be distortions of the results of other measurement due to poor positioning. This is why we don't actively recommend assays like this and also do not have any "optimized" protocols for growth studies that require continuous shaking.
Of course we leave it up to the customers to perform any protocol they would like, even if that means they would like to just use it as a very expensive microplate shaker. ;-)
Gregor is absolutely right in using the multiple reads per well function which will give you information about both the total OD in each well and also of the OD distribution across the well.
Please let me know if you need any further assistance with the reader as you continue your experiments.
Best,
Katrin
Hi Michael
Continuing with this discussion, I would like to know the range of expected values between 0 days and 2 days when you read following this protocol? I have done similar conditions but I don't see changes between 1 day and 2 days and the values were around 1.0 for both days, meaning no change between 1d and 2 days. I appreciate your comment. Thank you. Gregor W. Schmidt
Hi Nathalia Chica
Our raw baseline readings are usually at around 0.08. This depends mainly on the plate that you use and the media. The yeast itself should be diluted so much that you can not actually detect it at the beginning of the assay. When we start with an OD600 of 0.05-0.1 we can see the first changes in OD after 3-5 hours, and after 10-12 hours we have already reached stationary phase (e.g. for WT cells). The maximum raw OD600 readings go up to 1.3 (SDmin) or 1.6 (YPD).
We usually don't see any more changes in OD after the first day, unless we are working with a strain that is growing very slow or under slow growing media conditions. The time window during which you can see changes in OD under normal growth conditions is actually quite small (3-5 hours). Therefore we use a quite frequent measurement interval of 6 minutes to get sufficient data points for fitting the growth curve and extracting the growth parameters.
Best,
Greg
Hi Gregor W. Schmidt ,
Thank you for your answer. It has been very useful. I have seen also readings between 0.05 and 1.0/1.5 when cells are enough diluted and when cells reach the stationary phase correspondingly, and also that there are no changes in growth after 24h.
Best,
Nathalia
Hello. I just got growth curves in 96 well plates adapting a protocol from Hung, C. et al. A simple and inexpensive quantitative technique for determining chemical sensitivity in Saccharomyces cerevisiae. Sci Rep 8, 11919 (2018) .
The key factors are:
1) Do NOT shake your cells
2) Start with an inoculum low enough so that they all fall to the bottom before the log phase
3) use 200 microliters per well (Not sure if higher volume would be better, but lower is not)
The rationale of them is to create a lawn on the well and to read the optical density of the lawn.
I am attaching a file with the protocol (adapted to test expression of heterologous genes in two yeast strains) and a picture showing the effect of inoculum on growth curve for one of my cultures.
Dear Sylvia Varland
With raw readings I refer to the raw data acquired from the machine, without background corrections applied. With baseline readings I mean those readings before any growth becomes measurable, and maximum raw OD600 readings are the ones reached in stationary phase.
I would also expect your OD600 values to go beyond 0.36 in SD medium - we have observed maximum OD600 readings in our readers to reach 1.3 in SD minimal media. However, we have used prototrophic strains and 2% glucose - depending on your strain and media conditions the values could be very different. It also depends on the volume in each well - we typically use 200µl.
Good luck!
Greg
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