Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.
During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.
Sporulation in the budding yeast has been studied to provide insights into many aspects of biology, including the control of chromosome segregation during meiosis1, mechanisms of genetic recombination2, the control of development by cell signaling3, nutritional control of development4, the transcriptional regulation of development5, and the examination of spore formation6. Spore formation includes a novel cell division event involving the formation of new membrane compartments within the mother cell followed by the deposition of a protective spore wall6. These studies that examine sporulating cells often take advantage of the rapidly sporulating yeast strain SK1, which can undergo the process of sporulation in about 24 hr in a relatively efficient fashion7,8. Although optimization of sporulation conditions for budding yeast have been described9-13, these experiments examined sporulation on solid media or in larger scale liquid cultures where sporulation is carried out using culture tubes or flasks.
Here we describe a method for sporulating yeast in a 96 multiwell plate format. We find that for this method, aeration is critical for synchronous and efficient sporulation, and have devised techniques to ensure sufficient sporulation a small-volume multiwell format. Sporulating in a 96 multiwell plate format allows for cells to be screened using high-throughput techniques and reagents optimized for a multiwell plate format, such screening for high copy suppressors using a tiled library14-16.
1. Preparing for Sporulation
Note: The media described in this protocol are made using standard recipes and methods13,17. Table 1 gives the formulation for 1 L of the various media used in this protocol.
Bacto Peptone | Yeast Extract | Bacto Agar | Dextrose | Potassium Acetate | Glycerol | ddH2O | |
YPG plates | 20 g | 10 g | 20 g | – | – | 30 ml | 970 ml |
YPD plates | 20 g | 10 g | 20 g | 20 g | – | – | 1,000 ml |
YPD liquid | 20 g | 10 g | – | 20 g | – | – | 1,000 ml |
YPA liquid | 20 g | 10 g | – | – | 20 g | – | 1,000 ml |
Sporulation media | – | – | – | – | 10 g | – | 1,000 ml |
Table 1: Media formulations. Amounts are given for 1 L of media. Specifics about media ingredients can be found in the Materials Table.
2. Sporulating Cells in a 96 Multiwell Format
To assess this protocol, sporulation efficiencies obtained from sporulating cells in multiwell plates (as described above) were compared to cells sporulated using larger volumes in flasks (Table 2). The use of multiwell plates did not achieve the high efficiency seen when sporulating in flasks, where ~80% efficiency can be routinely seen. Sporulating in multiwell plates with proper aeration (provided by glass beads or stir bars) can achieve sufficient sporulation efficiencies greater than 50%, with the best results (66% efficiency) obtained using a 5 mm x 2 mm stir bar. A representative sporulation of two different strains (wild type and smk1Δ) is shown here (Figure 1). These cells are examined after 36 hr in sporulation media and visualized using a 96 well glass bottom plate.
Figure 1: Sporulating cells from a representative sporulation. Wild type and smk1Δ cells20 sporulated in a 96 multiwell plates. Cells were visualized using a 63X objective on an inverted microscope. Refractile tetrads (arrowheads) can be seen in the wild type culture, but not in the culture containing smk1Δ cells. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Sporulation was tested using either a 5 mm or 7 mm stir bar, each of which fits within the well of a 96 1.3 ml multiwell plate (Table 2). Using a 7 mm stir bar resulted in poor sporulation efficiency (28%) compared to the higher efficiency (66%) achieved using the 5 mm stir bar. The 7 mm stir bar in a well tended to interact with a stir bar in an adjacent well (Figure 1), preventing the bars from being able to properly stir and provide adequate aeration of the culture.
sporulating cells (%) | SEM (%) | |
flask in shaker | 82 | 1.8 |
no bead in shaker | 17 | 2.4 |
bead in shaker | 56 | 3 |
no stir bar on stir plate | 10 | 2.2 |
5 mm stir bar on stir plate | 66 | 3.9 |
7 mm stir bar on stir plate | 28 | 6.7 |
SEM = standard error of the mean |
Table 2: Sporulation efficiencies using different conditions. Sporulation was conducted in 96 well multiwell plates as described above, or, if noted, in 500 ml flasks containing 50 ml of sporulation media. Eight different wells were averaged for each condition carried out in a multiwell plate. Three different flasks were sporulated. All cultures are technical replicates, started from the same colony of mCherry-tagged Htb2 but otherwise wild type diploid SK1 yeast strain (LH90218). These cells were grown in a single flask of YPD and a single flask of YPA, and then divided to inoculate 96-well plates containing sporulation media, as described above. Sporulation efficiency was assayed by counting refractile spores 36 hr after transfer to sporulation media using Nomarski DIC brightfield microscopy.
Figure 2: Stir bars in 96 1.3 ml multiwell plates. Sporulation culture containing either (A) 5 mm x 2 mm stir bars (red) or (B) 7 mm x 2 mm stir bars (blue) in a 96 1.3 ml multiwell plate on a magnetic stir plate. Scale bar = 10 mm. Please click here to view a larger version of this figure.
Here we present a protocol for sporulating SK1 yeast in a 96 multiwell format. Aeration is key for efficient sporulation, which requires the use of either a stir bar or a glass bead in each well. When cells are sporulated in a 96 multiwell plate in a shaking incubator without either a bead or a stir bar, cells do not sporulate efficiently. Only a small increase in sporulation efficiency is seen when cells are sporulated without either a bead or a stir bar in a shaking incubator, compared to being at 30 °C without agitation (Table 1; 17% in shaker vs 10% placed on a stir plate). Similarly, the use of a 7 mm stir bar resulted in poor sporulation efficiency (28%), likely because the 7 mm stir bars did not aerate the culture properly due to interactions with stir bars in adjacent wells. This protocol describes conditions for sporulation using the synchronously and efficiently sporulating SK1 yeast strain7,8 used commonly to study sporulation; sporulation efficiencies that can be obtained with other yeast strains using this protocol should be examined before undertaking a large screen using these techniques.
Although a 5 mm x 2 mm stir bar results in a slightly better sporulation compared to the use of a glass bead (66% with 5 mm x 2 mm stir bar vs 56% with a glass bead), the cost required to purchase enough stir bars for each well can be prohibitive. The sporulation efficiencies obtained using a glass bead provides a low-cost solution that creates enough aeration for high-throughput screening in a 96 multiwell format16. Unfortunately, the addition of a stir bar or a glass bead did not achieve the very high sporulation efficiencies (typically ~80% or greater) seen when sporulating in flasks, and thus subtle sporulation phenotypes may be difficult to detect when screening in a 96 well format.
Unlike previously described sporulation methods9-13 which utilize solid media or larger liquid media volumes using flasks or culture tubes, this protocol describes conditions for efficient sporulation in a 96 multiwell format. The use of a 96 multiwell format is compatible with different types of high throughput screens that can be carried out to examine sporulation phenotypes. For example, chemical screens that affect processes in sporulation can be carried out using this technique, where sporulating cells are treated with different compounds that have been arrayed into a 96 well format. In this case, the cells to be sporulated would all be of the same genotype, and the compounds would be added at step 2.2 of sporulation. This protocol is also compatible with screening cells of different genotypes for sporulation phenotypes, such as yeast cells transformed with a high copy library or a yeast mutant strain collection. For this case, the strains would need to be arrayed first in a 96 well format and the protocol described starting at step 1.2 would be appropriate.
The authors have nothing to disclose.
This work was supported by a Joseph P. Healey grant from the University of Massachusetts Boston (L.S.H.) and R15 GM86805 from the NIH (L.S.H.). S.M.P. is supported in part by a Sanofi-Genzyme Fellowship at the University of Massachusetts Boston.
Nunc 1.3 ml DeepWell Plates | ThermoScientific | 260251 | Used for sporulation |
Nunc 2.0 ml DeepWell plates | ThermoScientific | 278743 | Used for presporulation growth, step 1.2.3 |
3 mm glass bead | Fisher | 11-312A | Used for sporulation |
5 mm x 2 mm stir bar, pack of 12 | Fisher | 14-511-82 | Used for sporulation |
96 well frogger | V&P Scientific | VP407 | needed for step 1.2 |
library copier | V&P Scientific | VP381 | needed for step 1.2; to be used with the frogger |
rectangular petri dish | ThermoScientific | 264728 | needed for step 1.2 |
Bacto Peptone | BD | 211677 | needed for media |
Yeast Extract | BD | 212750 | needed for media |
Bacto Agar | BD | 212750 | needed for media |
Dextrose | Fisher | D16-3 | needed for media |
Potassium Acetate | Fisher | P171-500 | needed for media |
Glycerol | Fisher | G33-500 | needed for media |
Black 96 well glass bottom plate | MatTek | PBK96G-1.5.5-F | needed for step 2.4 |