ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

1. Preparation of Agarose Gel Stock

1. Weigh out 2 g of high purity agarose.
2. Add the agarose to a 500 mL bottle and record their combined mass.
3. Calculate the target mass of the bottle plus 2 g agarose with 150 g of ultra-pure water, then add 150 g of ultra-pure water to within 0.1 g of this target mass.
4. Note the mass of the bottle plus the agarose and water.
5. Place the bottle in a microwave and make sure to fit the bottle with a loose cap on top to minimize water evaporation while heating the agarose.
6. Microwave the bottle in 15 – 20 s bursts followed by briefly swirling the bottle to mix the agarose and water. Repeat this procedure until the solution is boiling and the mixture is homogeneous.
   CAUTION: Take care to avoid scalding skin with steam escaping from the bottle. Also, the bottle will become hot to the touch and appropriate protection, such as an autoclave glove, is required to avoid injury.
7. After the molten agarose is homogeneous, weigh the bottle again and add ultrapure water to within 0.1 g of the original mass. This will replace any water lost due to evaporation.
8. Before the molten agarose solidifies, swirl to mix in the added water to ensure homogeneity.
9. Aliquot 15.2 g of agarose into 9 – 50 mL plastic tubes.
10. Refrigerate the tubes until needed.

2. Preparing ODELAY Agarose Media

1. Heat 400 mL of deionized water to boil in a covered beaker, on a hotplate under gentle stirring with a magnetic stir bar.
2. Add 2 mL of 10X media (e.g., Yeast Extract Peptone (YEP), or Complete Supplement Mixture (CSM)) to a 15.2 g agarose aliquot in a 50 mL conical tube from step 1.10.
   NOTE: CSM medium tends to stick to the glass slides. If using CSM media formulations, add 5 µL of 50% wt Polyethylene glycol (PEG) in sterile water to the medium formulation. The PEG prevents the agar from sticking to the glass slides during mold release and does not inhibit yeast growth.
3. Add additional 100X nutrient supplements, if needed, and use water to bring the total added volume from this step to 1 mL.
4. Weigh the agarose aliquot with added media and supplements before placing the 50 mL conical tube into boiling water from step 2.1.
5. After 16 min, take out the 50 mL tube and homogenize the mixture using a vortex. Keep the cap fastened tight on the 50 mL conical tube.
   NOTE: A lid on the beaker helps heat the whole tube evenly otherwise a film of solid agarose will form and possibly not melt. Also during this time, it is convenient to assemble the agarose mold (Figure 1).
6. Homogenize the mixture using a vortex.
7. Boil for an additional 2 min to ensure all agar is mixed and melted.
8. Weigh tube and replace any mass lost with ultra-pure sterile water.
9. Add 2 mL of 10X carbon-source reagent (e.g., 20% w/v glucose) and vortex to obtain agarose medium solution.
   NOTE: Separation of Glass Slides: When assembling the mold, care must be taken to make sure the long spacers are placed in the mold with the correct orientation. This is because as the laser cuts the acrylic, it tends to cut as a cone leaving the part with slightly angled sides instead of exactly 90° sides. Then when the mold is placed on the benchtop to release the glass from the agar, the edge of spacer should be at an acute angle with the agar. The acute angle helps compress the edge of the agar away from the upper piece of glass as the outside edge of the mold spacer is pivoted about the lower edge (see Figure 1). The result is a more consistent separation of the agar from the top piece of glass.
   NOTE: Mold release agents applied to the glass like oils, silicon sprays or even commercial window treatments should not be used because they will contaminate the surface of the agar and possibly inhibit growth. These methods take some practice to be consistent.
10. Clean four 2 in x 3 in x 1 mm thick glass slides with 70% ethanol and dry using forced air.
11. Clean acrylic molds with 70% ethanol, dry, and assemble as shown in Figure 1. Take the three bottom pieces, as shown, and assemble them so that the two identical pieces sandwich the third piece (Figure 1B). Clamp the base pieces on each side using two small binder clips (Figure 1C). Place the uprights into the mold making sure that the laser kerf is correctly positioned (Figure 1E).
12. Place the cleaned and dried glass slide on the mold and hold it in place while placing a second glass slide on the other side. Clamp the assembly with a larger binder clip (Figure 1D). Clamp down both slides with larger binder clips (Figure 1D). Make sure the upper binder clip is in contact with the glass slide overlapping with the acrylic.
13. Add remaining binder clips. Again, make sure the clamps contact the slide where it overlaps the acrylic (Figure 1D).
    NOTE: Before filling the assembled mold with molten agar, ensure the edges are sealed by pipetting about 70 µL of molten agar media along the inner side of the mold. This agar will quickly solidify and prevent any leaks.
14. Fill the mold with molten agar, making sure to avoid trapping air bubbles in the mold.
    NOTE: This can be accomplished by slowly pipetting the molten agar along the edge of the mold. After it is filled, allow the mold to cool for 40 min to 1 h at about 23 °C ambient temperature. If allowed to cool too long the agar may fracture in the mold.
    NOTE: Mold Separation: Proper removal of the mold from the cast agar is essential to ensuring a uniform solid agar pad for yeast growth. Failure to ensure efficient separation of the mold at this step will cause growth differences that are attributable to imperfections in the solid agar pad. Practicing this step a few times is recommended.
15. Start by removing the bottom binder clip. Remove the bottom part of the mold that consists of the three sandwiched pieces. Hold the slides by compressing them and then remove the binder clips. Be sure not to bump the mold sides while removing the binder clips. This will deform the media.
16. Place the mold on the edge of the bench top so that the side of the mold with the blue dot is facing up and in the lower left corner (Figure 1E). Place thumbs underneath the acrylic and the first finger on the top towards the inner edge of the mold.
17. Slowly and carefully push up with thumb against the mold, as if pivoting the mold spacer about its lower edge (Figure 1F). Apply constant but slowly increasing pressure. Users will see a break and air bubble appear along the line where the top glass covers the mold spacer.
18. After seeing the initial break line, continue pushing up with thumbs and apply constant pressure. The agar should start breaking away from the glass at this point (Figure 1F). Continue to pivot the mold upwards. This will lift the glass without sliding it. A line where the agar is peeling away from the glass should continue to move away from the initial break line.
    NOTE: At this point, the agar is sticking to both the bottom and top of the glass. This will occasionally occur with the left most edge. As long as the area deformed is not in the area where yeast is spotted, this should be fine.
19. As the glass comes completely free, grab it and remove it completely from the mold. Then remove the other mold piece using a similar motion to lift the piece free without moving the agar. Place the slides in a sterile tip box with some sterile high purity water in the bottom. Close the box and store at 4 °C O/N for use the next day. If stored for longer, the growth rates will become inconsistent.

3. ODELAY Culture Preparation

1. Lay out strains in a 96-well plate for O/N growth.
2. The next day, dilute 20 µL of overnight culture into 200 µL media for 220 µL total volume in a new plate.
3. Measure the optical density at 600 nm (OD600) of each culture in a plate reader. Using a plate reader and a liquid handling robot, follow the automated dilution protocol of steps 3.3.1 – 3.3.6. Manually dilute the cultures in the plate to approximately 0.1 OD600 (optional).
   1. On the plate reader, hit "Experiment" under "create new". Choose ODELAYDilution.exp and run the experiment. Enter experiment name as ODELAY "Date" "Time" "Experiment Name" iteration.
   2. Click on the statistics tab and then click on the Excel icon. Save the data to a thumb drive and transfer it to a liquid handling robot computer.
   3. Open the Robot's Layout and Method Editor. Open the Method file "ODELAYDilution_v1.med". Run the Executable "Convert_SynergyFiles.exe". Enter 0.09 for target OD600.
   4. Click on "Glu Correction" and "Gal Correction" to 0.05. Measure blank media in an identical plate. Click on "Generate File". Select the file in the method editor. Click on the stoplight icon to start the method to open the runtime dilution program.
   5. Make sure tubes are uncapped and plates have lids removed and dust covers are removed from tips. Load plates, tubes and tips onto the liquid handling robot's deck, as indicated by the deck layout.
   6. Click the "Play" button to run the dilution program. Select the correct number of 50 µL tips. Select the correct number of 300 µL tips. Wait about 12 min for the process to run.
4. Take the dilution plate from the robot, cover the tips, and recap the 15 mL media tubes. Culture the plate for 5 – 6 h at 30 °C.
5. About 1 – 2 h before starting the second dilution make sure to turn on the incubation chambers for the microscope. Allow them to equilibrate at °C or the temperature required for the experiment.
6. Repeat the dilution steps 3.3 – 3.4, but dilute cultures to an OD600 of 0.01 – 0.02 for spotting the cultures on agarose slides.
7. Cover the dilution plate with a metal freezer seal.
8. Sonicate the dilution plate in an ice bath for 30 s with the plate floating in ice water using a centrifuge metal bucket-fitting to hold the plate (Figure 2A). Transfer the sonicated cultures from the dilution plate to a flat bottom plate. Label 4 x 96-well flat bottom plates 1, 2, 3 and 4 (Figure 2B).
   1. Transfer 150 µL of the dilution plate from wells A01 – D06 into wells C04 – F09 of plate labeled 1. Then transfer 150 µL of the dilution plate from wells A07 – D12 into wells C04 – F09 of plate labeled 2. Then transfer 150 µL of the dilution plate from wells E01 – H06 into wells C04 – F09 of plate labeled 3. Finally transfer 150 µL of the dilution plate from wells E07 – H12 into wells C04 – F09 of plate labeled 4.
9. Move on to step 4, Spotting on Agar.

4. Spotting on Agar using an Automated Liquid Spotting Robot

1. Remove the agarose slides stored at 4 °C O/N (from step 2.19) from their humidified sterile pipette boxes.
2. If needed, trim the corners of the agarose medium with a clean razor blade to ensure that they will fit on the slide chamber.
3. Remove the slide clamp off the mount. Carefully place the agar plate into the recessed area of the stage chamber mount.
   NOTE: Make sure the orientation of the slide is consistent from experiment to experiment.
4. Check to make sure the clamp is flush with the bottom of the chamber. If it is crooked, then the slide may not be fully seated in the recessed area. Place the leveling spacer into the center plate position of the spotting robot. This spacer provides contact for the leveling screws so that the slide chamber can be leveled with the tips.
5. Place the plates on the table in order of their quadrant. Plates 1, 2, 3 and 4 ordered from left to right (Figure 2B).
6. Lay out the tips so that the inner 24 wells C04 – F09 are occupied with tips into 4 empty tip boxes. A fifth box will need to have one tip in the C10 position, as well as 4 tips with their ends cut off in the A01, A12, H01, and H12 positions. These 4 cut off tips provide stability for the tip plate clamp (Figure 2C and 2D).
7. Remove the top cover from the slide chamber mount.
8. Place the first tip punch into the start site of the robot control spotting program. This should punch a hole in the agarose which will later be used for aligning the origin coordinate. Place plate 1 on the liquid handling robot to spot the first quadrant. Make sure the lid is removed and continue the spotting program.
9. Check to make sure all spots are present. Also, wait ~30 s for them to dry. When the spots are about 1 mm in diameter, the program may continue. Empty the used tips into the biohazard container, and place fresh tips on the robot. Swap out plate 1 for the 2^nd quadrant plate.
10. Repeat for the third quadrant. And repeat again for the 4^th quadrant.
11. When the spots are dry, replace the slide chamber cover and flip the apparatus over.
12. Install the tubing connections with an air filter.
13. Place a glass slide on the top side of the chamber.
    NOTE: This slide is crucial for reducing the heat flux to the agar and minimizes the formation of condensation onto the objective cover slip side of the chamber. Do not forget this cover slip. Depending on the microscope configuration, this cover slip prevents heating from the illumination side from causing condensation on the objective side.
14. Position a fan to blow hot air onto the objective side of the chamber. This fan prevents condensation from forming on the cover slip. Set the air-flow rate through the bubbler to 10 mL/min.

5. Running ODELAY on Microscope

1. Run the script "ODELAY_Microscope_Control.m".
2. Click on the "Shutter" to open the transmitted light shutter and then the "Focus" button to initiate the high camera rate (Figure 3A, Red Arrows).
3. Click on "Go Origin" and move the stage to find the origin mark that was punched in the agar.
4. Then move slightly to the right to focus on yeast cells spotted in area E07.
5. Move back to the origin punch and center it in the field of view.
6. Set the origin to this value by pressing the "set" button (Figure 3A, Blue Arrows). Now move to position H18 and focus using the hex screw closest to that location. Then move to position L07 and focus using the hex screw closest to that location. Then move to position E07 and focus using the hex screw closest to that location.
7. Repeat steps 5.5 – 5.7 as necessary until those positions remain in focus.
8. Check the focus in the center and at the edges at spots E12, H12, L12. Adjust the autofocus range if the focus Z-values, as indicated by the Z-value, are greater than ± the autofocus range (e.g., set the autofocus range to 60 µm the Z-value for a center spot that is greater than ±40 µm).
9. Press the reset button as this will activate the button properties, and then press ODELAY (Figure 3A, Green Arrows). Choose the directory to save the data. Make sure that enough drive space is available.
   NOTE: The microscope collects data for 48 h, or until the program is closed.

6. Processing ODELAY Data

1. Open the ODELAY_IPT.exe or use the script ODELAY_Image_Processing_Tool_v7.m script (Figure 3C).
2. Prepare an *ODELAYExpDisc.xlsx excel spreadsheet.
3. Name the experiment in cell B1.
4. Select the Image Directory where the Image files E07 – H18 are stored.
5. Select the Data Directory where data will be written.
6. Write the date when the experiment was started into cell B04. Use the format MM/DD/YYYY.
7. Write the dilution time in cell C04. This time is roughly five min before the first image is collected. Use the format HH:MMpm where HH is for h and MM is for min.
8. Add in the strain names according to the source plate (step 3.1). Due to the way strains are spotted, they are rearranged on the ODELAY agarose slide. Cells in the spreadsheet B31-B126 are the source plate, while cells C31 – C126 are the spot locations in the ODELAY agar plate.
9. Then press the "Process Data" button and select the *ODELAYExpDisc.xlsx that was just prepared.
10. Wait 16-24 h depending on the computer system used to process data.
11. When the images are processed, a directory named "ODELAY Well Data" and a file "*_Index_ODELAYData.mat" will appear. Press the "Load Data" button and select the "*_Index_ODELAYData.mat" file that was just generated. This will load the data set that was just processed. The "*" in the file name will be listed according to the experiment name entered into the excel spreadsheet.
12. After loading the data, inspect it using the time bar found across the bottom, click on an image square to see growth curves for that spot, or find a well of interest in the list on the left and then load the images for that list.
13. Generate histograms of population growth parameters by pressing the "Violin Plot" button. This will generate a plot shown in Figure 4 or Figure 6.