Materials and Reagents

1. Single edge razor blades (e.g., VWR, catalog number: 55411-050 )
   
2. Double edge razor blades (e.g., Safety Razor Company, catalog number: 74-0002 )
   
3. Disposable Petri dishes (e.g., Corning, Falcon^®, catalog number: 351007 )
   
4. Double sided ½ inch wide ‘Scotch’ tape
   
5. Masking tape or similar (e.g., VWR, catalog number: 89097-920 )
   
6. 25 x 75 mm, 1.0 mm thick microscope slides (e.g., VWR, catalog number: 48300-025 )
   
7. 22 x 22-1.5 microscope cover glass (e.g., Fisher Scientific, Fisherbrand, catalog number: 12-541-B )
   
8. White onion (Allium cepa L.) bulbs; we use the Cometa cultivar
   
9. Distilled-deionized water (ddH₂O)
   
10. Sodium acetate trihydrate (e.g., VWR, catalog number: BDH9278-500G )
    
11. HEPES (e.g., VWR, catalog number: 97061-826 )
    
12. Hydrochloric acid (e.g., Merck, catalog number: HX0603-75 )
    
13. Acetic acid (e.g., Merck, catalog number: AX0073-6 )
    
14. HEPES buffer (20 mM, pH 6.8) (see Recipes)
    
15. Sodium acetate buffer (20 mM, pH 4.5) (see Recipes)
    

Equipment

1. Microwave oven, hot plate, or Bunsen burner
   
2. pH meter (e.g., Corning, model: Model 430 )
   
3. Analytical balance (e.g., Mettler Toledo, model: XPE105 )
   
4. Fine forceps (e.g., Dumont No. 5)
   
5. Flat tip forceps (e.g., Rose Scientific, catalog number: FF-001 )
   
6. Custom 3 mm slicing jig (see Notes section)
   
7. Microcomputer
   
8. Data acquisition hardware (e.g., Data Translation, model: DT9801 )
   
9. Constant-force extensometer (Figure 1) (Durachko and Cosgrove, 2009; Cosgrove, 2011; Cosgrove et al., 2017)
   
10. Stress/strain analyzer (Figure 2) (Cosgrove, 2011; Cosgrove et al., 2017)
    
    
    Figure 1. The constant-force extensometer. A vertically adjustable, open clamping chamber fixes the bottom of the wall sample and is capable of holding liquid with a capacity of roughly 150 microliters. A second clamp is applied to the upper portion of the wall sample and is connected to a balanced lever. Displacement of the lever and connecting rod is monitored using a linear variable displacement transducer (LVDT), e.g., Schaevitz 050HR. Weights are applied to the lever to modulate the tension on the sample. Custom software displays and records displacement and calculated extension rates. We use a bank of eight of these units connected in parallel to a microcomputer via a data acquisition module (Data Translation DT9801).
    
    
    Figure 2. The device used for stress/strain assays. A. The vertical stage is controlled by a PC serial port stepper driver (e.g., AutomationDirect STP-DRV-4850), stepper motor (e.g., Lin Engineering 4218L-01D-02), geared belt drive, and lead screw assembly. Stage position is monitored with an LVDT. Tension is monitored with an s-type load cell (e.g., Futek LSB200 100 g). Custom software controls the strain rate and tension. Two clamps hold the sample being measured. The upper clamp is fixed to the movable stage. The lower clamp is fixed to the load cell. B. Close up of peel affixed between clamps (peel stained blue to aid visualization for this image). C. Alternate view of device noting specific pieces.
    

Software

1. Custom software written using Microsoft Visual Basic is used for acquiring data and some data analysis. Microsoft Visual Basic may be freely downloaded at http://www.microsoft.com. Alternatively, many commercial software packages are available both free and for purchase to allow data acquisition and analysis

Procedure