Episode 6: Making Stains to Observe Plant Cells HD

Biological stains such as toluidine blue O (TBO) and iodine potassium iodide (I2KI) can make features of plant cells visible for microscopic study. This video shows how to prepare these two common stains. Full instructions appear below (click "show more"). TO MAKE TBO: 1. Stock Solutions for preparing Toluidine Blue O Let’s prepare 100 mL stocks of the 0.1 M citric acid (solution “A”) and 0.1 M sodium citrate (solution “B”). Detailed instructions below. A. To prepare 100 mL of 0.1 M citric acid: • Dissolve 1.921 g of citric acid in a beaker containing about 80 mL of dH20 (=distilled water) on a stir plate. • Transfer solution to a 100 mL volumetric flask and bring to volume (btv) with dH20. B. To prepare 100 mL of 0.1 M sodium citrate: • Dissolve 2.941 g of sodium citrate in a beaker containing about 80 mL of dH20 on a stir plate. • Transfer solution to a 100 mL volumetric flask and bring to volume (btv) with dH20. 2. Next step: Prepare citrate buffer To prepare citrate buffer of pH 4.0, use 25.5 mL of “A” above (the 0.1 M citric acid) and 24.5 mL of “B” above (the 0.1 M sodium citrate), and bring to a volume of 100 mL with dH20. Adjust pH to 4.0 by adding drops of 1N HCl (a dilute hydrochloric acid solution). 3. Final step: Preparation of the Toluidine Blue O Dissolve 50 mg Toluidine Blue O in 100 mL of citrate buffer (from preceding step, 2). Note: If larger volumes of Toluidine Blue O are desired, just increase the volumes/weights in steps 2 and 3 accordingly. TO MAKE I2KI: Iodine potassium iodide (I2KI) stain Let’s prepare 100 mL of this stain (recipe is doubled in the video). Detailed instructions below. 1. Dissolve 2 g potassium iodide (KI) in 100 mL dH20 (=distilled water). 2. Dissolve 0.2 g iodine in the KI solution (from Step 1 above), using a stir plate. This will take several minutes to a few hours, depending on volumes and masses, stir speed, and other factors. Plan accordingly. Caution: Handle iodine with care and do not breathe iodine vapors. Notes: Store out of light in amber glass dropper-bottle or wrapped in aluminum foil. Replace unused stain after one year since it becomes less effective with time. REFERENCES Stain recipes are based from those published in: Peterson, R.L., Peterson, C.A., and Melville, L.H. 2008. Teaching plant anatomy through creative laboratory exercises. NRC Press, Ottawa, Ontario. 164 pp.