Congo Red Staining: A Technique to Visualize Cellulose Deposits in Plant Stem Sections

Cellulose is an unbranched polymer of β-glucose units linked by β-1→4 linkage and arranged parallelly via intermolecular hydrogen bonds.

Cellulose serves as the major structural component of the plant cell wall, providing shape and rigidity to the cells and facilitating plant growth and development. The proportion of cellulose within the cell wall varies significantly throughout the plant, according to the cell type and growth stage.

To visualize cellulose deposition in the stem, take thin cross-sections of agarose-embedded stem sections.

Add Congo red fluorescent dye and incubate the sections.

Congo red molecules enter the cell wall and form hydrogen bonds with the hydroxyl groups of the cellulose, thereby getting adsorbed onto the polymer. This imparts a red color to the epidermis, cortex, and pith.

The dye molecules also diffuse within the complex cell walls of the xylem and interfascicular fibers, crossing the rigid lignin barrier, thus, imparting a red color to these tissues too.

Now, rinse the sections in water to remove excess stain. Transfer the sections onto a microscope slide and place a coverslip.

When visualized under a fluorescence microscope, the epidermis, cortex, central pith, xylem, and interfascicular fibers appear red, corresponding to their cellulose content.

For Congo Red staining, transfer the stem sections to a microcentrifuge tube, and add 1 milliliter of the 0.5% Congo Red solution. Gently pipette the 0.5% Congo Red solution up and down, without disturbing the sections, before incubating and washing the sections as described in the text protocol. Gently pipette the sections into a cut pipette tip, and onto a microscope slide, then, cover them with a coverslip. Observe the samples on the microscope slide under blue-light excitation using a 560-nanometer emission filter.