In the electron microscopy fixation protocol using glutaraldehyde, what is the correct sequence of steps?

• A. Glutaraldehyde → Phosphate buffer → Sucrose solution → Osmium tetroxide
• B. Glutaraldehyde → Osmium tetroxide → Phosphate buffer → Sucrose
• C. Phosphate buffer → Glutaraldehyde → Sucrose → Osmium tetroxide
• D. Osmium tetroxide → Glutaraldehyde → Phosphate buffer → Sucrose

Answer: (A)

In electron microscopy fixation, you first stabilize the cellular framework by fixing proteins with glutaraldehyde in a buffered solution. The phosphate buffer keeps the pH steady and the osmolarity close to physiological, so tissues don’t drift or distort during the first fixation. After this primary fixative step, it’s important to wash away residual fixative with buffer to prevent continued, uneven fixation.

Next, an osmotic stabilizer such as sucrose is used. This step helps balance the osmotic pressure and protects delicate membranes from shrinking or swelling during subsequent processing, setting the tissue up for the heavy-metal post-fixation.

Finally, osmium tetroxide is applied as the post-fixative. It cross-links lipids and adds electron density to membranes, yielding the strong contrast needed to visualize membrane structures clearly in the electron microscope.

Following this sequence—glutaraldehyde in buffer first, a buffer rinse, osmotic stabilization with sucrose, then osmium tetroxide post-fixation—helps preserve ultrastructure and maximize membrane contrast.