Fixation methods should be aimed at maximizing the rapidity of penetration of fixative into the tissue and the resulting interaction with and stabilization of cellular components.
1. Temperature -A higher temperature promotes more rapid penetration of fixative into the tissue but the risk of protein denaturation before fixation is greater. A lower temperature may minimize protein denaturation but penetration rate is slower. A good compromise is fixation at room temperature but drastic temperature changes may deform the tissue.
2. Surface Area/Volume -The S/V of the specimen should be as high as possible within the limits of the least manipulatable unit (usually about 1 mm3 ).
3. Agitation - Agitation (rotating tumbler) promotes diffusion and exchange of solutes.
4. Natural Barriers - Low permeable barriers such as bacterial and plant cell walls, and hard substances such as cartilage, chitin and keratin mitigate longer fixation times.