Which factor, if extended, can cause tissue to become over hardened during fixation?

• A. Prolonged fixation
• B. Short exposure
• C. Rapid dehydration
• D. Low temperature

Answer: (A)

Prolonged fixation causes tissue to become over-hardened because fixatives create cross-links between proteins; the longer the tissue sits in fixative, the more cross-linking occurs, stiffening the tissue and making it brittle. This excessive cross-linking can also hinder proper sectioning and later staining. Short exposure would leave the tissue underfixed, and rapid dehydration or low temperature are not the primary drivers of over-hardening during the fixation step (rapid dehydration occurs later in processing, and low temperature slows fixation rather than increasing hardening).