Histologists know this proverb by heart: “Tissues that didn’t fix well will not process well, will not section well, and will not stain well.” Everything in histology begins with proper fixation. When fixation is compromised, it creates a cascade effect through every subsequent step — processing, embedding, sectioning, staining, and ultimately interpretation. There’s another universal truth that is harder to digest but just as accurate: “Garbage in, garbage out.” We know this. We live this. And yet, fixation is often the one step we have the least control over.
When the Fixation Didn’t Go Well
Recently, our lab received several large pieces of swine heart ventricle from a new transplant research project. It was an exciting collaboration involving ex-vivo perfusion technology to revive a cardiac-arrested heart. The experiment ran all day and ended late at night.
The surgeon excised ventricular tissue measuring approximately 45 mm × 15 mm. The specimens were placed into a urine collection jar and covered with just enough 10% neutral buffered formalin (NBF) to submerge them. They were then left overnight in our drop-off box with instructions to process and embed the entire piece.
The next morning, the surface looked well fixed. But as every experienced histologist knows — fixation is not a surface phenomenon.
Because of the tissue thickness, we ran the specimens on our Long Processing Program (two- days process schedule). The tissue would had processed well if it was well fixed but the issue revealed itself during microtomy. As we trimmed deeper into the block, the core of the tissue was bright red and raw — completely unfixed. When we attempted flatten the section onto the floating water bath, the tissue section just separated from the paraffin quickly. The unfixed core fragmented and “exploded,” dispersing through the bath like confetti. The only temporary workaround was lowering the water bath temperature, but the damage was done — folds, fragmentation, and compromised morphology.
All because fixation was insufficient at the start.
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Large specimen in small container
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Under-fixed core of the tissue
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The Communication Challenge
Communicating the importance of fixation to surgeons and researchers can be one of the most challenging parts of our role.
The issues are rarely intentional.
They are often due to:
- Misconceptions
- Limited supplies
- Time constraints
- Cost considerations
- Or simply not knowing
In the scenario above, the surgeon had not prepared enough formalin and only had urine jars available when they harvested the tissue.
As it occurred to us often, we’ve received specimens fixed in formalin but was kept in the fridge overnight and transported in our lab on ice, worrying that the tissue would be spoiled if kept in room temperature, as if they were perishable food.
On one occasion, I was even challenged by a consultant pathologist who suggested that the “1:20 tissue-to-fixative ratio” or the even the “minimum 1:10 ratio” was a myth. Fortunately, we were able to reach consensus at the end and implement proper ratios for fixation on that project.
All these examples tell us that fixation practices vary widely — and not always under the histology lab’s control.
What Can We Do as Histologists?
While we may not control how tissues are harvested, we can influence education and protocol.
In the swine heart case above, we:
- Transferred the tissue into a larger container upon arrival to improve fixative volume
- Negotiated permission to trim the tissue longitudinally to reduce thickness
- Reinforced fixation guidelines with the research team
In our lab, we:
- Educate new students and researchers about optimal tissue thickness
- Discuss fixation temperature considerations
- Emphasize proper tissue-to-fixative ratios
- Provide written fixation protocols to all submitting users
- Train our histology team to assess fixation quality during grossing
Vigilance during accessioning and grossing can prevent downstream disasters.
Quick Fixation Facts
- Formalin penetrates tissue at approximately 1 mm per hour
- Optimal tissue thickness: 3–4 mm
- Complete protein cross-linking requires approximately 24 hours
- Recommended fixative for routine histology: 10% neutral buffered formalin
Other fixatives our lab use include:
- Bouin’s solution for tissue destined for Masson trichrome staining
- Osmium tetroxide for lipid preservation
- Clarke’s solution for frozen sections and smears
More Than a Technical Step
Fixation is not just a technical requirement — it is the foundation of diagnostic quality and research integrity. As histologists, we are not merely processors of tissue. We are stewards of morphology. We are quality advocates. We are educators. And sometimes, we are diplomats. If we continue to speak up, educate early, and intervene when needed, we protect not just our slides — but the science and patients behind them.
Written by NSH Member Napoleon Law HTL(ASCP)CM
Napoleon is an ASCP board-certified Scientist in Histotechnology who leads the Histopathology Core at the STTARR Innovation Centre, University Health Network, where he built a single-person lab into a fully staffed core facility. With over a decade of experience in preclinical research histopathology, he provides expert histology services and consultation across diverse research areas, supported by deep hands-on knowledge of tissue fixation, processing, and sectioning. His training in biology and veterinary technology further strengthens his expertise in animal anatomy, necropsy, and specialized tissue harvesting for complex research studies.