Skip to main content

Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact


• Background: Whole eyes fixed in 4% buffered formaldehyde (10% neutral buffered formalin) demonstrate a variety of artifacts, including separation of the neurosensory retina from the retinal pigment epithelium. We postulate that the osmolarity of 4% buffered formaldehyde causes contraction of the internal compartments of the eye leading to several artifactual changes commonly observed in routine histologic sections. • Methods: In part I of the study, enucleated animal eyes were examined histologically after immersion in different concentrations of formaldehyde. The variables of fixation and processing were kept constant except for the concentration and osmolarity of formaldehyde. In part II, enucleated animal eyes were used to empirically determine the optimal mixture of formaldehyde and glutaraldehyde for fixation based on subjective assessment of histologic sections. • Results: In the first part of the study, the post-fixed volume of the anterior chamber and vitreous increased as the concentration (and osmolarity) of formaldehyde decreased. In part II of the study, fixation of whole eyes was optimal with a mixture of 1% buffered formaldehyde and 1.25% glutaraldehyde. The neurosensory retina was less likely to detach from the retinal pigment epithelium, and the anterior chamber retained a more normal shape with this fixative. • Conclusions: Volume contraction of whole eyes fixed in 4% buffered formaldehyde is caused by the relatively high osmolarity of the fixative. Immersion fixation of whole eyes for 36 h (or longer) in 1% buffered formaldehyde/1.25% glutaraldehyde reduces tissue distortion without compromising cellular preservation.

This is a preview of subscription content, access via your institution.


  1. Apple DJ, Rabb MR (1985) Ocular pathology. Clinical application and self-assessment. Mosby, St Louis, p 4

    Google Scholar 

  2. Carson FL (1990) Histotechnology. A self-instructional text. ASCP Press, Chicago, pp 10–11

    Google Scholar 

  3. Folberg R, Campbell JR, Guzak SV, Luckenbach M, Margo CE, Proia AD, Schachat AP (1993) Ophthalmic pathology and intraocular tumors. Basic and clinical science course. American Academy of Ophthalmology, San Francisco, p 14

    Google Scholar 

  4. Foos RY (1978) The eye and adnexa. In: Coulson WF (ed) Surgical pathology. Lippincott, Philadelphia, pp 1405–1406

    Google Scholar 

  5. Fox CH, Johnson FB, Whiting J, Roller PP (1985) Formaldehyde fixation. J Histochem Cytochem 33:845–853

    Google Scholar 

  6. Hopwood D (1982) Fixation and fixatives. In: Bancroft JD, Stevens A (eds) Theory and practice of histological techniques. Churchill Livingstone, Edinburgh, p 27

    Google Scholar 

  7. Karcioglu ZA, Haik BG (1987) Tissue diagnosis: intraocular tumors. In: Karcioglu ZA (ed) Laboratory diagnosis in ophthalmology. Macmillan, New York, p 53

    Google Scholar 

  8. Luna LG (1968) Manual of histologic staining methods of the Armed Forces Institute of Pathology, 3rd edn. McGraw-Hill, New York, p 3

    Google Scholar 

  9. Margo CE, Grossniklaus HE (1991) Ocular histopathology, a guide to differential diagnosis. Saunders, Philadelphia, p 328

    Google Scholar 

  10. Margo CE, Karcioglu ZA (1987) Diagnostic electron microscopy. In: Karcioglu ZA (ed) Laboratory diagnosis in ophthalmology. Macmillan, New York, pp 120–130

    Google Scholar 

  11. Menocal NG, Ventura DB, Yanoff M (1980) Eye technics. Routine processing of ophthalmic tissue for light microscopy. In: Sheehan DC, Hrapchak BB (eds) Theory and practice of histotechnology, 2nd edn. Mosby, St Louis

    Google Scholar 

  12. Torczynski E (1981) Preparation of ocular specimens for histopathologic examination. Ophthalmology 88:1367–1371

    Google Scholar 

  13. Yanoff M (1973) Formaldehyde-glutaraldehyde fixation. Am J Ophthalmol 76:303–304

    Google Scholar 

  14. Yanoff M, Fine BS (1967) Glutaraldehyde fixation of routine surgical eye tissue. Am J Ophthalmol 63:137–140

    Google Scholar 

  15. Yanoff M, Zimmerman LE, Fine BS (1965) Glutaraldehyde fixation of whole eyes. Am J Clin Pathol 44:167–171

    Google Scholar 

Download references

Author information

Authors and Affiliations


Rights and permissions

Reprints and Permissions

About this article

Cite this article

Margo, C.E., Lee, A. Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact. Graefe's Arch Clin Exp Ophthalmol 233, 366–370 (1995).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI:


  • Formaldehyde
  • Glutaraldehyde
  • Retinal Pigment Epithelium
  • Anterior Chamber
  • Histologic Section