Histology FAQ

Staining, Histochemistry and Histotechnology

(Frequently Asked Questions)


Dr. John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada



FAQ Home > Fixation, freezing etc

Alternatives to mercury-containing fixatives


What is the best substitute for B-5 fixative, without mercuric chloride?

  [ B-5 is: Water                                   90 ml
               Formalin (40% HCHO)            10 ml
               Mercuric chloride                   6 g
               Sodium acetate (anhydrous)   1.25 g

The sodium acetate brings the pH into the 5.8-6.0 range. Fix by immersion, 12-24 hours, then transfer to 70-80% alcohol. See Lillie RD & Fullmer HM 1976 Histopathologic Technic and Practical Histochemistry. New York: McGraw-Hill, pp 52-53.]


We recently completed a "blind comparison" of B-5 substitutes. We need to find something, as our water treatment plant had notified us that as part of a Zero Discharge Program they would be monitoring our mercury output.  Of course, we were capturing our mercury ... but we still had measurable amounts in our discharged water. The treatment plant immediately zeroed in on our department, and without delay asked if we used mercury fixatives! We agreed that we would cease, or absolutely contain our mercury by June 1, 1998. I felt it better to cease using mercury, so that any future mercury found in the discharge water from the hospital could be blamed on another source!! We had all of our sink traps cleaned, and tested ... no mercury coming from us!!!

For our study, we used our standard B-5, Z-5, and Z-fix from Anatech, IBF from Surgipath, our 10% NBF, and B-plus fixative from BBC. We used tonsil and lymph nodes for the study, and placed small pieces of tissue in each of the fixatives, and gave them to the pathologists labeled as fixative 1, fixative 2 etc. The pathologists were given an evaluation sheet with each case, and asked to rank the fixatives from 1-6, with 1 being the best.  When we had tested a sufficient number of cases, the evaluations were tallied, and lo and behold ... B-5 won!  I wasn't surprised, and neither were the pathologists.  We all agreed that we would use the second place winner.

This was B-Plus Fix which is sold by BBC (800-635-4477, or write to PO Box 609, Stanwood, WA98292). However, all the solutions that we tested were acceptable. One surprising result was that our 10% NBF came in 3rd, very close to our 2nd place winner. We have been using our substitute since March, and are pleased with the results so far...  However, the pathologists are missing their B-5, which they still refer to as the gold standard.

Sheila Tapper
St. Mary's / Duluth Clinic Health Systems
Duluth, MN
(STapper [AT]smdc.org)