Re: NBF penetration rate

From:Bryan Hewlett

Brett, Rob and Patrick,

My apologies for the lengthy reply, but much confusion exists regarding this

Formaldehyde is one of the most rapidly penetrating fixatives used.
Unfortunately, it is one of the slowest to fix the tissue. This paradox was
finally explained by Burnett in 1982.

An excellent description of the properties of formaldehyde may be found in
John Kiernan’s book.

The penetration rate of formaldehyde in mm/hr is a variable thing. It
depends on how the data is obtained. It may also vary slightly depending on
tissue type. The penetration rate of formaldehyde fixatives has been
extensively studied, often with conflicting results. The penetration of
non-coagulating fixatives is difficult to measure.

The original experiments of Medawar utilized plasma clots with an indicator
to mark depth of penetration. Medawar showed that fixatives obey the
diffusion laws, that is, the depth penetrated was proportional to the square
root of time. Medawar determined a coefficient of diffusibility for each
fixative, the Medawar constant K. Using the equation d = K√t, where d is
distance penetrated in mm, t is time in hours and K the Medawar constant for
the fixative in question, it is possible to determine the penetration rate.

Medawar determined K = 5.5 for formaldehyde. Using this value NBF would
penetrate 27.5 mm in 25 hours. Plasma clots are easier to penetrate than
solid tissues, so the rate is probably less.

Baker chose a gelatin/albumen gel to more closely mimic solid tissue and
determined that K = 3.6 for formaldehyde, or 18 mm in 25 hours. Baker also
pointed out that the actual penetration into tissue would probably be less,
possibly due to the resistance of lipid containing cell membranes. He quotes
the data of Tellyesnicsky (1926) who, mainly using liver tissue samples,
indicated a more conservative K = 0.78 for formaldehyde. That would
translate to 3.9 mm in 25 hours.

>From d = K√t, it follows that fixatives penetrate more quickly into small
samples of tissue compared to large ones. The initial rate of penetration
into tissue is extremely rapid.

The first layer of cells (20 μm) takes a second or so (70 mm/hr). Using
Bakers K =3.6, the following examples will illustrate further;

1 hour = 3.6 mm,

4 hours = 7.2 mm, averaged to (1.8mm/hr),

9 hours = 10.8 mm (1.2mm/hr),

16 hours = 14.4 mm (0.9mm/hr),

25 hours = 18 mm (0.72mm/hr),

100 hours = 36 mm (0.36mm/hr).

So much for penetration rate.

The real issue is fixation rate, i.e. penetration rate plus binding time.

Fox used 14C labeled formaldehyde to study the covalent binding time for rat
kidney tissues. At a temperature of 25°C, the amount of formaldehyde bound
to tissue increased with time until equilibrium was achieved at 24 hours. At
37°C the reaction was faster and equilibrium was reached at 18 hours. A
later study by Helander also used 14C labeled formaldehyde to study binding
time for the fixation of rabbit liver. At 25°C. equilibrium was achieved at
25 hours. The correlation of results between these two studies is
impressive. Particularly in view of the fact that Fox used 16mm thick
sections of fresh rat kidney, whereas Helander used 4 mm cubes of fresh rat
liver. The virtually identical equilibrium times achieved by each study
indicate that penetration time is not a factor in the kinetics of the
reaction. Despite the fact that thin slices of tissue will be penetrated
faster than thicker slices, it would seem that the binding time is the
limiting factor for tissue stabilization. The later study by Helander,
quoted by Patrick, used rat brain and kidney at twice the thickness (8mm) of
the original study, a factor to be taken into account when comparing the

Failure to recognize the importance of formaldehyde binding time, is the
leading cause of the tremendous intra and inter- laboratory variability in
IHC performance. A clinical laboratory’s so called ‘routine formaldehyde
fixation’, actually consists of allowing the tissue to fix for variable
periods of time, dictated by the start time of the tissue processor!

For both 1 mm thick core biopsies and 4mm thick tissue slices, the minimum
stabilization time is 24-25 hours at ambient temperatures. The minimum
stabilization time does not, unfortunately, denote complete fixation time.
The initial cross-links are still relatively weak and reversible; stronger
cross-linking continues to occur over time. Complete fixation is thought to
take at least 7 days. Even after this time cross-links continue to form

Werner, quoting the two papers above, considers cross-linking complete in
24 -48 hours, but also expresses concern about the ‘overfixation’ due to
excessive cross-linking, which may occur if fixation is allowed to exceed
24-48 hours. I agree with Werner, in that excessive cross-linking may mask
some epitopes, but in my experience this does not occur with the vast
majority of antibodies in clinical use until 5 -7 days of fixation. Even
then, providing the IHC has been optimized, with the majority of antibodies
fixation up to 4 weeks is acceptable. A far more serious problem is short
<24 hour fixation. Formaldehyde fixation begins at the periphery of the
tissue. The initial layers of cells bind all of the available
formaldehyde(<0.1%) and start the ‘clock’. Methylene glycol continues to
rapidly penetrate the tissue and, over hours, more formaldehyde is generated
from methylene glycol. If this process is interrupted before completion, the
formation of addition compounds will be incomplete, easily reversed and full
stabilization by cross-linking will not occur. Depending upon the time of
interruption, the periphery may show adequate cross-linking, whereas the
remainder of the tissue is fixed by coagulant alcohol during processing.
This may have disastrous effects upon IHC staining. This will occur whether
the tissue is a small biopsy or a 4 mm slice.


Burnett MG. The mechanism of the formaldehyde clock reaction: Methylene
glycol dehydration. J Chem, educ. 1982; 59, 160

Kiernan J.A., Histological and Histochemical Methods: Theory and Practice,
3rd Edition, 1999. Oxford: Butterworth-Heinemann. ISBN # 0-7506-3106-6.

Medawar PB. The rate of penetration of fixatives. J R Microsc Soc. 1941; 61,

Baker JR. (1958).Principles of Biological Microtechnique, Methuen & Co. Ltd,
pp 37-40.

Tellyesnicsky, K., (1926). Article on ‘Fixation’ in R. Krause’s Enzyklopädie
der mikroskopischen Technik, vol.2. Berlin (Urban & Schwarzenberg).

Fox CH., Formaldehyde fixation. J Histochem. Cytochem. 1985; 33,
845 -853

Helander, KG. Kinetic studies of formaldehyde binding in tissue.
Biotechnique and Histochemistry. 1994; 69, 177 -179

Helander, K.G. Formaldehyde binding in Brain and Kidney: A kinetic study of

The Journal of Histotechnology. 1999; 22(4), 317-318.

Werner M., Effect of formalin fixation and processing on

Am J Surg Pathol. 2000; 24(7), 1016 -1019



Bryan R. Hewlett
Technical Specialist
Anatomical Pathology
Hamilton Regional Laboratory Medicine Program
Ontario, Canada

----- Original Message -----
From: "Connolly, Brett M" 
To: "'HISTONET' (E-mail)" 
Sent: Wednesday, June 05, 2002 11:50 AM
Subject: NBF penetration rate

> Can someone give me the tissue penetration rate of  10% NBF in mm/hr. at
> room temp.
> Thanks,
> Brett
> Brett M. Connolly, Ph.D.
> Merck Research Laboratories
> Dept. of Neuroscience
> WP26A-3000
> PO Box 4
> West Point, PA 19486
> PH 215-652-2501
> fax. 215-652-2075
> e-mail.
> --------------------------------------------------------------------------
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