10/11/2017 Stainsfile - 10% Neutral buffered formalin

10% Neutral Buffered

Formalin
(4% Neutral Buffered Formaldehyde)

10% Neutral Buffered Formalin Litre 5 Litres 50 Litres

Strong formalin 100 mL 500 mL 5 L
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Tap water 900 mL 4.5 L 45 L
Sodium dihydrogen phosphate, monohydrate (NaH2PO4.H2O) 4.0 g 20.0 g 200 g Google Translate

Disodium hydrogen phosphate, anhydrous (Na2HPO4) 6.5 g 32.5 g 325 g Instructions

Neutral buffered formalin, usually simply shortened to NBF, has become the standard
fixative for use in a diagnostic setting. It is more effective than the simple formalin mixtures
as the phosphate salts present make it unlikely that erythrocytes will be damaged, and the
neutral pH inhibits the formation of formalin pigment. The phosphates will adjust the pH to
about 7.0 as the "neutral" infers, but there is no need to adjust it to this level if it is slightly
different.

Although the formation of formalin pigment is inhibited it is not stopped altogether, and it
may slowly form in very bloody tissues, or in tissues stored for a long time in NBF without it
being changed.

NBF is useful as a fixative for museum and photography specimens as it permits restoration
of natural colour to the specimen.

Time
This fixative should be applied overnight as a minimum, but fixation is not complete until
applied for a few days, and a week or two is not too long. For thorough fixation the proteins
in the tissue need to be crosslinked, but it is well known that simple formalin mixtures
discolour tissues well before they crosslink the proteins. For that reason visual observation
does not give any indication as to the degree of fixation, and discolouration should never be
used as an indicator that it is complete.

It is essential that the time in fixative be noted, and sufficient time be allowed for the
chemical reactions to occur. Time measured in a few hours is not adequate, and the lack of
crosslinking in tissues treated for such a short time will not give adequate protection to the
tissue from the fixation effects of dehydrating ethanols. Smaller pieces of tissue do not fix
appreciably faster than larger pieces. Fine needle biopsies require the same length of time as
3 mm thick slices of solid organs.

On a practical basis in a diagnostic laboratory the slowness of simple formalin fixation is a

distinct drawback. If the constraints of making a diagnosis are a major problem, then
consideration should be given to increasing the temperature of the fixative solution. It must
be emphasised that, although increasing the temperature of the fixative may increase the
speed of fixation, it will cause a reduction in the quality of the final stained section and there
may also be effects on some staining or immunohistochemical reactions. The temperature
increase should be kept to a minimum consistent with accomplishing the goal of faster
fixation, since heat itself is a means of fixation. If increased temperatures are used then
formaldehyde fumes will be generated, more so as the temperature is increased, so fixation
should be done in a fume chamber to protect personnel.

Complete organs are often placed in containers filled with NBF. Although formalin
penetrates fairly rapidly, it will not reach the centre of a large mass for some time and the
delay may permit some autolysis in the middle of the tissue. Large organs should be
described as soon as possible, then sliced into 1 cm or so slices to allow the fixative to gain
access. Similarly, intestines should be opened and cleaned of any contents, then returned to a
container large enough to permit the fixative to contact all the tissue surfaces, or pinned out
on a board with rust free pins and placed into the fixative.

Tissues may be stored in this solution for long periods, but the fixative should be changed at
least every six months.

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Aftertreatment
NBF contains salts which have limited solubility in high concentrations of ethanol. For that
reason the tissues should be transferred to a dehydrant containing 60% ethanol, or less, for a
short time in order to give the salts an opportunity to be removed. If transferred directly to
95% or absolute ethanol the phosphates will most likely precipitate on and in the tissue,
causing difficulties in sectioning, such as tearing and scoring. Processing machines should be
flushed periodically with water to remove accumulated salts.

Secondary fixation
Other fixatives may be applied after formalin fixation, and some of their characteristics will
be obtained. It must be realised that secondary fixation in any fixative will not give the same
results as would have been obtained if the secondary fixative had been applied to fresh tissue.

Reference
Drury, R.A.B. and Wallington, E.A.,
Carleton's Histological Technique, Ed. 5,
Oxford University Press, Oxford, UK.

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Last updated January 2009

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