3/12/97
Can someone explain the relation between the tonicity and osmolarity
of a fixation solution?
Gary Dietrich Chinga
garyc@stud.ntnu.no
depression. In simple terms it is the total concentration of solutes
(including ions) in a solution, and this does not depend on whether the
solutes can cross cell membranes or not. Tonicity relates to the osmotic
GRADIENT due to solutes that affects a semi-permeable membrane ( i.e.
a membrane that is permeable to water); ONLY solutes that do not cross
the membrane contribute to this effect. The two characteristics are
different and obviously have vastly different consequences on
membranes. Weak bases, even though ionized, have some measurable
permeability and so their contribution to tonicity must be much less than
their contribution to osmolarity. Osmium is hydrophobic and quite
permeable through membranes, so contributes *nothing* to an osmotic
gradient that can disrupt the membranes (even assuming it doesn't alter
their permeability to tonic agents like sucrose). So osmium contributes
nothing to tonicity, but certainly does contribute to osmolarity.
I personally wonder how to express the tonicity of compounds that are
somewhat permeable, such as weak bases or short carboxilic acids &
aldehydes. I guess the concept of tonicity either doesn't apply or is
operational in such cases, depending on the time scale of interest. Can
anyone comment or give a reference to a good detailed textbook?
Something about reflection coefficients?
Richard Thrift
Richard_Thrift@depotech.com
Tonicity is a relative, unitless comparison of one solution to
another in which the first solution (presumably the water in your tissue
sample) is hypertonic, hypotonic or isotonic to the second solution
(presumably your fixative). Osmolarity is an absolute scale (usually in
some type of pressure unit) which describes the concentration of osmolytes
in a single solution. So you need to know the osmolarity of your tissue so
you can set the osmolarity of your fixative to be isotonic with respect to
the tissue.
Robert R. Wise
Plant Physiologist and Director, UWO Electron Microscope Facility
Department of Biology
University of Wisconsin Oshkosh
Oshkosh, WI 54901
(414) 424-3404 tel
(414) 424-1101 fax
wise@uwosh.edu
pressure) of a solution. In a rough sense it is the measure of the amount
of solute in a solution, but the degree of dissociation of the solute
affects its osmolarity, so osmolarity is not necessarily a direct measure
of molar concentration. (For example, a 1 molar solution of NaCl will
have *approximately* twice the osmotic pressure [osmolarity] of a 1 molar
solution of sucrose, since the NaCl dissociates into 1 molar Na+ and
1 molar Cl-.)
Two solutions (not necessarily of the same solute or of a single solute)
are isosmotic if they have the same osmolarity (osmotic pressure). If two
isosmotic solutions are placed on opposite sides of a semipermeable
membrane, the osmotic pressure on each side is the same and there is no
*net* movement of water across the membrane. However, if the osmolarity
of the two solutions is not the same, then water will move across the
membrane from the hypo-osmotic solution (more dilute) to the hyperosmotic
solution (less dilute) until the osmotic pressure on each side is equal
(a number of other factors affect this as well, but let's ignore them
here).
Tonicity refers to the response of *cells or tissues* to the solutions in
which they are immersed. If cells are placed in a hypertonic solution,
net movement of water will be out of the cell, causing the cell to
shrivel. If cells are placed in a hypotonic solution, net movement of
water will be into the cell, causing the cell to swell or burst. Tonicity
is useful only in reference to a particular cell or tissue.
Thus, the microscopist wishes to add sufficient solute(s) to the fixation
solution so that the solution has the correct osmolarity (measured in
milliosmols) so that it will have the desired tonicity with respect to the
cells that are being exposed to the fixative. [There is debate whether
the solution should be slightly hypertonic or hypotonic, but then that is
another subject about which we may choose to debate.]
To summarize, osmolarity is a measurement of solute concentration
(measurement can be made in a beaker). Tonicity is a comparison of
osmolarities between a cell and the solution to which it is exposed.
I hope that this does not leave readers more confused than they were
before.
Donald L. Lovett e-mail: lovett@tcnj.edu
Assoc. Professor, Dept. of Biology voice: (609) 771-2876
The College of New Jersey fax: (609) 771-2674
Trenton, NJ 08650-4700
perpetuates the myth that tonicity is related to the difference between
two OSMOLARITIES on opposite sides of the membrane. That is ONLY
true if the membrane involved is impermeable to ALL of the solutes
contributing to the osmolarity. Since formaldehyde is soluble in both
benzene and chloroform I expect it to be quite permeable across cell
membranes. Thus it will contribute to osmolarity, but should contribute
little to tonicity. The tonicity of your fixative should be adjusted with other
components, and the final osmolarity of an isotonic fixative will be approx
300 mOsm PLUS the value of osmolarity contributed by the formaldehyde
present.
To reiterate, IF a compound is permeable across the membrane it
contributes to osmolarity but does not contribute to the osmotic
GRADIENT. Thus it does NOT contribute to tonicity.
Caveats: Practically speaking, the rate at which a permeable solute
crosses the membrane will affect the transient forces on the membrane.
I assume tonicity is ill-defined unless the system is at equilibrium. The
situation becomes even more complicated if the compounds added (such
as aldehydes or alcohols) alter the permeability of the membrane for
other buffer components present. And if differences in rates of
diffusion through a chunk of tissue are involved, are we really still talking
about tonicity?? That calls for empirical, not theoretical, optimization of
the recipe used !!
On that note, how many of you microscopists have actually compared
e.g. confocal images of cell cultures fixed with buffers of different
tonicity with equivalent images of live cultured cells? The time to diffuse
through a monolayer of cells should be negligible. What do you find is
optimum?
Richard Thrift
Richard_Thrift@depotech.com
with your comments. I had tried to focus on the difference between
the two terms and simplify my response with the caveat:
>
> "(a number of other factors affect this as well, but let's ignore them
here)."
>
I also agree that the response of the cell to the solution (as evaluated
by trial and error) is the most important aspect to microscopists,
irrespective of how one names or measures the composition of the solution.
Donald L. Lovett e-mail: lovett@tcnj.edu
Assoc. Professor, Dept. of Biology voice: (609) 771-2876
The College of New Jersey fax: (609) 771-2674
Trenton, NJ 08650-4700
In reguards to the question of tonicity, does anyone know the best
percentage of sucrose to have in a primary Zamboni fixative(2% para and
15% picric acid in sorensons) to fix cell cultures in order to minimize
any cell distortion.
Thanks in advance
Bob Underwood
Morphology core
University of Washington
underwoo@u.washington.edu
among pH, osmolality and concentration of fixative solutions, Stain
Technology 42:175-182 (1967). You need to know what osmolality you want in
the fixative solution. Around 300 milliosmols (range 200-400) seemed to be
most common when I had to work it out too many years ago. In general the
contribution of the fixative can be ignored. So, work out the osmolality of
the buffer and add sucrose to bring the osmolality up to the total. For
instance, Sorensen's is approx 105 at 0.05M, approx 210 at 0.1M. Sucrose is
obvious (eg 0.2M = 200). It may be an idea to add some CaCl2 or MgCl2.
Diana van Driel
Dept Ophthalmology
Sydney University C09
AUSTRALIA 2006
dianavd@eye.usyd.edu.au
osmotic effect of glutaraldehyde during fixation". J. Ultrastr. Res.
56:339-350, 1976. A very interesting and through study.
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane Piscataway, NJ 08854
voice: (908)-235-4583; fax -4029 e-mail: mcauliff@umdnj.edu