Adv Physiol Educ 42: 626 – 635, 2018;
doi:10.1152/advan.00094.2018.
HOW WE TEACH Generalizable Education Research
Learning (by) osmosis: an approach to teaching osmolarity and tonicity
Predrag Vujovic,1 Michael Chirillo,2 and Dee U. Silverthorn3
1
Department for Comparative Physiology and Ecophysiology, Institute for Physiology and Biochemistry, Faculty of Biology,
University of Belgrade, Belgrade, Serbia; 2Department of Neuroscience, University of Texas at Austin, Austin, Texas; and
3
Department of Medical Education, Dell Medical School, University of Texas at Austin, Austin, Texas
Submitted 11 May 2018; accepted in final form 11 September 2018
Vujovic P, Chirillo M, Silverthorn DU. Learning (by) osmosis:
an approach to teaching osmolarity and tonicity. Adv Physiol Educ 42:
626 – 635, 2018; doi:10.1152/advan.00094.2018.—Understanding os-
molarity and tonicity is one of the more challenging endeavors
undertaken by students of the natural sciences. We asked students who
completed a course in animal physiology to submit an essay explain-
ing what they found most perplexing about this subject, and what
in-class activities proved most useful to them. Students had difficulty
distinguishing osmolarity from tonicity and determining tonicity
based on the solution’s composition. The most useful activities were
questions requiring simultaneous consideration of both osmolarity and
tonicity. Problems that require calculating osmotic concentration and
the volumes of body fluid compartments after administration or loss of
various solutions emphasize the significance of osmolarity and tonic-
ity in the context of systemic homeostasis and clinical medicine. We
hope that our approach to teaching osmolarity and tonicity will prove
useful to physiology lecturers who are looking for new ways of
introducing this complicated topic to their health professions students.
IV fluid therapy; osmolality; osmolarity; osmosis; tonicity
INTRODUCTION
Truly understanding osmolarity and tonicity is one of the
more challenging endeavors undertaken by students of the
natural sciences.1 In 2012, Cheng and Durairajanayagam (3)
reported that 41% of students participating in the International
Intermedical School Physiology Quiz were unable to predict
correctly what would happen to the volume of an erythrocyte
when placed in an isosmotic solution of urea. So how can we
teach osmolarity and tonicity more effectively? One strategy
for improving student understanding is to let them experience
the effects of different solutions on erythrocyte hemolysis, a
classic experiment in physiology [see, for example, the recent
Sourcebook paper by Goodhead and MacMillan (5)]. But
teaching osmolarity and tonicity in a classroom setting can be
tough. Nonetheless, mastering these concepts is essential for
those interested in pursuing careers in medicine, physiology
research, and other related fields. For example, health profes-
1
The survey mentioned in this paper was conducted informally among
undergraduate students at the University of Belgrade to improve the lecturing
process for the Systems Physiology course. Permission to use collected data for
publication was granted by the Dean’s office of the University of Belgrade,
Faculty of Biology.
Address for reprint requests and other correspondence: P. Vujovic, Dept. for
Comparative Physiology and Ecophysiology, Institute for Physiology and
Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16,
11000 Belgrade, Serbia (e-mail: predragv@bio.bg.ac.rs).
626 1043-4046/18 Copyright © 2018 The American Physiological Society
Downloaded from www.physiology.org/journal/advances (103.003.046.027) on September 25, 2019.
sionals need to understand that not all isosmotic intravenous
(IV) fluids are isotonic (15).
The approach we present here has been simplified for teach-
ing osmolarity and tonicity to undergraduate health professions
students. It is specifically designed to assist them in the proper
selection of IV fluid therapy in clinical settings. As with any
simplification of a complex physiological topic, the nuances of
the topic are glossed over to facilitate student comprehension.
The selection of appropriate IV solutions is not an exact
science, and it does not require understanding or application of
the van’t Hoff equation or reflection coefficients (1, 4, 18). The
clinician needs to choose IV solutions based on their ability to
increase effective circulating volume, to expand or contract
cell volume, and to affect osmolarity. Having the skills to
calculate approximations of these changes using broad assump-
tions helps prevent iatrogenic errors in clinical settings.
The difficulties students and teachers alike encounter while
learning osmolarity and tonicity are at least partially accounted
for by the differing approaches taken by many physiology
texts. A lack of consistent terminology and detail often leaves
room for confusion and misconceptions (Table 1).
Because osmolarity and tonicity are fundamental and often
confusing, we thought it important to review the topic for
instructors, as well as outline some teaching approaches we
have found effective in the classroom. We start with our
definitions of the two terms, then give you a chance to check
your understanding of the basics of osmolarity and tonicity
through a few short questions and a brief review. We then
present several aspects of this topic that our students find most
confusing. Finally, we outline our approach to teaching osmo-
larity and tonicity and give examples of questions that we have
used to develop our students’ understanding of the topic.
Clarifying definitions. The varying presentations of tonicity
in Table 1 are tricky to understand at first glance. Let us start
by clarifying the definitions of osmolarity and tonicity. Our
definitions apply to biologically relevant situations only, so we
are ignoring solutions such as concentrated acids and bases.
One note on units: in this paper, milliosmole is expressed in
units of mosmol, whereas milliosmolar (mosmol/l) is ex-
pressed as mosM. (Note the capitalizations.)
Osmolarity is a measure of the concentration of osmotically
active particles in a solution. It is sometimes called a “colli-
gative” property of the solution by chemists because it depends
on the number of particles in a volume of solution rather than
the identity of the particles. Osmolarity is closely related to
molarity, a concept that most students learn in introductory
chemistry. One mole of any substance has Avogadro’s number
 TEACHING OSMOLARITY AND TONICITY 627

Table 1. Varying definitions of tonicity in a sample of physiology textbooks

Michael D. Johnson, Human Biology: Concept and Current Issues (8th Edition) (9). “Tonicity refers to the relative concentrations of solutes in two fluids.
Extracellular fluid that is isotonic has the same solute concentration as the intracellular fluid. When cells are placed in a hypertonic solution, one with a
concentration of solutes higher than the intracellular fluid, water diffuses out of the cells and the cells shrink.”
Cindy L. Stanfield, William J. Germann, Principles of Human Physiology (3rd Edition) (17). “Whereas a solution’s osmolarity is based solely on its total
solute concentration, its tonicity is determined by how it affects cell volume, which depends not only on the solute concentration but also on the solute
permeability of cell membranes. A solution is said to be isotonic when it does not alter cell volume. This refers to the cell’s final volume. Under certain
conditions a cell may shrink or swell initially, even if the solution is isotonic.”
Richard W. Hill, Gordon A. Wyse, Margaret Anderson, Animal Physiology (7). “If a cell membrane or epithelium is impermeable to a solute and the solute
is more concentrated on one side than the other, the solute creates a persistent difference of osmotic pressure across the cell membrane or epithelium.”
Arthur C. Guyton, John E. Hall, Medical Physiology (10th Edition) (6). “If a cell is placed into a hypotonic solution that has a lower concentration of
impermeant solutes, water will diffuse into the cell, causing it to swell; water will continue to diffuse into the cell, diluting the intracellular fluid while
also concentrating the extracellular fluid until both solutions have about the same osmolarity.”
Walter F. Boron, Emile Boulpaep, Medical Physiology: A Cellular and Molecular Approach (2nd Edition) (2). “The difference between effects of mannitol
and urea on the final cell volume illustrates the need to distinguish between total osmolality and effective osmolality (also known as tonicity).”
Kenneth S. Saladin, Anatomy & Physiology: The Unity of Form and Function (4th Edition) (14). “Tonicity is the ability of a solution to affect the fluid
volume and pressure in a cell. If a solute cannot pass through a plasma membrane, but remains more concentrated on one side of the membrane than on
the other, it causes osmosis.”

(6.02 ⫻ 1023) of particles. Molarity (M) of a solution is an
expression of concentration, with one mole of solute per liter of
solution.
However, the molarity of a solution is not always the same
as the solution’s osmolarity. This is because some solutes, such
as ionic compounds like NaCl, dissociate into separate parti-
cles (e.g., Na⫹ and Cl⫺) when dissolved in water. For an ideal
solution, the solution’s osmolarity equals its molarity times a
dissociation factor, the number of ions formed from one
particle of the solute when placed in water:
osmolarity共osM兲 ⫽ molarity共M兲 ⫻ dissociation factor
Every solute has a dissociation factor, or osmotic coefficient
(5). For any substance that does not dissociate into ions when
dissolved in water, such as glucose or urea, the dissociation
factor is 1 and the solution’s osmolarity is the same as its
molarity (e.g., 1 M glucose ⫽ 1 osM glucose).
The dissociation factor for an ideal solution of NaCl is 2,
given that the compound is made up of 2 ions. However, many
ionizable substances do not completely dissociate in water. For
example, the particles formed when NaCl dissolves in water
include Na⫹, Cl⫺, and some undissociated NaCl. The calcu-
lated dissociation factor for NaCl at 25°C is 1.8 particles per
NaCl (13). This means that a 1 mM solution of NaCl would be
a 1.8 mosM solution. We prefer to use the term “dissociation
factor” when teaching, because students understand the rela-
tionship between dissociation of ionic compounds and osmo-
larity.
You can measure the osmolarity of a solution using a
machine called an osmometer. The most common commercial
osmometers measure either freezing point depression or vapor
pressure of a sample of solution (12, 16). An educational
activity for student laboratories with access to an osmometer is
to have the students calculate how to make a 150 mM solution
of NaCl, make it using good laboratory technique, then mea-
sure the osmolarity of the resultant solution. (Answer: Use
8.775 g NaCl/liter of solution. Predicted osmolarity using the
dissociation factor of 1.8 would be 270 mosM.)
Osmolarity is an important property of any biological solu-
tion. But just as knowing the molarity of a solution does not tell
us its osmolarity, knowing the osmolarity of a solution does not
tell us its tonicity. This is because tonicity is a term that
requires two compartments: the solution being described and a
cell. In addition to knowing the concentration (osmolarity) of
the solution, you must know the composition of the solution:
what the solutes are and whether or not they can enter the cell.
Solutes that enter a cell by any means (simple diffusion,
protein-mediated transport, and so on) are said to be “penetrat-
ing” solutes. Solutes that do not enter the cell are said to be
“nonpenetrating” solutes.
The tonicity of a solution predicts the effect of the solution
on cell volume at equilibrium and depends on the relative
concentrations of nonpenetrating solutes in the cell and the
solution. At equilibrium, water movement into the compart-
ment with the higher starting concentration of nonpenetrating
solutes will increase that compartment’s volume. Net water
movement stops when the concentrations of nonpenetrating
solutes in the cell and solution are equal.
The cell’s volume change in response to the solution tells us
the tonicity of the solution:
• If cell volume at equilibrium has increased, the solution is
said to be hypotonic to the cell.
• If cell volume at equilibrium has decreased, the solution is
said to be hypertonic to the cell.
• If cell volume at equilibrium has not changed, the solution is
said to be isotonic to the cell.
In other words,
• If the cell has a higher initial concentration of nonpenetrat-
ing solutes than the solution, at equilibrium water will have
moved into the cell. The solution is hypotonic to the cell.
• If the solution has a higher initial concentration of nonpen-
etrating solutes than the cell, at equilibrium water will have
moved out of the cell. The solution is hypertonic to the cell.
• If the cell and solution have equal concentrations of non-
penetrating solutes, at equilibrium there will be no net
movement of water. The solution is isotonic to the cell.
Because clinicians are usually selecting IV fluids based on
their effect on intracellular fluid (ICF) and extracellular fluid
(ECF) volumes, describing tonicity in terms of cell volume
change is the most useful approach for teaching health profes-
sions students.
Freely penetrating solutes in a solution can be ignored for
the purpose of determining tonicity. They will contribute to the

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628 TEACHING OSMOLARITY AND TONICITY
osmolarity of the solution but distribute throughout the cell-
solution system as if the cell membrane were not present. They,
therefore, do not ultimately contribute to water movement
between compartments. This is discussed more below.
There are several important points to keep in mind about
tonicity:
• Tonicity has no units like mM or mosM. It is a comparative
term that predicts changes in cell volume at equilibrium after
exposure of the cell to a solution.
• By convention, we speak of the tonicity of a solution relative
to a given cell. We never speak of the cell being hypo-/
hyper-/isotonic to the solution.
• With one exception, knowing only the osmolarity of the
solution tells you nothing about the tonicity of the solution.
The exception is that all hyposmotic solutions are hypotonic.
• Tonicity describes what has happened to cell volume at
equilibrium but does not tell you what happens to cell
volume on the way to equilibrium. We will return to this
concept later.
• It is important to be clear about the cell that is the frame of
reference when discussing tonicity. Solutes that can enter
one type of cell may not be able to enter a different type of
cell. One example where this is true involves the disaccha-
ride sucrose (table sugar). Mammalian cells do not have
transporters for sucrose, so sucrose is a nonpenetrating
solute for mammals. But plant cells do have sucrose trans-
porters, making sucrose a penetrating solute for plants.
When teaching health professions students, we assume that
the idealized cell is a typical human cell, and we use urea and
NaCl as our prototypical solutes. Urea is the classic example of
a penetrating solute. It freely crosses most cell membranes,
especially the erythrocyte membrane, via diffusion through
urea transporters and (to a small degree) through the phospho-
lipid bilayer (10). NaCl is the primary nonpenetrating solute of
the ECF. Its ions carry a charge, making it difficult for them to
freely pass through the phospholipid bilayer of the cell mem-
brane. As noted later, while some Na⫹ does leak into cells,
Na⫹-K⫹-ATPases pump Na⫹ out at roughly the same rate,
making this solute functionally impermeable. We also make
the assumption that all solutes in the cell are nonpenetrating
and will not leave the cell as long as the cell membrane is
intact.
Test your understanding. Below is a set of basic questions
meant to check your understanding of osmolarity and tonicity.
For each of the following questions, there are five assumptions
we will make to simplify the matter:
1. Our idealized cell has an internal concentration of 300
mosM. (We will continue this assumption through the
rest of the text, unless otherwise noted.)
2. The solutes present in the cell are assumed to be nonpen-
etrating and cannot leave the cell.
3. NaCl is a functionally nonpenetrating solute that behaves
as if it cannot cross the cell membrane. (As noted above,
in reality, Na⫹ that leaks into the cell is removed by the
Na⫹-K⫹-ATPase at a rate that closely matches the rate of
leakage in.)
4. Urea is a freely penetrating solute that easily crosses the
cell membrane. Because of this, urea will distribute
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throughout body compartments until its concentration is
equal in all compartments.
5. Water freely crosses all cell membranes, dividing the
intracellular and extracellular compartments.
Try to answer these questions based on your understanding
of osmolarity and tonicity.
1. You place a cell with an internal concentration of 300
mosM in a 400 mosM solution. What happens to the
volume and osmolarity of the cell at equilibrium?
Answer: The first part of this question asks about cell
volume and, therefore, addresses tonicity of the solution.
Tonicity depends on the relative concentrations of nonpen-
etrating solutes in the solution and cell. There is no way to
tell what will happen to the cell’s volume from this question
as written, because all you are told is that the solution is 400
mosM. To predict tonicity, you must know if the solution
solutes are penetrating or nonpenetrating, and the question
did not give you that information.
The second part of the question asks about the osmolarity of
the cell at equilibrium. This is a question about concentra-
tion, not cell volume. The 400 mosM solution is hyperos-
motic to our idealized cell. Thus you can predict that the cell
will become more concentrated, either because water moves
out into the solution, or because penetrating solute moves
into the cell. You do not know the solutes in the solution,
and you do not know the relative volumes of the cell and the
solution. Without this information, you cannot say what the
actual osmolarity will be at equilibrium. You can only say
that osmolarity has increased.
2. Which would have a greater effect on the volume of a cell
at equilibrium: solution A composed of 300 mosM NaCl
and 200 mosM urea, or solution B composed of 200
mosM NaCl and 300 mosM urea?
Answer: This question asks about cell volume and is, there-
fore, addressing tonicity, which depends on the concentra-
tions of nonpenetrating solutes. The nonpenetrating solute in
these solutions is NaCl, so we compare the NaCl concen-
trations in the solution to the cell’s osmolarity, assumed to
be due only to nonpenetrating solutes. At equilibrium, water
will have moved to equalize the nonpenetrating solute con-
centrations. We can ignore the urea, which is penetrating and
will not cause water to shift between compartments once
equilibrium is reached.
• Solution A has 300 mosM NaCl compared with the
cell’s nonpenetrating solutes at 300 mosM. The nonpen-
etrating solute concentrations are equal in cell and
solution, so there is no gradient to cause net water
movement. Cell volume at equilibrium does not change,
making solution A isotonic to the cell.
• Solution B has 200 mosM NaCl compared with the
cell’s nonpenetrating solutes at 300 mosM. The nonpen-
etrating solute concentration is higher in the cell, so
water moves into the cell until the nonpenetrating solute
concentrations are equal. Water movement into the cell
increases the cell volume at equilibrium, making solu-
tion B hypotonic to the cell.
So although both solutions have an osmolarity of 500
mosM and are hyperosmotic to the cell, hypotonic
solution B exerts a greater effect on the volume of the
cell at equilibrium than isotonic solution A.
 TEACHING OSMOLARITY AND TONICITY
This example shows how a hyperosmotic solution can
be isotonic or hypotonic, depending on the concentra-
tion of nonpenetrating solutes in the solution. Saying “a
cell will ultimately shrink after being placed in a hy-
perosmotic solution” is not always correct, although this
is frequently heard in the classroom. A cell will shrink
when placed in a hypertonic solution. This example is
useful for showing students that, while osmolarity and
tonicity are related, they are not the same thing.
3. Given the same solutions as in the previous question (300
mosM NaCl/200 mosM urea or 200 mosM NaCl/300
mosM urea), which solution would have a greater effect
on the osmolarity of the cell at equilibrium?
Answer: This question, like the second part of question 1,
asks about the osmolarity of the cell at equilibrium. If you
place a 300 mosM cell in a 500 mosM solution, you know
that the final concentration of the cell will increase. Because
solutions A and B have equal osmolarities, you know that
they will have the same effect on the final concentration of
the cell.
One important take-home message from these three ques-
tions is that a student should be able to look at the concentra-
tions of penetrating and nonpenetrating solutes in a solution,
figure out its relative osmolarity and tonicity compared with a
cell, and recognize how the solution will affect the volume and
osmolarity of a cell at equilibrium. For example, given our
hypothetical 300 mosM cell, describe the osmolarity and to-
nicity of solution C with 200 mosM NaCl and 500 mosM urea.
The answer is that solution C at 700 mosM is hyperosmotic but
hypotonic, because the solution has 200 mosM nonpenetrating
solutes vs. 300 mosM in the cell.
How can a hypotonic solution like C increase the osmolarity
of the cell? If the cell takes up water and swells, you might
expect that the cell’s internal concentration would decrease
(concentration ⫽ solute/volume). However, this explanation
Osmotic
equilibrium reached
hypotonic
Cell placed in
Cell volume
Cell volume
the solution
isotonic
hypertonic
Time
Solution B
300 mosM NaCl +
200 mosM urea
Cell placed in Osmotic
Cell volume
Cell volume
the solution equilibrium reached
Time
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629
fails to account for the penetrating solute urea, which is also
able to get into the cell. Urea, spreading by diffusion through-
out the solution and cell, will ultimately add to the overall
solute in the cell. Thus the cell in solution C will swell, but also
become more concentrated due to movement of urea into the
cell down its concentration gradient.
An important concept to emphasize with students is that the
cell’s final concentration will follow that of the osmolarity of
the solution, regardless of the solution’s effect on cell volume.
If you add a cell to a hyperosmotic solution, its osmolarity will
increase, regardless of whether the cell swells, shrinks, or stays
the same volume. The cell’s concentration will always follow
that of the solution. Placing a cell in a hyposmotic solution will
cause the cell’s concentration to decrease. Placing a cell in an
isosmotic solution will not change the cell’s concentration.
Student points of confusion. Considering that understanding
osmolarity and tonicity seem to require more effort on the part
of the student, we thought it important to uncover what aspects
students perceive as most complicated or confusing. We asked
the students who had completed a course in Animal Physiology
at the University of Belgrade (Serbia) during the last 4 aca-
demic years (2014 –2017) to submit a short essay explaining
what they found most challenging about osmolarity and tonic-
ity and what in-class activities proved most useful to them.
After analyzing essays submitted by over 80 students, we
found that confusion stems from one or more of the three main
issues:
1. Distinguishing tonicity from osmolarity
2. Determining tonicity based on the solution’s composition
3. Distinguishing units expressing amount vs. concentration
of solutes (e.g., osmol vs. osmol/l or osM)
Given these very common points of confusion, we de-
signed a set of teaching activities that enable students to
fully understand and apply the concepts of osmolarity and
tonicity.
Solution A Osmotic
200 mosM NaCl + equilibrium reached
200 mosM urea
Cell placed in
the solution
Fig. 1. The time-dependent effects of three
hyperosmotic solutions of different tonicities.
Solution A contains 200 mosM NaCl and 200
mosM urea (400 mosM solute); solution B has
300 mosM NaCl and 100 mosM urea (again,
400 mosM solute); and solution C is 400
Time mosM NaCl (once again, 400 mosM solute).
Considering that water molecules move faster
Solution C across the membrane than solutes, all of the
solutions will initially cause a cell to shrink.
400 mosM NaCl
However, once the osmotic equilibrium is
reached, the cell’s volume will increase in
Cell placed in hypotonic solution A, stay the same in iso-
the solution tonic solution B, and decrease in hypertonic
solution C.
Osmotic
equilibrium reached
Time
630 TEACHING OSMOLARITY AND TONICITY

Solution A Solution B

Osmotic equilibrium
reached

Fig. 2. The task that requires students to pre-

dict a solution’s osmolarity and tonicity based
Cell placed in Cell placed in

Cell volume
on how the solution affects the cell’s volume

Cell volume
the solution. the solution.
before reaching osmotic equilibrium. Consid-
ering that the cell’s volume did not change
after being placed in solution A, this solution
is isosmotic and isotonic. In the second sce-
nario, the cell’s volume increased once the Osmotic equilibrium
osmotic equilibrium was reached, indicating reached
the solution is hypotonic. Since the cell’s
volume was continuously increasing, solution Time Time
B could be either hypo- or isosmotic.

ANSWER: Solution A is isotonic ANSWER: Solution B is hypotonic but

and isosmotic. can be either hyposmotic or isosmotic.

A starting point in the classroom. Beginning with clear and
concise definitions of the terms establishes a distinction be-
tween osmolarity and tonicity. Whereas most students are
quick to understand that osmolarity is a concentration, most of
them struggle with differentiating osmolarity from tonicity.
Osmolarity and tonicity are certainly related, but they are not
the same thing as noted above, and this should be stressed in
the classroom. According to our students’ reports, they found
it helpful to remember that tonicity is defined by the effect a
solution has on cell volume at equilibrium, and that tonicity is
determined by comparing the concentrations of nonpenetrating
solutes in the solution and the cell. In these essays, students
also mentioned they found it especially difficult to remember
that tonicity describes how the solution affects cell volume at
equilibrium. Furthermore, the fact that a hyperosmotic solution
can be hypo-, iso-, or hypertonic proved perplexing to many of
them.
One of the tasks that seems very effective at clarifying these
two muddy points is asking students to graph the time-depen-
dent effects of three hyperosmotic solutions, all with the same
concentration of 400 mosM, on the volume of an idealized cell:
• Solution A contains 200 mosM NaCl and 200 mosM urea
(400 mosM solute)
• Solution B contains 300 mosM NaCl and 100 mosM urea
(400 mosM solute)
• Solution C contains 400 mosM NaCl (400 mosM solute)
We ask students to draw the cell’s change in volume from
the moment of placing the cell in the solution until osmotic
equilibrium is reached. The complexity of this task is reflected
in the fact that, although the three solutions have the same
osmolarity (400 mosM), they exhibit different tonicities due to
varying concentrations of nonpenetrating and penetrating sol-
utes (Fig. 1).
To simplify the matter, students are instructed to assume that
the cell’s osmolarity is 300 mosM and that the cytosol only
contains nonpenetrating solutes. To successfully complete this
task, students have to demonstrate that they understand the
following points:
1. The solution’s tonicity depends on the solution’s concen-
tration of nonpenetrating solutes (e.g., NaCl) relative to
that in the cell, not on the total osmolarity of the solution.
2. Tonicity provides the information on how the solution
affects the cell’s volume once osmotic equilibrium is
reached; that is, when the osmolarities of the solution and
the cytosol become equal.
3. Water molecules move faster through the cell membrane
than particles of solute do. Therefore, a cell placed in a
hyperosmotic solution will always shrink initially, re-
gardless of the solution’s tonicity.
This type of problem-solving can be equally as effective
when presented in reverse. For example, students can be asked
to predict a solution’s osmolarity and tonicity based on the
effect it has on the volume of a cell, as depicted in a graph (Fig.
2). To successfully complete these assignments, students must
simultaneously consider both the osmolarity and tonicity of a
solution.
Osmolarity and tonicity in context. To ensure that students
completely understand the significance of osmolarity and to-
nicity in the light of systemic homeostasis, a lecturer might

Table 2. Starting fluid volumes table

Starting Table Total Body Fluid (TBF) Extracellular Fluid (ECF) Intracellular Fluid (ICF)

Amount of solutes (mosmol) 30 liters ⫻ 300 mosmol/l ⫽ 9,000 10 liters ⫻ 300 mosmol/l ⫽ 3,000 20 liters ⫻ 300 mosmol/l ⫽ 6,000
mosmol mosmol mosmol
Volume (liters) 30 liters 1/3 ⫻ 30 liters ⫽ 10 liters 2/3 ⫻ 30 liters ⫽ 20 liters
Osmolarity (mosM ⫽ mosmol/l) 300 mosM 300 mosM 300 mosM
Students use the equation, solute amount/volume ⫽ osmolarity, to calculate missing values. Calculated values are indicated by bold type.

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 TEACHING OSMOLARITY AND TONICITY
design tasks that put these processes in different physiological
contexts while giving them relevance. For example, it is
essential that pre-health professions students understand that
the appropriate choice of IV or oral fluid therapy depends on
the particular clinical scenario they will encounter.
An in-class activity that lends itself well to showing how
different solutions influence osmotic concentration and com-
partment volumes is to have students calculate numerical
values after adding a solution to the body. To successfully
complete such an assignment, we ask our students to do the
following:
1. Compare the solution’s osmolarity to the osmolarity of
the body or cell.
2. Determine the solution’s tonicity.
3. Predict how adding this solution will affect the volume
and osmolarity of body fluids.
a. Body concentration: increase, decrease, or no change?
b. Total body volume: increase, decrease, or no change?
c. ECF volume and osmolarity: increase, decrease, or no
change?
d. ICF volume and osmolarity: increase, decrease, or no
change?
4. Perform calculations that mathematically demonstrate
how the solution affects the body volumes and osmolarity
(described more below).
5. Check whether their theoretical predictions (step 3) are in
accordance with their calculations (step 4).
Let us take a solution and add it to an idealized body with
simple volumes and concentration to work through these steps.
For our example, students are asked to describe the effects of
adding 1 liter of a 100 mosM NaCl and 300 mosM urea
solution to a person whose normal total body volume is 30
liters, with two-thirds of body fluid volume (20 liters) inside
the cells. We work through each of the above steps in se-
quence.
1. The solution is hyperosmotic to the body because the
solution has a concentration of 400 mosM, and our
idealized body has a concentration of 300 mosM.
2. The solution is hypotonic to the body because the
solution has a concentration of nonpenetrating solutes of
100 mosM, and the concentration of nonpenetrating
solutes in the body is 300 mosM.
3a. The concentration of all body compartments will in-
crease, because the solution is hyperosmotic. This is
important for the student to understand. The body com-
partments are in osmotic equilibrium. Thus increasing
the concentration of the body with a hyperosmotic
solution will increase the concentration of all compart-
ments.
Table 3. Adding volume and nonpenetrating solutes to the
total body
Add Volume and Total Body Extracellular Intracellular
Nonpenetrating Solutes Fluid (TBF) Fluid (ECF) Fluid (ICF)
Solutes (mosmol) 9,000 ⴙ 100 ⴝ 9,100
Volume (liters) 30 ⴙ 1 ⴝ 31
Osmolarity (mosM)
The nonpenetrating solute amount and volume are added to the starting
values for the total body. Calculated values are indicated by bold type.
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631
Table 4. Calculating new total body osmolarity after adding
volume and nonpenetrating solutes
Total Body Extracellular Intracellular
Calculate New Osmolarity Fluid (TBF) Fluid (ECF) Fluid (ICF)
Solutes (mosmol) 9,100
Volume (liters) 31
Osmolarity (mosM) 9,100/31 ⴝ 293.5 293.5 293.5
The new total body osmolarity is calculated using solute amount/vol-
ume ⫽ osmolarity. Because the ECF and ICF are in osmotic equilibrium, the
total body osmolarity value is copied into the osmolarity boxes for those
compartments. Calculated values are indicated by bold type.
3b. Total body volume will increase, because we are adding
volume to the body.
3c. ECF volume will increase because we are adding vol-
ume to the body and because the NaCl will remain in the
ECF, keeping some water with it. ECF osmolarity goes
up because we added a hyperosmotic solution to the
body.
3d. ICF volume will increase because the solution is hypo-
tonic. ICF osmolarity also goes up because we added a
hyperosmotic solution to the body and it contained a
penetrating solute.
It might initially be difficult for students to think their way
through steps 1–3 above, as some of the predictions seem at
first to make no sense. How can the ICF concentration increase
if the volume of the ICF is increasing? Mathematically describ-
ing how the body compartments are affected (step 4) can
demonstrate these processes in a concrete manner. We call the
series of tables used for the calculations in step 4 “box
problems,” and they are shown in Tables 2–7.
The first step is filling out a chart that contains the initial
values of an idealized and simplified body (Table 2). Columns
include total body fluid (TBF) as well as the ECF and ICF
compartments. Each column contains rows for solute amount,
compartment volume, and compartment osmolarity. Unknown
values can be calculated by using solute amount/volume ⫽ os-
molarity.
The starting table uses volume and osmolarity values as they
are before the solution is added. Our example assumes that
normal total body volume is 30 liters, and that two-thirds of the
volume (20 liters) are inside the cells. The initial body osmo-
larity is 300 mosM. For students, using numbers like 300
mosM and volumes of 10, 20, and 30 liters simplifies the work.
As students fill in the chart, they must differentiate between
the amount of solute and the concentration of solute as well as
the units for amount (mosmol) and concentration (mosM).
Students are instructed to always fill in the TBF column first.
The problem asks the effect of administering 1 liter of a
solution composed of 100 mosM NaCl and 300 mosM urea. In
Table 5. Adding nonpenetrating solute
Total Body Extracellular Intracellular
Add NaCl to ECF Fluid (TBF) Fluid (ECF) Fluid (ICF)
Solutes (mosmol) 9,100 3,000 ⴙ 100 ⴝ 3,100 6,000
Volume (liters) 31
Osmolarity (mosM) 293.5 293.5 293.5
NaCl is nonpenetrating, so the entire amount of NaCl adds to the ECF
solutes. Calculated values are indicated by bold type.
632 TEACHING OSMOLARITY AND TONICITY
Table 6. Calculating new ECF and ICF volumes
Calculate New ECF and ICF Volumes Total Body Fluid (TBF)
Solutes (mosmol) 9,100
Volume (liters) 31
Osmolarity (mosM) 293.5
Students use the new osmolarity to calculate the ECF and ICF volumes. Calculated values are indicated by bold type.
the second step (Table 3), students add the volume of the
solution and the amount (mosmoles) of nonpenetrating solute
in the solution to the starting conditions. (The milliosmoles of
urea are held in reserve and added later, once water has
redistributed.) This step requires students to calculate the
amount of solute to add, based on the solution’s concentration
and volume. In this example, 1 liter of a solution with 100
mosM NaCl was added. Students calculate that 1 liter of 100
mosmol/l NaCl ⫽ 100 mosmol NaCl.
Students use the changed volume and solute values to
calculate the new TBF osmolarity (Table 4). They apply their
understanding of osmotic equilibrium in the body by recogniz-
ing that the calculated total body osmolarity will be the same
as the osmolarities of both the ECF and ICF compartments.
Students then need to figure out that the added 100 mosmol
of NaCl stay entirely in the ECF because NaCl is nonpenetrat-
ing and cannot enter cells (Table 5). The amount of solute in
the ICF remains unchanged at this point in the calculations.
The revised values for the ECF and ICF volumes can now be
calculated by dividing the amount of solute each compartment
contains by its osmolarity (Table 6).
The last part of the problem is to add the amount of the
penetrating solute contained in the solution to the body. In this
case, the 1 liter of the solution had 300 mosM urea, in addition
to the 100 mosM NaCl. This means that 300 mosmol of urea
(300 mosmol/l ⫻ 1 liter) were added to the body. Because urea
is a freely penetrating solute, it will not cause water to shift
between the ECF and ICF compartments. The urea contributes
to the osmolarity of the solution but not its tonicity.
Students first add the urea to the total body solute cell and
calculate the new total body osmolarity (Table 7). The total
body osmolarity can once again be copied to the ECF and ICF
columns because the two compartments are always in a state of
osmotic equilibrium. Considering that the entire volume of the
solution was already added in the preceding step, the values for
the TBF, ECF, and ICF volume have not changed and may be
copied from the previous chart.
Finally, based on the ECF and ICF volumes and new
osmolarity, the amount of solutes contained in each of the two
subcompartments can be calculated. If concentration ⫽
amount/volume, then amount ⫽ volume ⫻ concentration. De-
Table 7. Adding penetrating solutes
Add Urea to Get Total Body Extracellular Intracellular
Final Osmolarity Fluid (TBF) Fluid (ECF) Fluid (ICF)
Solutes (mosmol) 9,100 ⴙ 300 ⴝ 9,400
Volume (liters) 31 10.6 20.4
Osmolarity (mosM) 9,400/31 ⴝ 303.2 303.2 303.2
Penetrating solutes will not cause water to shift between the ECF and ICF
compartments, so the urea is added to the total body solute amount. Calculated
values are indicated by bold type.
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Extracellular Fluid (ECF) Intracellular Fluid (ICF)
3,100 6,000
3,100/293.5 ⴝ 10.6 liters 6,000/293.5 ⴝ 20.4 liters
293.5 293.5
pending on the student population, this final calculation can be
skipped because clinically we are usually only interested in the
compartment volumes and their osmolarity. Once the tables are
complete, students check whether their theoretical predictions
(step 3) are in line with the calculations (step 4). The predic-
tions were that osmolarity of all compartments would increase
and that volume of all compartments would increase. By
comparing Table 2 with Table 7, a student should observe the
following:
1. Body osmolarity increased while the ICF volume in-
creased. This is in accordance with the initial predictions:
the solution added to the body was hyperosmotic and
hypotonic. Because the solution was hypotonic, the ICF
volume increased. (In our tables, we see ICF volume
increase from 20 to 20.4 liters). It might seem that this
would dilute the ICF, causing the ICF concentration to
decrease; however, the solution contained enough urea to
make it hyperosmotic. Urea, a penetrating solute, passes
through cell membranes and adds to the solute in the
cells, increasing the overall concentration of the ICF from
300 to 303.2 mosM.
2. The mosmoles of penetrating solute can be distributed
between ECF and ICF in proportion to their respective
compartment volumes after the volumes changed from
the addition of nonpenetrating solute. This puts an equal
concentration of urea into each compartment. Tell stu-
dents to avoid thinking “distributes equally,” because that
makes them inclined to divide the urea amount into two
equal batches rather than distributing the urea proportion-
ately according to compartment volume.
3. A change in total body osmolarity is reflected in a similar
change in ECF and ICF osmolarity. ECF and ICF will
always be in the state of osmotic equilibrium (in other
words, the osmolarity of all compartments are equal).
Insisting that students consistently copy the symbols for
units (mosmol, l, mosM) in each of the tables helps them
memorize the units and prevents them from mistaking the
amount for the concentrations of solute.
The complexity of these box problem assignments can be
taken to the next level by creating a variety of real-life
scenarios in which a person is subjected, for instance, to sweat
loss from increased physical activity followed by the inappro-
priate choice of a rehydration drink. In addition to following
the usual set of instructions for filling in the charts, students
demonstrate the ability to calculate the osmotic concentration
of a solution and deduce how the wrong choice of sport drink
can lead to a disturbance in osmotic homeostasis (Fig. 3).
Glucose as a solute. The examples above used NaCl as the
prototype nonpenetrating solute and urea as the freely pene-
trating solute. But what about glucose, clinically known as
dextrose? Many IV solutions contain glucose, either as the sole
solute (for example, 5% dextrose in water, or D5W) or in
 TEACHING OSMOLARITY AND TONICITY 633

David, a 38-year-old man with a desk job, decided to try “hot yoga” (performed in a 105° F room) to lose
weight. A yoga session at this temperature promotes profuse sweating. After the first session David noticed a
4% body mass reduction, but he was unaware that it was entirely due to water and NaCl lost through sweating.
Create David’s pre- and post-yoga body fluid tables. Assume the following: David’s pre-yoga body weight
was 100 kg and 50% of that weight was water. His initial ECF volume was 17 L. His plasma osmolarity
changed from 300 mosM pre-yoga to 310 mosM post-yoga. 1 kg body fluid = 1 L. Calculated values in the
tables are shown in bold type. TBF = total body fluid

Pre-yoga TBF ECF ICF Post- TBF ECF ICF

yoga
Solute 15,000 5100 9900 Solute 14,260 5100 – 740 9900
(mosmol)
(mosmol) = 4360
Volume (L) 50 17 33 Fig. 3. This is the example of a box problem
Volume 50 – 4 = 46 14.1 31.9 assignment that helps students understand the
(L) importance of osmolarity and tonicity within
Osmolarity 300 300 300
Osmolarity 310 310 310 the context of systemic homeostasis. This
(mosM)
particular task illustrates how the inappropri-
(mosM)
ate choice of a rehydration drink following
Answer: TBF column: 4% body mass loss = 0.04 sweat loss from increased physical activity
Answer: TBF water is 50% of 100 kg, or 50 kg x - may disturb osmotic homeostasis. Students
x 14,260 post-yoga = 740 mosmol NaCl lost from follow the steps laid out in the different parts
ECF. of Table 2. For simplicity, sweat was assumed
to include only nonpenetrating solute lost
After the yoga session ended, David drank 1 L of a diet sports drink that contained 200 mM NaCl and from the ECF. In reality, sweat is made
5% artificial sweetener K (M.W. = 398 g/mol; does not dissociate). The sweetener is absorbed in the mostly of sodium with smaller components of
gut, but cannot enter body cells due to the lack of appropriate membrane transporters. What is the total chloride, potassium, lactate, and urea. This
osmolarity of this drink? question could be made more complex by
including these substances in the question
x x stem.
• 5% K = 5 g K / 100 mL solution = ? g of K / 1000 mL. 1 L (1000 mL) contains 50 g sweetener K
• 50 g / 398 g/mol = 0.126 mol = 126 mmol in 1 L = 126 mosmol of the sweetener K in 1 L
• 400 mosmol NaCl + 126 mosmol sweetener K = 526 mosmol /1 L drink = 526 mosmol added
All solutes in the drink are nonpenetrating. What is David’s plasma osmolarity after the drink?
Post-drink TBF Answer: The sports drink made his TBF
osmolarity increase to 315 mosM. It is
Solute (mosmol) 14,260 + 526 = 14,786 not necessary to calculate the ECF and
Volume (L) 46 + 1 = 47 ICF columns because TBF osmolarity is
the same as ECF, plasma, and ICF
Osmolarity 315 osmolarities.
(mosM)

combination with NaCl (for example, 5% dextrose in half-
normal saline). As it turns out, glucose as a solute must be
treated as a special case for the purpose of working problems.
Students will state that glucose is a penetrating solute be-
cause they know that it enters cells. However, it is not freely
penetrating because, in most cells, glucose that enters is im-
mediately phosphorylated by hexokinase to glucose-6-phos-
phate (G-6-P). Phosphorylated compounds are not able to
leave the cell, so the glucose that enters adds to the cell’s pool
of nonpenetrating solutes when it becomes G-6-P.
Table 8. Calculating plasma ion changes
ECF Conditions Following
Initial ECF 1 liter of 100 mosM NaCl
Conditions and 300 mosM Urea
Amount of K⫹ (meq) 4.5 meq/l ⫻ 10 liters ⫽ 45 45
Volume (liters) 10 (from Table 2) 10.6 (from Table 7)
Plasma [K⫹] (meq/l) 4.5 4.2
This table demonstrates how to calculate changes in plasma ion concentra-
tions. For this example, you are told that the subject from Table 2 had a starting
plasma K⫹ concentration ([K⫹]) of 4.5 meq/l. Plasma [K⫹] are equal to ECF
plasma concentration, so you can calculate the amount of K⫹ in the extracel-
lular fluid (ECF). After the subject is given 1 liter of 100 mosM NaCl and 300
mosM urea, the ECF volume increases (as shown in Table 7), but the amount
of K⫹ does not change. Adding the intravenous fluids decreases the plasma
[K⫹] from 4.5 to 4.2 meq/l. Calculated values are indicated by bold type.
In a normal person given a load of glucose by mouth or
IV, over a period of several hours 100% of the administered
load will enter the cells. In addition, through normal aerobic
metabolism, much of the administered glucose will be
processed from G-6-P to CO2 and water. Thus the net result
of giving a pure glucose IV over time is the same as giving
plain water, making glucose IV solutions hypotonic, even if
they are isosmotic. Failure to recognize hypotonicity and the
metabolism of glucose to water can result in iatrogenic
hyponatremia when D5W is used as a routine maintenance
solution (8).
When administering a solution containing glucose in the box
problems described above, we use a “freeze frame” approach.
Students are asked to calculate changes in ECF and ICF
volumes and osmolarities at the time when X% of glucose has
entered the cell. They must recognize that 100 ⫺ X% of the
glucose has remained in the ECF, and they must distribute the
appropriate amounts of glucose into each compartment to do
their calculations.
One variant of glucose as a solute involves the distribution
of glucose in a patient with type 1 diabetes mellitus, where
absence of insulin prevents glucose uptake into most cells. In
these situations, glucose remains in the blood and effectively
becomes a nonpenetrating solute. A similar situation occurs in
hyperglycemic hyperosmolar states (11). These two scenarios

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634 TEACHING OSMOLARITY AND TONICITY
help students link changes in tonicity to physiologically sig-
nificant disruptions of normal function.
Extending the concept. The box problems described above
are a simple example of the principle of mass balance: take
what was in the body at the start, then add and subtract solutes
and volume to find what is in the body now. The box problem
approach can be extended to include calculating the concen-
tration of specific components of the different compartments.
For example, a patient comes in dehydrated with an elevated
plasma potassium concentration. If you replace his volume loss
with an IV of isotonic NaCl, what will happen to his plasma
K⫹ concentration? Or a patient comes in with acute hypona-
tremia from water intoxication. How will administering a small
volume of hypertonic saline alter both her plasma Na⫹ con-
centration and the distribution of water between the ECF and
ICF?
Answering these questions requires using the box problem
strategy with the addition of a set of boxes that are specific to
the ion (Table 8). In the ion-specific tables, students calculate
the amount of ion in the ECF from the plasma ion concentra-
tion and the ECF volume. This is a good place to reinforce that
we can substitute plasma ion concentration as a surrogate for
ECF ion concentration (unless significant amounts of ion are
bound to plasma proteins, as is the case for Ca2⫹).
The K⫹ example shown in Table 8 is based on the subject
being given 1 liter of 100 mosM NaCl and 300 mosM urea
from the set of Tables 2–7 calculations. Students should
recognize that the amount of K⫹ in the ECF does not change
post-IV. But there is a new ECF volume after giving the IV, so
the plasma K⫹ concentration will change.
In the example of hyponatremia treated with a bolus of
hypertonic saline, after students work out the effects of the
saline IV on the body, they use a second set of Na⫹ boxes to
add Na⫹ from the IV to the ECF Na⫹ amount. The revised Na⫹
amount divided by the post-IV ECF volume yields the new
ECF Na⫹ concentration.
DISCUSSION
Our students felt that several different approaches to osmo-
larity and tonicity were effective in overcoming misconcep-
tions with this often confusing subject. Approaches included
simple definitions, real-world situations, and the simultaneous
consideration of both osmolarity and tonicity. Although we
have not conducted formal research into the efficacy of our
approach, we have observed over the years that students
achieve different levels of mastery. Some students learn the
algorithm for working the box problems and can answer
quantitative problems successfully, but are unable to transfer
their understanding to answer conceptual questions, such as:
“Given a 300 mosM cell, design a solution using NaCl and
urea that is isosmotic and hypotonic.” A few students never
master the task of converting percent solutions to molarity and
then to osmolarity. Our evidence of efficacy is successful
completion of test questions similar to the ones in Fig. 3 and
Table 8 and questions asking students to predict changes in
ECF and ICF volume and osmolarity in scenarios they have
never studied. For more examples of box problems that can be
used to test students’ understanding, readers can navigate to
Osmolarity and Tonicity at SYSTEMSphys.org (http://test.
systemsphys.org), a website put together by the authors.
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The application of osmolarity and tonicity is fundamental to
understanding physiology. It is also crucial for the selection of
appropriate IV fluid therapy in different situations, such as
dehydration, hemorrhage, and hyponatremia. A thoughtful ap-
proach to teaching osmolarity and tonicity promotes the devel-
opment of problem-solving skills, builds student self-confi-
dence for answering problems they have never seen before, and
enables students to reason their way through a variety of other
physiological processes. We hope the approach we take to
teaching osmolarity and tonicity as described in this paper may
prove useful to physiology lecturers who are looking for new
ways of introducing this complicated topic to their students.
ACKNOWLEDGMENTS
The authors thank Dr. Jan Machart (College of Natural Sciences, University
of Texas at Austin) for permission to adapt her “hot yoga” problem to the
version shown in Fig. 3.
Present address: M. Chirillo, Internal Medicine-Pediatrics Residency Pro-
gram, Department of Internal Medicine, University of Utah School of Medi-
cine, Salt Lake City, UT.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
P.V., M.C., and D.U.S. conceived and designed research; P.V., M.C., and
D.U.S. performed experiments; P.V., M.C., and D.U.S. analyzed data; P.V.,
M.C., and D.U.S. interpreted results of experiments; P.V., M.C., and D.U.S.
prepared figures; P.V., M.C., and D.U.S. drafted manuscript; P.V., M.C., and
D.U.S. edited and revised manuscript; P.V., M.C., and D.U.S. approved final
version of manuscript.
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