In the Classroom

Dilution Confusion: Conventions for

Defining a Dilution
Laurence A. Fishel
Fertility & Cryogenics Lab, 8635 Lemont Road, Downers Grove, Illinois 60516
larry@fclab.us

Two conventions for preparing dilutions are used in clinical frequently use the proportional method for describing mixtures
laboratories. The first convention defines an a:b dilution as of reagents or dilutions of samples. This is particularly true in the
a volumes of solution A plus b volumes of solution B. The second instructions that are provided by manufacturers accompanying
convention defines an a:b dilution as a volumes of solution A enzyme-linked immunosorbent assay (ELISA) and other types of
diluted into a final volume of b. In most commercial diagnostic commercial kits for performing assays to test for infectious
kits, if the instructions state, for example, that a 1:5 dilution is to diseases or other abnormal health conditions. But among these
be prepared for a particular reagent before it is to be used in the types of laboratory personnel, there is no standardized method
assay, somewhere in the text there is an explicit instruction on for performing dilutions. A useful reference is available that
how this is to be done, for example, 100 μL of stock reagent A describes both dilution conventions (1).
plus 400 μL of B diluent. However, anyone not reading carefully
may easily make one or more dilution errors (e.g., there are often Why is Each System Used?
several reagents that require separate dilutions), and this in turn
Most chemists and physical scientists would agree that the
could affect the sensitivity and results of the assay. Many
best method to specify reagent concentrations is using molarity,
scientists and laboratory personnel learn to prepare dilutions
molality, or normality. However, for a variety of reasons, this is
early in their careers, but do not retain or recall the exact
not always done in the world of commercial diagnostic test kits.
citations. To them, what they do eventually becomes a matter
In some cases, this appears to be to protect proprietary informa-
of common sense.
tion that, if made known, could enable domestic or foreign
A brief foray via the search engine Google into the prepara-
commercial competitors to gain an advantage over the original
tion of dilutions revealed considerable confusion. Inquiries
manufacturer. In clinical laboratories, human or veterinary
concerning the preparation of dilutions in laboratories were,
samples such as blood (plasma, serum) frequently need to be
therefore, made of several scientists from the fields of biochem-
diluted before being used in an analytical assay. This is generally
istry, clinical chemistry, and related areas. Each scientist who was
specified in the manufacturer's assay instructions. It is important
surveyed was asked to describe how to prepare a 1:1, a 1:2, and a
to note that, although chemistry professors may not use or teach
1:9 dilution. Additional information was gathered from libraries
the proportional dilution method, it is quite certain that should
and online (the Internet). The following two responses are
they become ill, their blood or other samples will be processed by
typical of those received from the inquiries.
a clinical laboratory using one or the other, or even both dilution
• Consensus of two professors: A 1:1 dilution means addition of
conventions. This, in turn, may result in significant errors in the
an equal volume of one solution to another. Therefore, a 1:2
assay results, with potentially serious adverse health consequences.
solution is addition of 1 vol of one solution to 2 vol of another,
Specific examples of the two dilution conventions are
and 1:9 means 1 vol of one solution added to 9 vol of another
described below. These examples are taken directly from com-
solution. One of the professors later noted he “Googled 1:2
mercial clinical assays to provide instructors, students, and
dilution and found that most entries suggest that it means the
potential end users concrete examples of exactly what they may
mixing of equal volumes of 2 solutions (1 plus 1 = 2). However,
encounter in the clinical laboratory or commercial manufactur-
what does a 1:1 mean? I have no idea...”.
ing environment. This may enable instructors to devise realistic
• “I interpret n:m to mean relative volumes n þ m. Or a dilution
examples to use in their own courses or textbooks.
of n/(n þ m). So 1:1 is 2 and 1:9 is 10... It's just common
sense to me.”
Examples of Dilutions in Commercial ELISA Kits

Who Uses Which System and When?
Introductory and analytical chemistry courses teach sys-
tematic concentrations (molarity, molality, normality) and do
not generally present the concept of proportional dilutions (1:1,
1:2, 4:7, etc.). Only in organic chemistry, where thin-layer
chromatography is discussed, is a method commonly mentioned
for describing the proportions of reagent solutions used in a
solvent mixture (a:b).
In contrast to the world of chemistry, pharmacists, micro-
biologists and other types of biologists, as well as clinical chemists,
The BioRad Laboratories Genetic Systems GS HIV-1/
HIV-2 PLUS O EIA kit (2), which is FDA-approved, very
clearly states how a dilution is to be performed: “Dilute speci-
mens and controls 3:4 in the Specimen Diluent. Specimens and
controls may be prediluted 3:4 in the Specimen Diluent prior to
addition of the diluted specimen or control to the well (for
example, dilute 150 μL of specimen in 50 μL of Specimen
Diluent and then transfer 100 μL to the well)...” Thus, there is no
possibility of error if the instructions are followed. It is worth-
while to note that in this case, 3:4 dilution is either 75% as
concentrated or 43% as concentrated, depending on the dilution

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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 87 No. 11 November 2010 Journal of Chemical Education 1183
10.1021/ed1001762 Published on Web 08/27/2010
 In the Classroom
convention used. This is obviously a significant difference that
could have profound effects on the performance of the kit and
the results should the instructions not be followed precisely.
An ELISA kit from The Binding Site (3) is of interest
regarding dilutions: The Bindazyme Human Anti-phosphati-
dylserine IgA Enzyme Immunoassay Kit MK051 is manufac-
tured in the United Kingdom. It is also FDA approved. On page
5 of the package insert, several instructions stand out, including:
Sample dilution: Dilute 10 μL of each sample with 1000 μL
of sample diluent (1:100) and mix well.
Note that the sample dilution is 10 μL sample þ 1000 μL of
diluent. This is a final volume of 1010 μL. It is labeled as 1:100.
This is an example of the dilution convention that a:b is defined
as a þ b, not a diluted into a final volume of b. The dilution is
therefore a/(a þ b), or 10/(10 þ 1000), and thus, a dilution
factor of 1010/10, or 101. Although in this case the difference in
results if the alternate dilution convention were followed might
be insignificant, in many cases, the results could be profound.
It is important to note that 1:100 in these instructions is not the
same as 1:100 in many other package inserts, such as the BioRad
HIV-1/HIV-2 Plus O ELISA kit discussed above.
Other kits are not so explicit in their instructions. For
example, the ACTIVE MIS/AMH ELISA (DSL-10-14400) kit
from Diagnostic Systems Laboratories, Inc. (4), which is not
FDA-approved, does not explicitly state how dilutions are to be
performed.
“Assay Procedure: ...For pediatric samples: Dilute 1:10...
before assay...”
Discussion
From a formal perspective, dilutions should correctly be speci-
fied as being v:v, w:v, or w:w, with v being volume and w being
weight, with any volume changes that occur upon mixing being
appropriately taken into account. In all cases, instructors need to
instill in students the need to always specify what these numbers
describe. For example, a 1:7 proportion could be by volume v:v, by
weight w:w, or some mixture of the two measures w:v.
Most clinical chemistry laboratory kits do not generally list the
exact concentrations of the components of the stock solutions or
reagents that require dilution before use, and must be used exactly as
per the manufacturers' instructions. Therefore, it is not possible to
dilute the various solutions based on their molar compositions. Blood
( plasma, serum), in particular, must be diluted using one of the two
conventions described here. In all cases, it is critical that a statement at
the beginning of the experimental portion of each protocol explicitly
describe how dilutions are to be performed.
The convention a:b meaning “a þ b” has historically been
used and continues to be used in many different fields of inquiry
including chemistry, biology, social research, and statistics. Of
particular interest is the proportion 1:1. In the realm of thin-
layer chromatography, solvent systems are routinely described
using such a convention. The scientific literature from around
the world is filled with examples supporting this view. Three
examples follow:
• “The solvents diethyl ether-hexane (1:1), chloroform-hexane
(34:66), chloroform-methylene chloride-hexane (17:23:60)...” (5)
• “...developed in vertical ascending TLC chambers using chloro-
form, ethanol, water, triethylamine (35:35:7:35, v/v/v/v) as the
solvent system.” (6)
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1184 Journal of Chemical Education Vol. 87 No. 11 November 2010
• An example from social science research: in the description of
“A Comparative Experiment in Sampling Methods”, four
groups of high school graduates in the state of Minnesota
(USA) were compared out of a total of 24,395 (7). The method
used and described here was “stratification proportionate to the
total number in the population in each of the four subclasses.”
For this analysis, it was first necessary to “compute the propor-
tions of the four subclasses.” They were (N1) boys (outside 3
cities of first class), (N2) girls (outside 3 cities of first class), (N3)
boys (inside 3 cities of first class), and (N4) girls (inside 3 cities
of first class). The number of boys and girls in each group
respectively were: 6668; 10,180; 3205; 4522. The author then
set up the following ratio:
N1:N2:N3:N4 = 6668:10,180:3025:4522
In contrast to the comparison or dilution method just
described, the dilution convention now being employed by many
groups involved in clinical diagnostic assays defines 1:1 as the
initial concentration, that is, no dilution. This is detailed in
books such as Pharmaceutical Calculations (8), Remington: The
Science and Practice of Pharmacy (9), and Tietz Textbook of
Clinical Chemistry and Molecular Diagnostics (10). Thus, for a
dilution of a:b, a is diluted to a total volume of b. This is by no
means a trivial matter, as making an incorrect dilution of reagents
or specimens may significantly affect the assay results and thus
the diagnosis of a patient's disorder.
What Can Be Done To Fix the Problem?
There is an urgent need to fix the problem of having two different
dilution conventions in use simultaneously. A solution would be for an
organization such as the American Chemical Society (ACS) or
International Union of Pure and Applied Chemistry (IUPAC) to
establish a rule that would thereafter be the standard and sole method
for describing a dilution using proportions. Undergraduate students
who intend to pursue advanced or professional training in fields such
as pharmacy, medical technology, microbiology, and human and
veterinary medicine, as well as clinical chemistry are required to enroll
in introductory chemistry courses, and usually also advanced courses in
inorganic, organic, and physical chemistry. It is during the early phases
of their scientific training that they should be taught an unambiguous
standardized method for defining a dilution.
This will eventually eliminate the problem of dual dilution
conventions. Until this has been accomplished, a more immedi-
ate fix is urgently needed to inform clinical chemists, medical
technologists, microbiologists, physicians, and manufacturers of
diagnostic test kits of this problem, so that they can take the
necessary steps to prevent significant laboratory errors that may
result in the misdiagnosis of human and veterinary diseases. A
first step would be for manufacturers to state the exact definition
of a proportional dilution explicitly at the beginning of the
experimental portion of each laboratory protocol (e.g., 1:3 is
defined as 1 part A þ 2 parts B). This urgently needs to be
implemented, where not already being done.
Acknowledgment
I thank Hisham Greiss for inviting me to participate in
establishing Fertility & Cryogenics Lab, helpful discussions; and
allowing me to review more than 50 commercial ELISA and other
clinical diagnostic assays. I also thank Richard A. Graff, William
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pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc.

Allison, Chris Bystroff, Nancy Konigsberg Kerner, and William
W. Wells, for helpful discussions.
Literature Cited
1. Robyt, J. F.; White, B. J. Biochemical Techniques: Theory and
Practice; Waveland Press: Prospect Heights, IL, 1987; pp 30-31.
2. Bio-Rad Laboratories Headquarters, 1000 Alfred Nobel Drive,
Hercules, CA 94547. U.S. License No. 1109; revised: February
2008; Product No. 32588. http://www.bio-rad.com
3. The Binding Site Ltd., P.O. Box 11712, Birmingham, B14 4ZB,
U.K. http://www.bindingsite.co.uk (accessed Aug 2010).
4. Beckman Coulter, Corporate Headquarters, 445 Medical Center Blvd.,
Webster, TX 77598-4217. Website: http://www.beckmancoulter.com
5. Fried, B.; Sherma, J. Thin-Layer Chromatography: Techniques and
Applications, 2nd ed.; Revised and Expanded. Marcel Dekker, Inc.:
New York, 1986; p 82.
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In the Classroom
6. Fuchs, B.; Bischoff, A.; Su. ss, R.; Teuber, K.; Schu. renberg,
M.; Suckau, D.; Schiller, J. Anal. Bioanal. Chem. 2009, 395,
2479–2487.
7. Johnson, P. O. Statistical Methods in Research, 2nd ed.; Prentice
Hall: New York, 1950; pp 202-205.
8. Ansel, H. C. Pharmaceutical Calculations, 13th ed.; Wolters
Kluwer, Lippincott Williams & Wilkins: Philadelphia, PA, 2010;
pp 81-101.
9. Schnaare, R. L.; Prince, S. J. Metrology and Pharmaceutical
Calculations. In Remington: The Science and Practice of Pharmacy,
21st ed.; Troy, D. B., Ed.; Wolters Kluwer, Lippincott Williams &
Wilkins: Philadelphia, PA, 2006; pp 99-126.
10. Bermes, E. W., Jr.; Kahn, S. E.; Young, D. S. Introduction to
Principles of Laboratory Analyses and Safety. In: Tietz Textbook of
Clinical Chemistry and Molecular Diagnostics, 4th ed.; Burtis, C. A.,
Ashwood, E. R., Bruns, D. E., Eds.; Elsevier Saunders: St. Louis,
MO, 2006; pp 3-39.
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