Serial Dilutions Tutorial Lab Ruth Allen, Ph.D.

Preparation of dilutions
All laboratory work is dependent on the preparation of reagents at exact
concentrations. Often, a reagent is available in a concentration that is higher than the
concentration needed for the assay being performed. In this case, the reagent must be
diluted to the appropriate concentration.
For example, suppose you have a solution of IgG

in phosphate buffered saline

!"#$%
&
at a concentration of mg'ml but need a solution of IgG at a concentration of (.
mg'ml for the assay that you are performing. )ou will need to dilute your mg'ml
solution of IgG (*fold !i.e. from .( mg'ml to (. mg'ml%. In order to do this, you
would need to increase the volume of your solution by a factor of ( i.e. your mg of
IgG would need to be contained in a volume of ( ml rather than a volume of ml !since
mg'( ml is e+ual to (. mg'ml%. )ou would thus achieve the appropriate dilution by
adding , ml of "#$ to ml of your mg'ml solution of IgG !so that your mg of IgG is
now contained in a total volume of ( ml%.
If you think about this, you will reali-e the following relationship exists between
the initial and desired concentrations, the dilution factor and the initial and final volumes
of the solution.
Initial concentration !/
I
% Final re+uired volume !0
F
%
***************************** 1 2ilution factor 1 *********************************
Final concentration !/
F
% Initial volume !0
I
%
3his relationship can be rearranged to derive the following e+uation.
0
F
1 !/
I
x 0
I
%'/
F
where
0
F
1 the final total volume of the solution !this includes the initial volume%
/
I
1 the initial concentration of the solution
0
I
1 the initial volume of the solution
/
F
1 the final desired concentration of the solution
Another version of this e+uation is.
0
A
1 4!/
I
x 0
I
%'/
F
5 * 0
I
where
0
A
1 the volume that must be added to the initial volume in order to obtain the desired
final concentration.

IgG is the most common class of antibody found in blood.

&
"hosphate buffered saline !"#$% is a common buffer used in experimental immunology.
As an example of the use of this e+uation, consider a situation in which you have
(.6 ml of a & mg'ml solution of an antigen and you re+uire a final concentration of (.7
mg'ml. #y following the formula above, you would determine the volume of buffer you
would need to add to your initial (.6 ml in order to dilute your & mg'ml solution to a (.7
mg'ml solution as follows.
/
I
1 the initial concentration of the solution 1 & mg'ml
0
I
1 the initial volume of the solution 1 (.6 ml
/
F
1 the final desired concentration of the solution 1 (.7 mg'ml
0olume to add !0
A
% 1 4!/
I
x 0
I
%'/
F
5 * 0
I
1 4!& x (.6%'(.75 8 (.6
1 4'(.75 8 (.6
1 &.6 8 (.6
1 & ml
)ou would thus add & ml of buffer to your original (.6 ml. )our initial (.6 ml contained
mg of antigen. #y adding a further & ml to this, you will have mg of antigen in &.6
ml, which is e+ual to (.7 mg of antigen per ml.
Of course, you could also arrive at the same result by intuitively reali-ing that a
dilution from & mg'ml to (.7 mg'ml is a five*fold dilution and will therefore re+uire a
five*fold increase in total volume of the solution i.e. an increase from (.6 ml to &.6 ml.
In some cases, you may not want to use all of your initial material in preparing
your dilution. In the case above, for example, you may need to have available some of
your antigen at & mg'ml and some at (.7 mg'ml. /onsider again this relationship.
Initial concentration !/
I
% Final re+uired volume !0
F
%
***************************** 1 2ilution factor 1 *********************************
Final concentration !/
F
% Initial volume !0
I
%
3his relationship can be rearranged in a different manner from that shown above
to derive the following e+uation.
0
I
1 !/
F
x 0
F
%'/
I
where
0
I
1 the initial volume of the solution
/
F
1 the final desired concentration of the solution
0
F
1 the final total volume of the solution !this includes the initial volume%
/
I
1 the initial concentration of the solution
9sing this version of the e+uation will allow you to determine what starting volume you
will need in order to obtain a desired final volume at a specific concentration. In the case
above, for example, suppose that you need only ml of the (.7 mg'ml antigen solution.
)ou could calculate the initial volume as follows.
/
F
1 the final desired concentration of the solution 1 (.7 mg'ml
0
F
1 the final total volume of the solution !this includes the initial volume% 1 ml
/
I
1 the initial concentration of the solution 1 & mg'ml
Initial volume !0
I
% 1 !/
F
x 0
F
%'/
I
1 !(.7 x %'&
1 (.7'&
1 (.& ml
3hus, you would begin with (.& ml of your & mg'ml solution and add sufficient buffer to
bring the total volume up to your desired ml i.e. you would need to add (.: ml of
buffer.
Of course, you could also arrive at the same result by intuitively reali-ing that
since you need a five*fold dilution, you will need to start with an initial volume that is
one fifth of your desired final volume i.e. an initial volume of (.& ml.
Preparation of very large dilutions
$ometimes, you may need to prepare very large dilutions. For example, suppose
you have ml of a (( mg'ml solution of IgG and you need ml of a ;g'ml solution of
IgG for the assay you wish to perform. 9sing the e+uation above, you would calculate
this as follows.
/
F
1 the final desired concentration of the solution 1 ;g'ml
0
F
1 the final total volume of the solution !this includes the initial volume% 1 ml
/
I
1 the initial concentration of the solution 1 (( mg'ml 1 ((,((( ;g'ml
Initial volume !0
I
% 1 !/
F
x 0
F
%'/
I
1 ! x %'((,(((
1 '((,((( ml
1 (.(((( ml
1 (.( ;l
3hus, to obtain ml at the desired concentration of ;g'ml, you would need to dilute
(.( ;l of your initial IgG solution to the final volume of ml. /learly, you cannot
possibly measure (.( ;l accurately< =ven if you made (( ml of your final solution,
you would still have to accurately measure only ;l of your initial solution<
>hen large dilutions such as this are needed, a better approach is to prepare your
final dilution in more than one step. 3his is facilitated by the fact that dilution factors can
be multiplied. In other words, if you prepare a ((*fold dilution of a solution and then
prepare a further (*fold dilution of your ((*fold diluted solution, your overall dilution
factor would be ,((( !i.e. (( x (%.
In the example above, you need to prepare a ((,(((*fold dilution. ?ather than
performing this in one step, a more appropriate approach would be to first prepare a
,(((*fold dilution and then prepare a ((*fold dilution from your first dilution. For
example, you could first dilute 6 ;l of your initial IgG solution to a final volume of 6 ml.
3his would be a ,(((*fold dilution, giving a solution with an IgG concentration of (.
mg'ml !i.e. (( ;g'ml%. )ou could then dilute ( ;l of your (. mg'ml solution to a final
volume of ml. 3his would be a further ((*fold dilution and would give you the
desired final IgG concentration of ;g'ml.
>hen making very large dilutions such as this there are many possible ways to
arrive at the final desired concentration. 3he best approaches will be those that maintain
accuracy by not re+uiring the ali+uotting of extremely small volumes and that maintain
efficiency by not using exceptionally large volumes of your diluting buffer
@
.
"erform the following exercises in order to familiari-e yourself with the steps
re+uired in making large dilutions.
@
Another word for the buffer used to dilute solutions is diluent.
Preparation of serial dilutions
In many immunological assays it is necessary to prepare a series of dilutions of a
sample or reagent. An example would be a set of solutions in which each successive
solution is twice as dilute as the solution preceding it. $uch a set of solutions is referred
to as a two*fold dilution series.
A dilution series is prepared by simply performing successive identical dilutions.
In the case of a two*fold dilution series, an ali+uot of the initial !neat% solution is first
mixed with an e+ual volume of buffer to form a A strength solution. An ali+uot of the A
strength solution is then mixed with an e+ual volume of buffer to form a B strength
solution. An ali+uot of the B strength solution is then mixed with an e+ual volume of
buffer to create a

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:
strength solution, and so on.