Dilution Techniques

There are many instances where it is necessary to dilute solutions:

1. Measure the quantity of a substance using calibrated procedures.
2. When the concentration of a particular substance in a specimen is too
high to be accurately determined
3. When only a small volume of specimen is available, it may be
necessary to dilute the original specimen.

Dilution of a solution could be achieved using various techniques. The

choice of the technique depends on the original concentration of the stock
solution, the determination value (needed target final concentration) and
the analysis and interpretation methods.

Following are the different dilution techniques:

I. Single Dilutions
Single dilutions are usually expressed as a ratio, such as 1:2, 1:5 or 1:10
or as a fraction, 1/2, 1/5 or 1/10. These ratios or fractions refer to 1
unit of the original specimen diluted to a final volume of 2, 5, or 10.
For example to make a 1:5 dilution, 1ml of a specimen is added to 4ml of
diluent (Figure 1) or 0.5ml of a specimen to 2ml of diluent.
Then if the specimen concentration is for instance 500mg/dl, then the
concentration of the 1:5 dilution should show
100mg/dl after determination. This is calculated as 1 ml specimen
follows 500mg/dl x 1/5 = 100mg/dl.
Usually, the determination of the concentration is
performed on the dilution and we have to calculate
the concentration of the undiluted serum. This is 4 ml
diluent
done by multiplying the determination value by
the reciprocal of the dilution fraction = dilution
factor. In this case, when the determination value
for the 1:5 dilution is 100mg/dl then the
concentration of the undiluted specimen is Figure 1
100mg/dl x 5 (dilution factor)= 500mg/dl, since the original specimen
was five times more concentrated then the diluted specimen tested in the
procedure.
 II. Serial Dilutions
Preparing a dilution series is used in a wide range of procedures,
including serologic ones, blood banking, microbiology and preparation of
standard curves for calibration of analytical instruments, where a range of
dilutions of a particular sample is required.
There are different approaches to prepare a serial dilution:

1. Linear Dilutions
The aim of linear dilutions is to prepare a series of single dilutions
separated by an equal amount. Ex: 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg/ml. Here
each dilution is prepared alone directly from the stock solution or the
patient’s serum as in the single dilution procedure. To determine the
volume of stock solution required for each dilution, we use the formula
C1 V1 = Cs Vs
Vs = C1 V1
Cs
Where: C1 = target sample concentration
V1 = volume of the sample
Cs = stock solution concentration
Vs = volume of the stock solution required

Linear dilutions are usually used to prepare a calibration curve for

spectrophotometric assay of protein concentration or an enzyme assay.

2. Harmonic dilution series

The dilutions in this series take the values of the reciprocals of
successive whole numbers ex. 1/1, 1/2,1/3, 1/4, 1/5, etc.
The individual dilutions are simply achieved by adding a constant unit
volume (V) of the specimen or stock solution to each tube, then a
stepwise increase in the volume of diluent in the successive tubes ex. by
adding 0, 1, 2, 3, 4 and 5 times the volume (V) of the diluent.

3. Logarithmic dilution series

The aim of logarithmic dilution is to prepare a series of dilutions
separated by constant proportions called step intervals. A complete
dilution series usually contains five to ten tubes.
Logarithmic dilution is useful when a broad range of concentrations is
required notably for titration of biologically active substances
(antibodies, cytokines, hormones …). The most common examples are:
 a.. Doubling Dilutions:
Each concentration is half that of the previous one i.e. two fold step
interval or log 2 dilution series (Figure 2, upper dilution).
First, in tube 1 make up the most concentrated solution at twice the
volume (V) required and put in
all tubes except 1 a volume (V)
of diluent. Measure out half of
this volume into the next tube,
mix thoroughly and repeat. For
as many doubling dilutions as
are required. If volume (V) is
1ml then tube 2 containing 1ml
of the diluent and receives 1ml
of undiluted specimen resulting in
a 1/2 dilution of the specimen. A
1ml portion of the 1/2 dilution is
transferred to tube 3 resulting in a
1/4 dilution of the specimen
(1/2 x 1/2), etc… 1 ml of the
final tube is transferred to the
last tube and kept in case higher dilutions are needed. The dilution factor
will be 2, 4, 8, 16 etc. and the dilutions obtained will be 1/2, 1/4, 1/8,
1/16 etc.
Note that each tube is diluted twice as much as the previous tube and that
Figure 2
the final volume in each tube is the same.
The nondiluted specimen may also be given a dilution value of 1/1.

b. Decimal dilutions
Each concentration is one tenth that of the previous one i.e. tenfold step
interval, log10 dilution series (Figure 2, lower dilution)..
First, in tube 1 make up the most concentrated solution required, with at
least a 10% excess and nine times the volume (V) in all tubes.
Measure one tenth of the volume required into the next tube, mix
thoroughly and repeat the dilution fraction will be 10, 100, 1000 … and
the dilutions obtained will be 1/10, 1/100 and 1/1000…

General Remarks
1. Solutions must be thoroughly mixed before measuring out volumes for
the next dilutions. This is achieved by vortexing or by repeated drawing
and draining cycles by a pipet.

2. Use a fresh measuring vessel for each dilution to avoid contamination

or wash your vessel thoroughly between dilutions.

3. Clearly label the tubes before starting dilutions.

4. Diluent are usually distilled water, PBS or saline. But other dilution
buffers could be used depending on the reaction buffer.

5. The antibody titer is defined as the reciprocal of the last dilution in

which the antibody is still detectable (showing a reaction with the
antigen).

6. High viscosity liquids are difficult to dispense, allow time for all the
liquid to transfer

7. Organic solvents may evaporate rapidly, making measurements

inaccurate, work quickly, seal containers without delay.

8. Solutions prone to frothing (e.g. protein and detergent solutions) are

difficult to measure and dispense, avoid forming bubbles due to
overagitation or quick transfer.

9. Suspensions may sediment, thoroughly mix them before dispensing.