Serial Dilutions
MLAB 1335 Immunology/Serology
Terry Kotrla
Introduction
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Many of the laboratory procedures involve
the use of dilutions.
It is important to understand the concept of
dilutions, since they are a handy tool used
throughout all areas of the clinical
laboratory.
These dilutions have to be considered as
they make a quantitative difference in what
is going on.
Serial Dilutions
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A serial dilution is any dilution where
the concentration decreases by the
same quantity in each successive
step.
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Serial dilutions are mutiplicative.
What Does This Mean??
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If a solution has a 1/10 dilution the
number represents 1 part of the
patient sample added to 9 parts of
diluent.
So the volumes used would be 10-1=
9.
This represents 1 part patient sample
added to 9 parts of diluent.
Dilutions
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If a 1/8 dilution of the stock solution is
made followed by a 1/6 dilution what
is the final dilution.
The final dilution is: 1/8 x 1/6 = 1/48
These type of dilutions are trickier and
not used very frequently in the clinical
lab.
Doubling Dilutions
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“Doubling dilutions” are very popular.
This is a series of ½ dilutions. Each
successive tube will ½ the amount of
the original concentrated solution.
If this is done 6 times this is what you
would end up with:
Doubling Dilution 6 Times
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1st dilution = 1 /2
2nd dilution = 1 /2 x 1 /2 = 1/4
3rd dilution = 1/4 x 1 /2 = 1/8
4th dilution = 1/8 x 1 /2 = 1/16
5th dilution = 1/16 x 1 /2 - 1/32
6th dilution = 1/32 x 1 /2 = 1/64
This results in a series of dilutions, each a
doubling dilution of the previous one
Dilution Factor
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The dilution factor is the final uses the
formula volume/aliquot volume.
EXAMPLE: What is the dilution factor if you
add 0.1 mL aliquot of a specimen to 9.9 mL
of diluent?
– The final volume is equal to the aliquot volume
PLUS the diluent volume:
0.1 mL + 9.9 mL = 10 mL
– The dilution factor is equal to the final volume
divided by the aliquot volume:
10 mL/0.1 mL = 1:100 dilution
Practice
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Problem: What is the dilution factor
when 0.2 mL is added to 3.8 mL
diluent?
Set Up The Problem
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dilution factor = final volume/aliquot
volume
0.2 +3.8 = 4.0 total volume
4.0/0.2 = 1:20 dilution
Problem Continued
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Remember that serial dilutions are
always made by taking a set quantity
of the initial dilution and adding it
successively to tubes with the same
volume.
So each successive dilution would be
multiplied by the dilution factor.
Problem Continued
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So in the above problem all successive
tubes would have 3.8 mLs of diluent.
You would then transfer 0.2 of the
initial diluted sample into the next
tube, mix transfer 0.2, mix and so on.
If you had 4 tubes what would be the
final dilution of tube 4?
Solving the Problem *Calculate DF of tube 1
Tube
1
2
3
4
Aliquot
0.2
0.2
0.2
0.2
Diluent
3.8
3.8
3.8
3.8
*4/0.2
1/20x1/20
1/400x1/20
1/8000x1/20
1:20
1:400
1:8000
1:160,000
Math
Dilution
Solving the Problem
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Or if you simply wanted to know the
dilution of the final tube you could just
multiply them together:
1/20 x 1/20 x 1/20 x 1/20 = 1:160,000
Next
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What if we added the indicator system to
the test system?
You must create additional rows, if you are
showing your work, to take into
consideration the dilutional effect that will
have on the dilution.
When an indicator is added it is CRITICAL to
remove an aliquot from the last tube.
Adding Indicator Cells
Tube
1
2
3
4
Dilution
RBCs
1:20
1:400
1:8000
1:160,000
0.2
0.2
0.2
0.2
Volume
DF
Math
3.8
3.8
3.8
3.8
4x20
4x400
4x8000
4x160,000
Dilution
1:80
1:800
1:32,000
1:640,000
4/1
Serial Dilutions
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When performing serial dilutions in the
clinical laboratory one must know the
dilution of each tube so a chart is always
created to indicate the dilution of each tube.
If you know the dilution factor you multiply
each successive tube by it and can easily
determine the concentration of each tube to
create your chart.
Titers
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TITERS are reported out as the
reciprocal of the last tube giving a
positive reaction.
So if tube 2 was the endpoint, the
dilution is 1:800 the titer is reported
out as 800/1= 800.
Other Applications
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Sometimes when analyzing a blood
sample the results are above the
linearity (or accuracy) of the machine.
When this happens the blood must be
diluted, retested, then the result
multiplied by the dilution factor.
Example
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A blood glucose of 800 mg/dL was
obtained.
According to the manufacturer the
highest glucose result which can be
obtained on this particular instrument
is 500 mg/dL.
The sample must be diluted.
Example
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The serum was diluted 1:10 and
retested.
The result is 80 mg/dL.
THIS IS NOT THE REPORTALBE
RESULT!
You must multiply by the dilution
factor of 10.
10 x 80 = 800 mg/dL