Serial Dilutions

MLAB 1335 Immunology/Serology

Terry Kotrla
Introduction

 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

 A serial dilution is any dilution where

the concentration decreases by the
same quantity in each successive
step.
 Serial dilutions are mutiplicative.
What Does This Mean??

 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

 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

 “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

 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
 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

 Problem: What is the dilution factor

when 0.2 mL is added to 3.8 mL
diluent?
Set Up The Problem

 dilution factor = final volume/aliquot

volume
 0.2 +3.8 = 4.0 total volume
 4.0/0.2 = 1:20 dilution
Problem Continued

 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

 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

Math *4/0.2 1/20x1/20 1/400x1/20 1/8000x1/20

Dilution 1:20 1:400 1:8000 1:160,000

Solving the Problem

 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

 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 1:20 1:400 1:8000 1:160,000

RBCs 0.2 0.2 0.2 0.2

Volume 3.8 3.8 3.8 3.8

DF 4/1

Math 4x20 4x400 4x8000 4x160,000

Dilution 1:80 1:800 1:32,000 1:640,000

Serial Dilutions

 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

 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

 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

 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

 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