IMMUNOLOGY AND SEROLOGY LAB

LECTURE 1: DILUTION
MIRALYN MADEL QUIRIT, RMT
AUGUST 17, 2021
For updates and corrections → @mar4rii on Twitter

DILUTION 5
● Weakend solutions = 20
● Involves a particular solution with a definite concentration =
1
● Process of adding additional solvent to a solution to 4

decrease its concentration

● A mixture having 2 components:
○ Substance to be diluted/solute (serum, red EXAMPLE NO. 2
cells, urine, acids, etc)
○ Diluent/solvent (distilled water, saline solution, Dilute 3 mL of a serum with 25 mL of saline. What is the
buffer, or indicated chemical reagent) dilution?
● Mixing a solute with the diluent results to the reduction of
a particular solution
● Common solute in Serology: Serum or other body fluids

● Total volume of solution = solute + solvent

● When performing dilution, get the volume of solute and - answer can be expressed as a fraction or with the use of a
add it with the volume of your diluent colon
● RATIO: the relationship of the solute and the solvent
𝑣𝑜𝑙. 𝑠𝑜𝑙𝑢𝑡𝑒 What is the resulting dilution when 0.5 mL of a serum is
𝑅𝑎𝑡𝑖𝑜 = 𝑣𝑜𝑙. 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 added with 1.5 mL saline?
● DILUTION FACTOR

SINGLE DILUTION
● Stock solution + distilled water = single dilution
● One step process of adding diluent to the stock solution
● Example: Dilute 1mL of serum with 9mL of saline

0.5/2.0 is an acceptable dilution, but it is recommended

to expressed it as a whole number
1𝑚𝑙
= 1+9𝑚𝐿
1
= 10
● If 1/10 is the dilution what is the ratio?
𝑣𝑜𝑙. 𝑠𝑜𝑙𝑢𝑡𝑒
𝑅𝑎𝑡𝑖𝑜 = 𝑣𝑜𝑙. 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
1
𝑅𝑎𝑡𝑖𝑜 = 9

EXAMPLE NO 3
EXAMPLE NO 1

Make 250 mL of a 1/10 dilution of serum in saline.

Five milliliters of serum is diluted up to 25mL with saline. What is
the serum dissolution? What is the serum to saline ratio?
given: TV - 250 mL, Dilution – 1/10
𝑠𝑜𝑙𝑢𝑡𝑒 problem: how much volume of serum is used?
𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
5𝑚𝐿
= 25𝑚𝐿
1
= 5
If the total volume of the solution is 25, what is the total volume
of the solvent?
Total volume = solute + solvent
25mL = 5mL + x
25mL - 5mL = x
20mL = x 25 mL serum is added with saline to bring the total volume
up to 250 mL/
What is the serum to saline ratio?
𝑣𝑜𝑙. 𝑠𝑜𝑙𝑢𝑡𝑒 Problem: How much saline (solvent) is needed to add to 25 mL
𝑅𝑎𝑡𝑖𝑜 = 𝑣𝑜𝑙. 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
(serum) in order to make a 250 mL volume of a solution having
a 1:10 dilution?

1
TV = 25 + y
y = 250 – 25
ysolvent = 225 mL

to check:
serum = 8 mL
8 mL + 32 mL solvent = ⅕ dilution
TV = 40 mL

SERIAL DILUTION
● We do series of the steps
● We repeat the procedure more than once.
- by using 25 mL serum, added with 225 mL saline, we will ● Production of solutions having different concentrations of
yield a 1:10 dilution with a total volume of 250 mL. the same substance.
● The concentration of the solution being diluted decreases
● Make 1/10 dilution of serum in saline. with increased dilution.

EXAMPLE NO. 7

- You can choose to use other values but just reduce it to its Ex: When is the dilution if you add 1 mL aliquot of a specimen to
simplest form. 9 mL of diluent?

● If you want to have a total volume of 70 mL:

Step 1 : D= 1/10
All successive tubes would have 9 mL of diluent. You
7 mL – serum would then transfer 1 mL of the initial diluted sample into the
63 mL - solvent next tube, mix, transfer 1 mL to the next, mix and so on.
● Prepare a series of tubes (ex. 3 tubes)
Given : 1/10 dilution ● In the successive tubes, you are going to add the
● No definite volume of solvent same volume of the solvent used in the first step
○ Serum = 2 mL ● In series of tubes, you have to use the same volume
of diluent that you used in the first step
● Transfer 1ml of the initially diluted sample to the next
tube, so on and so forth
10= 2 + y
10 - 2 = y
Y= 8 mL (solvent)
CAN BE USE THIS FORMULA FOR BIGGER VALUES
● Solute = 20
● TV= 1/10

X = 10 (20)
X= 200 (TV)

solvent= 180 mL
● You prepared 5 tubes
● Stock solution (solute)
● Mix 1 ml of your solute and 9ml of diluent
= 1/10 ○ Dapat same ang volume ng diluent in all of the
tubes
○ Place 9ml of diluent in all of the tubes prepared
● In step 1: you will get a volume from your stock solution,
EXAMPLE NO. 4
from the problem, it needs 1 ml. Transfer to Tube 1 that
contains 9 ml of the diluent
○ Total volume of the solution now becomes 10
Determine the amount of serum in 40 mL of a ⅕ dilution of
ml
serum saline.
○ Having a dilution of 1/10
● Get from tube 1 and transfer it to tube 2
○ Use the same volume of solute that you’re
going to transfer from tube 1 to tube 2 = 1 ml
● Tube 2 total volume becomes 10ml
○ Dilution changes (increases) = 1/100
2
 ○ The color also changes; becomes lighter
○ The concentration of substance decreases, but
the value for dilution increases
● Repeat the same process to the succeeding tubes
○ Tube 3 total volume becomes 10mL with a
dilution of 1/1000 (increases)
○ Concentration decreases (lighter blue)
● In serial dilution, the concentration of the substance
decreases in the successive tubes whereas the value
for dilution increases.
● Final step: Discard volume
○ Volume of solute we set
○ Discarded vol: 1ml
○ Get 1ml from the last tube and discard
● Tubes will be subjected for testing
● As we transfer 1 ml from the previous tube to the next,
the volume of the previous tubes will decrease. (from 10
ml to 9ml)
● If you had 5 tubes, what would be the final dilution of
tube 4?
○ 1/10000
○ How did we come up with that value?

SERIAL DILUTION (ex no. 7)

1 2 3 4 5

Volume of
1ml 1ml 1ml 1ml 1ml
solute

Volume of
0ml 9ml 9ml 9ml 9ml
diluent

Total volume 10ml 10ml 10ml 10ml 10ml

Tube dilution 1/10 1/10 1/10 1/10 1/10

Final dilution 1/10 1/100 1/1000 1/10000 1/100000

● Tube dilution - focuses on the dilution of the tube itself without considering the initially diluted sample
● Final dilution - must determine first the tube dilution
○ Multiply the tube dilution with the previous final dilution

MIXED DILUTION
● Pag mixed dilution, iba ang volume ng diluent sa Tube 1, iba din ang volume ng diluent sa Tubes 2 and onwards

● Transfer 1mL of serum in the first tube
● Sa first tube, naglagay ka ng 9mL of the diluent for the
first tube and 4mL of diluent on Tubes 2,3,4, and 5.
● Form tube 1, you get 4mL and transfer it to the
succeeding tubes
● Get 4 mL from the last tube then discard
● Problem:
○ A serial dilution is made by placing 9mL
diluent in the first tube of a series and 4mL in
each of the remaining four tubes. 1mL of
serum is added to the first tube and 4mL from
the first tube is transferred to the second tube
and then to each succeeding tube. The last
4mL transferred and discarded.
○ Process ng pag transfer is still the same
● The same principle still applied na as the tube
progresses,
The concentration decreases but the value of the dilution
increases
● We are still going to use the same table in the
computation
● So how do we do it in the computation?
○ Place the corresponding values in the table
○ Then compute for the tube dilution and the final
dilution the same thing we did in the basic serial
solution

3
 MIXED DILUTION

1 2 3 4 5

Volume of 1mL 4mL 4mL 4mL 4mL

solute

Volume of 9mL 4mL 4mL 4mL 4mL

diluent

Total 10mL 8mL 8mL 8mL 8mL

volume

Tube 1/10 1/2 1/2 1/2 1/2

dilution

Final 1/10 1/20 1/40 1/80 1/160

dilution

● Then we get the dilution of Tube 1

D = 1/10

● For Tube 2

D = 4/8 converted to ½
(the same for the succeeding tubes)

● For the computation of the final volume

○ The final dilution of tube 1 is the tube itself so it will be 1/10
○ The final dilution of Tubes 2 to 5 will be the same kung ano ginawa sa serial dilution kanina.
■ Multiply the tube dilution of the next tube to the Final dilution of the previous tube
■ 1/10 * ½ = 1/20

EXAMPLE NO. 8

● You are given a series of 5 tubes, each of which contains 2 mL of diluent. 0.5 mL is added to the first tube and 0.5 mL is carried out in
the remaining tubes. What is the dilution of the mixture in tube 3 and tube 5?

SERIAL DILUTION

1 2 3 4 5

Volume of 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL

solute

Volume of 2mL 2mL 2mL 2mL 2mL

diluent

Total 2.5mL 2.5mL 2.5mL 2.5mL 2.5mL

volume

Tube 1/5 1/5 1/5 1/5 1/5

dilution

Final 1/5 1/25 1/125 1/625 1/3125

dilution

4
 EXAMPLE NO. 9

● You are given a series of 10 tubes, each of which contains 4mL of diluent. 1 mL of fluid is added to the first tube and dilution using
0.5mL is carried out in the remaining tubes. What is the serum concentration in tubes 4 and 8?

SERIAL DILUTION

1 2 3 4 5 6 7 8 9 10

Volume of 1mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL
solute

Volume of 4mL 4mL 4mL 4mL 4mL 4mL 4mL 4mL 4mL 4mL
diluent

Total volume 5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL

Tube dilution 1/5 1/9 1/9 1/9 1/9 1/9 1/9 1/9 1/9 1/9

Final dilution 1/5 1/45 1/405 1/3645 1/32805 1/295245 1/2657205 1/2391484 1/4.65x10^ 1/5.16x10
5 -9 ^-10

DILUTION as TEST ENDPOINTS

● TITERS
○ To determine the value of the titer
○ Endpoint reaction can be: (most common)
■ Agglutination
■ Precipitation
○ Perform serial dilution

● Serially diluted sample = becuase the dilution increases DILUTION FACTOR

in the succeeding tubes and the concentration decreases
● 4 tubes with reactions = 1,2,3 ,4
○ 5 & 6 = no reaction; disregard 1
● Which among the 4 has the smallest amount of Titer dilution =
8
concentration that produces a particular endpoint
reaction? 1
○ Tube 4 = has the least amount of Dilution factor = or 8
8
concentration; masyadong diluted kaya mahina
na ang reaction; this tube is still counted Dilution factor = reciprocal of the dilution
because it still has observable reaction
○ Titer = 1/160 Ex. ASO test
■ Used for the computation of the - when performing semi-quantitative, u will use a dilution factor of
concentration of the antigen ot the 200 iu/L (concentration of the reagent in the kit)
antibody
● RED CELLS = most common indicator
200 x 8 = 1,600 iu/L (reported concentration of ASO)
○ It is necessary to use indicator for us to
visualize the reaction of the antigen and
antibody - Reporting is in the form of numbers
○ The common observation is agglutination - Semi-quantitative because we did not exactly measure the
antibodies, we just did the titer determination and multiply it with
the factor of the test