IMMUNOLOGY-SEROLOGY LAB 3rd Year - 2nd Semester | Rona Maye G.

Trinidad, RMT, MSMT | Prelims

c. Diluent Diluent = Total vol – Solute

Exercise 2: Serial Dilution
Diluent = 4mL – 0.2mL
DILUTIONS Diluent = 3.8 mL
• Necessary to make a less concentrated solution from a 2. A 1:10 dilution of patient serum is necessary to run a
reagent such as an acid or a buffer in order to use the serological test. There is a 0.2 mL of serum that can be
reagent in a particular procedure used. What amount of diluent is necessary to make
• If the relative proportions of antigens and antibodies present this dilution using all of the serum
are not similar, the reaction cannot be detected a. Given: 0.2mL serum, 1:10 dilution,
• Water or saline, is added to the concentrate to make the b. “X” = Total Volume
reagent the proper strength for testing c. Total vol
1 = 0.2 mL 0.2 = Solute
SOLUTE SOLVENT / DILUENT 10 x 1:10 = Dilution used
The medium making up the
The material being diluted x = (10)(0.2 mL)
rest of the solution
1
Ø The relationship between these two is a ratio that can also
x = 2 mL Total vol
be expressed as fraction d. Diluent
1 : 20 Diluent = Total vol – Solute
Solute Total Volume Diluent = 2mL – 0.2mL
(1 part Solute) (19 parts of diluent) Diluent = 1.8 mL

1 / Dilution TAKE NOTE

= Amount of Solute Ø Percentage is simply a different way of expressing a
Total Volume dilution
Ø 10% solution = 1/10
Total Volume = Solute + Diluent
DETERMINATION OF THE CONCENTRATION OF
TITER ANTIBODY (TITER) FOR A SPECIFIC ANTIGEN
• Indicator of an antibody’s strength • It involves 2 steps:
• Reciprocal of the highest dilution of the patient’s serum
in which the antibody is detectable 1. Preparing a serial dilution of the antibody
• The higher the titer present, it can be clinically significant is containing solution (serum)
the amount of antibody present in the serum of the patient 2. Adding an equal volume of antigen suspension
to each dilution

SERIAL DILUTION
• Simply a series of simple dilutions which amplifies the
dilution factor quickly beginning with a small initial quantity
of material
• Two-fold or ten-fold serial dilution – commonly
encountered in the laboratory

Ø Ex. The third tube showed the highest dilution wherein there
is a positive result. The titer is 1,000
Ø Dilution (ex. 1:20)
Ø Reciprocal (ex. 20:1 or 20 / 1)
o Titer that is to be reported is 20

Problem
1. 4 mL of a 1:20 dilution is needed to run a specific
serological test. How much serum (solute) and how
much diluent are needed to make this dilution?
a. “X” = Amount of serum
(Ten-fold serial dilution)
b. Solute:
1=x 4mL = Total volume
20 4mL 1:20 = Dilution used

x = (1)(4mL)
20
x = 0.2 mL serum

JRFR | 3MT04 | TUA-CMT

TOPIC: SERIAL DILUTION
• Discard 0.2 mL in order to add an indicator by the end of
the preparation
• Final dilution = 1:32 dilution

Titer is the last tube in which a positive reaction is visible

• Ex: The last tube with a positive result is in tube 4, the titer
is 16

Problem
3. What is the final dilution of serum obtained from the
following serial dilutions: 1:4, 1:4, 1:4, 1:4, 1:4, 1:4 (6
test tubes)?
Solution: 46 = 4,096
(4 x 4 x 4 x 4 x 4 x 4)

(Two-fold serial dilution) Final dilution = 1:4,096

• What do you want to initially get in the dilution, is the
amount of diluent to be used for the preparation
• You can predict that the volume of the diluent is also
(1.0mL)
• Steps:
a. Place the amount of diluent in all of the tubes (1mL)
b. Add the solute (from the patient sample) – 1mL, by
aspirating directly from the sample to test tube no. 1
c. Test tube no. 1 : 1mL of serum + 1mL of saline = 2mL
d. Mix it using a pipette, aspirate an aliquot of 1mL from
test tube no.1, to be transferred to test tube no. 2
e. Test tube no. 2 = 1mL aliquot of TT1 + 1mL of saline =
2mL
a. Multiply 2 (initial dilution) by 2 (previous
factor) = 1:4 dilution for TT2
f. Mix it using a pipette, aspirate an aliquot of 1mL from
test tube no. 2, to be transferred to test tube no. 3
g. Test tube no. 3 = 1mL aliquot of TT2 + 1mL of saline =
2mL
a. Multiply 2 (initial dilution) by 4 (previous
factor) = 1:8 dilution for TT3
h. Mix it using a pipette, aspirate an aliquot of 1mL from
test tube no. 3, to be transferred to test tube no. 4
i. Test tube no. 4 = 1mL aliquot of TT3 + 1mL of saline =
2mL
a. Multiply 2 (initial dilution) by 8 (previous
factor) = 1:16 dilution for TT4
j. Mix it using a pipette, aspirate an aliquot of 1mL from
test tube no. 3, to be transferred to test tube no. 4
k. Test tube no. 5 = 1mL aliquot of TT4 + 1mL of saline =
2mL
a. Multiply 2 (initial dilution) by 16 (previous
factor) = 1:32 dilution for TT5
Titer = 4,096
PROCEDURE
1. Label the 5 tubes using numbers 1 to 5.
2. Using the automatic pipette place 500 uL of 0.85% saline
to all tubes.
3. Using a new tip for the pipette, add 500 uL of antibody A
to tube number 1. Place the tube in a vortex.
4. Using a new tip, draw up to 500 uL from tube number 1 and
transfer to tube number. Place again the tube in a vortex.
5. Repeat step number 4 up to the last tube.
6. Discard 500 uL from the last tube.
7. Using new tip for the pipette, add 500 uL of 3% Red Blood
cell suspension to each tube.
8. Mix well by using a vortex mixer or by simply tapping the
tubes
9. Place your sets of tubes in the refrigerator 30 minutes.
10. Remove from refrigerator, centrifuge for 20 seconds.
11. Read immediately for agglutination by gently shaking the
tube to dislodge the red blood cell button.
Note: If the tubes are shaken too roughly false negative
reactions can occur.
INTERPRETATIONS
1. Clumping of the red blood cells is positive. A smooth,
uniform appearance of red blood cell suspension is
negative.
2. The last tube showing agglutination is the endpoint of
the test. The titer is reported out as the reciprocal of the last
dilution showing a positive result.

Ø A tube with a highest dilution of a positive result, where the OBSERVED RESULTS:
succeeding tube would be getting a negative result Positive: With agglutination
Negative: Without agglutination
DOUBLING DILUTION
• Amount of serum is cut in half with each dilution A. Dilution: Calculate the dilution of tube number 1.
B. Based on the value of tube number 1, determine the dilution
Discard 0.2 mL

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 values from tubes 2 to 5 and record it on the table.
C. Titer: Write the titer for each tube.
Diluent 0.2 mL 0.2 mL 0.2 mL 0.2 mL 0.2 mL
Serum 0.2 mL 0.2 mL 0.2 mL 0.2 mL 0.2 mL
Dilution 1:2 1:4 1:8 1 : 16 1 : 32
• Add an equal amount of diluent from tube 1 to tube 5
• Add the amount of serum
• Multiply the factor of the previous dilution by the initial
dilution
JRFR | 3MT04 | TUA-CMT