DILUTIONS IMSE 311

Post-Laboratory Discussions
Prepared by:JILLIAN MARIE M. Cuevas, RMT, MSMT
Presented By: Rochelle Darlucio-Yabut, RMT, MPH
EXPERIMENT NO.
Serial Dilution to Detect Cold Reacting Antibodies
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 *IgM - will detect on lab activity

- capable of reacting on cold environment

“
e.g. ABO blood group - example of IgM antibodies - capable of reacting to cold environment

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 “ collect type A blood

MATERIALS

blue reagent for blood typing

- clumping/agglutination if in contact w/
type A blood

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 “ (test tube #, group #, section)

PROCEDURES

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 PROCEDURE
1. Label ten test tubes 1-10 (test tube #, group #, section)
2. Place 0.25 ml of saline in each of the ten tubes. saline will act as diluent
3. Use a clean serological pipette to draw up 0.25 ml antibody A. Add the antibody to tube #1 by carefully lowering
and raising the solution into the pipette to tube #1 by carefully lowering and raising the solution into the pipette
three times to mix, being careful to avoid creating bubbles in the mixture aspirate,
dispense
do not create bubbles

Transfer 0.25ML from tube #1 and transfer to tube #2, to tube #3, then #4, until #10

0.25 ml Discard
antibody A 0.25 ml
1 2 3 4 5 6 7 8 9 10 from tube
solute 10
- always in the 1:10
dilute the bleach
antibody mix - aspirate dispense each tube solution
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0.25 ml of saline
 composition:

PROCEDURE
1. SALINE - diluent
2. ANTIBODY A - solute
3. RCS -
(needed to have reaction)

- to determine the strength of

4. Use clean serological pipette to add 0.25 ml of 3% red blood cell suspension to each tube anti-A but not included in the
enhance agglutination reaction (cold reacting
5. Mix well and place in refrigerator for 30 minutes Ab) computation
6. Remove from refrigerator, centrifuge for 20 seconds. Read immediately for agglutination by gently shaking the tube to
dislodge the red blood cell button
concentrated
antibody A
(dark)

diluted as
we go along
TITER (light color)

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 read under direct light
source
4+ = 1 large
clump/1 solid
clump (usually
color blue solution)
3+ = 1 large
clump +
medium-sized
clump
2+ =
medium-sized
clump
1+ = small clumps
(usually red color
solution)
0 = no
aggt./negative
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 “
The last tube showing
agglutination is the
endpoint of the test. Interpretation
of Results
The titer is reported out as
the reciprocal of the last
dilution showing a positive
result.
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CALCULATING
DILUTION
 DILUTION FORMULA
*FRACTION OR RATIO
Amount of Solute
▸ 𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 =
Total Volume (amt of solute + amt of diluent)
0.25 mL saline = DILUENT
0.25 mL antibody A = SOLUTE
*Where TOTAL VOLUME= Amount of solute + diluent 0.25 mL RCS = not included in
computation
0.25 mL
Dilution =
0.25 mL + 0.25 mL
= 0.25 1
= or 1:2
0.50 2
 SERIAL DILUTION
Transfer 0.25ML from tube #1 and transfer to tube #2, to tube #3, then #4, until #10

0.25 ml 0.25 ml of saline Discard

antibody A 0.25 ml
1 2 3 4 5 6 7 8 9 10 from tube
10
𝐼𝑁𝐼𝑇𝐼𝐴𝐿
1 1 1 1 1 1 1 1 1 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 2 2 2 2 2 2 2
2 2 2
FINAL 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024
DILUTION
Final Dilution (TT1) = Initial Dilution (TT1) = 1/2
Amount of Solute ID (TT2) X FD (TT1) = 1/2 X 1/2 = 1/4
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = ID (TT3) X FD (TT2) = 1/2 X 1/4 = 1/8
Total Volume ID (TT4) X FD (TT3) = 1/2 X 1/8 = 1/16
0.25ML 5 1 ID (TT5) X FD (TT4) = 1/2 X 1/16 = 1/32
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = = 0.50𝑀𝐿 = = --> INITAL DILUTION
12 0.50ML 10 2
 SERIAL DILUTION
Transfer 0.25ML from tube #1 and transfer to tube #2, to tube #3, then #4, until #10

0.25 ml 0.25 ml of saline Discard

antibody A 0.25 ml
1 2 3 4 5 6 7 8 9 10 from tube
10
𝐼𝑁𝐼𝑇𝐼𝐴𝐿
1 1 1 1 1 1 1 1 1 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 2 2 2 2 2 2
2 2 2 2
1 1 1 1 1 1 1 1 1 1 1 1 1
X X X X 1 1 X 1 1 X 1 X X X
2 2 2 4 2 8 2 16 2 32 2 64 2 128 2 256 2 512
𝐹𝐼𝑁𝐴𝐿 1 1 1 1 1 1 1 1 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = = = = = = = = =
4 8 16 32 64 128 256 512 1𝟎𝟐𝟒

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 SERIAL DILUTION
endpoint
4+ 4+ 3 2+ 1+ 1+ 1+ - - -

1 2 3 4 5 6 7 8 9 10
𝐼𝑁𝐼𝑇𝐼𝐴𝐿
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 1 1 1 1 1 1 1 1 1 1
2 2 2 2 2 2 2 2 2 2
FD
𝐹𝐼𝑁𝐴𝐿 1 1 1 1 1 1 1 1 1 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁
2 4 8 16 32 64 128 256 512 1𝟎𝟐𝟒

𝑫𝑰𝑳𝑼𝑻𝑰𝑶𝑵 𝑭𝑨𝑪𝑻𝑶𝑹 = 𝟐 --> reciprocal of initial dilution

14 reciprocal of final dilution --> TITER = 128 (always a whole number)
 “For many serology
tests, it is the serum
that is concentrated;
it may be necessary to
dilute it with saline in
order for a visible
reaction to occur.”

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TYPES OF
DILUTION
 SIMPLE DILUTION

DILUTION Amount of Solute

𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 =
▸ SOLUTE material being diluted Total Volume
▸ DILUENT medium making up the rest of the solution

A 1:20 dilution implies 1 part of solute and 19 parts of diluent.

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 SIMPLE DILUTION
Given:
2 mL - total volume
1:20 - dilution
EXAMPLE: Serum (solute) = ?
Diluent = ?
2 mL solution of a 1:20 dilution is needed to run a specific
serological test. How much serum and how much diluent are
needed to make this dilution? x
1
Amount of Solute =
20 2 mL
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = cross multiply
Total Volume
X (20) = 1 (2mL)
Diluent = 2 mL - 0.1 mL = 1.9 mL amt of diluent X=2
= 0.1 mL amt of solute
20
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 Given:
SIMPLE DILUTION 1.5 - dilution
0.1 mL - solute
diluent - ?
1 0.1 mL
= = X (1) = 4 (0.1 mL) = 0.4 mL amt of diluent
EXAMPLE:
5-1 x

A 1:5 dilution of patient serum is necessary to run a serological test.

There is 0.1 mL of serum that can be used.
What amount of diluent is necessary to make this dilution using all
of the serum?

Amount of Solute 1 Amount of Solute

𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = =
Total Volume Dilution − 1 Amount of Diluent
- use this formula to check your answer
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 SIMPLE DILUTION
Given:
500 mL - total volume
1:10 or 1/10- dilution
EXAMPLE: Glacial - ?
Diluent - ?
A volume of 500 mL of a 10% solution of acetic acid is needed.
How much glacial acetic acid is needed and how much diluent
should be used?
1 X
Amount of Solute =
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = 10 500 mL
Total Volume
TV = amt of solute + amt of diluent
amt of diluent = TV - amt of solute X (10) = 500 X 1 = 50 mL amt of solute (glacial
= 500 mL - 50 mL acetic acid)

= 450 mL amt of diluent

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 COMPOUND DILUTION

For example, if a 1:500 dilution is necessary, it

would take 49.9 mL of diluent to accomplish this
in one step with 0.1 mL of serum

0.1ml
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 =
50ml
2 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = 0.002 = =
1000ml 500ml
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 COMPOUND DILUTION
0.4 ml NSS

0.1 ml
SERUM
1 2 Amount of Solute
3 𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 =
Total Volume
𝐼𝑁𝐼𝑇𝐼𝐴𝐿 1 1 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 0.1ML 1
5 10 10 𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = = 0.2𝑀𝐿 = TUBE 1
𝐹𝐼𝑁𝐴𝐿 1
0.5ML 5
1 1 1 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁
5
X X 0.1ML 1
10 5 10 50
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = = 0.1𝑀𝐿 = TUBE 2
1 1 1 ML 10
= =
50 500 0.1ML 1
𝐷𝐼𝐿𝑈𝑇𝐼𝑂𝑁 = = 0.1𝑀𝐿 = TUBE 3
22 1 ML 10
 SERIAL DILUTION

A Medical Technologist placed 10 ml of diluent on 5 tubes.

She placed 2 ml of serum on tube no. 1. Then she transferred 2 ml from tube no.1 to tube no.2,
repeating steps up to tube no.5.
She discarded 2 ml from tube no.5 and added, 2 ml of red cell suspension in each tube.

TUBE NO. INITIAL DILUTION FINAL DILUTION RESULT

TUBE 1 1/6 1/6 4+
TUBE 2 1/6 1/36 2+
TUBE 3 endpoint 1/6 1/216 1+
TUBE 4 1/6 1/1296 No aggt.
TUBE 5 1/6 1/7776 No aggt.
21 titer - 216 | dilution factor - 6