How to perform a two fold dilution in a clinical laboratory

It describes basic dilution techniques

1. Name: Negash Alamin
Occupation: Clinical labscientist
Topic: Serology
Email: [email protected]
Date: 31/5/18

2. How to perform a two fold dilution in clinical laboratory?

N.B This is just an example the level of serum or plasma may be more than 50 microliter
depending on the procedure.

Principle: Six test tubes are arranged with each holding 50 microliters of normal saline and 50
microliter of plasma is added to the first tube taking its total volume 100 microliter. Consecutive
dilution is performed until the last tube and the final mixture from test-tube six is discarded.

N.B Dilution is used to differentiate convalescence period from active infection by measuring the
level of antibodies. It is used together with specific and nonspecific tests to provide effective
diagnosis as opposed to 'presumptive diagnosis' using nonspecific tests.

Materials:
1-Test tubes
2-Micropippette
3-Normal saline
4-Test tube rack
5-Patient plasma

Procedure:
1- Add 50 microliters of normal saline in all tubes by forward pipetting.
2- Add patient serum 50 microliters in first tube. This will give a total volume of 100 microliters.
3- Now, take 50 microliters of mixture from test tube one to the second tube, third tube, fourth
tube and so on until the sixth tube and discard the mixture of the sixth tube not to a seventh tube
(because there is no seventh tube) but to waste sink.

3. Result: Now, we calculate sample volume (amount of serum in each tube and its dilution
strength).
General formula: Dilution strength= sample volume/ Total volume
Tube-1: 50/100=1/2 dilution strength
Sample volume (patient’s serum in the mixture) = 50 microliter.
Tube-2: 25/100= 1/4 dilution strength
Sample volume=25 microliters
Tube-3: 12.5/100=1/8 dilution strength
Sample volume=12.5 microliters
Tube-4: 6.25/100= 1/16 dilution strength
Sample volume=6.25 microliters
Tube-5: 3.125/100 =1/32 dilution strength
Sample volume= 3.25 microliters
Tube-6: 1.5625/100= 1/64 dilution strength
Sample volume= 1.5625 microliters


N.B Reverse pipetting occurs when the button on the micropipette is pressed all the way down
before inserting it in our sample and releasing it halfway to suck sample once it is in our sample.
Forward pipetting occurs when we press the button on the pippette halfway before inserting in
the sample tube and releasing it to suck sample; then, it is pressed all the way down to discard
our sample.