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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. This represents 1 part patient sample added to 9 parts of diluent. . So the volumes used would be 10-1= 9.

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. .

This is a series of ½ dilutions. If this is done 6 times this is what you would end up with: .Doubling Dilutions    Doubling dilutions are very popular. Each successive tube will ½ the amount of the original concentrated solution.

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 .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 mL aliquot of a specimen to 9.9 mL = 10 mL The dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL + 9.Dilution Factor   The dilution factor is the final uses the formula volume/aliquot volume.1 mL = 1:100 dilution . EXAMPLE: What is the dilution factor if you add 0.9 mL of diluent? The final volume is equal to the aliquot volume PLUS the diluent volume: 0.

8 mL diluent? .Practice  Problem: What is the dilution factor when 0.2 mL is added to 3.

8 = 4.2 +3.0 total volume 4.Set Up The Problem    dilution factor = final volume/aliquot volume 0.0/0.2 = 1:20 dilution .

. So each successive dilution would be multiplied by the dilution factor.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.

Problem Continued    So in the above problem all successive tubes would have 3. If you had 4 tubes what would be the final dilution of tube 4? . mix and so on.2.8 mLs of diluent. You would then transfer 0.2 of the initial diluted sample into the next tube. mix transfer 0.

2 3.Solving the Problem *Calculate DF of tube 1 Tube Aliquot Diluent Math Dilution 1 0.2 3.8 *4/0.8 1/400x1/20 1:8000 4 0.2 1:20 2 0.2 3.8 1/8000x1/20 1:160.8 1/20x1/20 1:400 3 0.2 3.000 .

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.

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.Next    What if we added the indicator system to the test system? You must create additional rows. . if you are showing your work.

8 4x400 1:800 0.Adding Indicator Cells Tube Dilution RBCs Volume DF Math Dilution 1 1:20 2 1:400 3 1:8000 4 1:160.8 4x8000 1:32.8 4/1 4x20 1:80 0.000 0.000 0.2 3.2 3.8 4x160.000 .2 3.2 3.000 1:640.

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.

retested. . then the result multiplied by the dilution factor.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.

The sample must be diluted.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.

THIS IS NOT THE REPORTALBE RESULT! You must multiply by the dilution factor of 10.Example      The serum was diluted 1:10 and retested. 10 x 80 = 800 mg/dL . The result is 80 mg/dL.