1. By: EID ALATAWI Serial Dilution

2. 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. DEFINITIONS: Solutions : homogeneous mixture of two or more substances physically mixed together in a uniform way. Solute: substance being dissolved. Solvent: part of a solution doing the dissolving. Soluble: when a substance dissolves in another substance. Aliquot: a measured sub-volume of original sample. Diluent: material with which the sample is diluted.

3. Dilution: - It is the process of reducing the concentration of a solute in solution, usually simply by mixing with more solvent. To dilute a solution means to add more solvent without the addition of more solute. A serial dilution: - It is any dilution where the concentration decreases by the same quantity in each successive step. Serial dilutions are always made by taking a set quantity of the initial dilution and adding it successively to tubes with the same volume. Serial dilutions are multiplicative, so each successive dilution would be multiplied by the dilution factor.

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

5. 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 - 1/32 6th dilution = 1/32 x 1 /2 = 1/64 This results in a series of dilutions, each a doubling dilution of the previous one

6.  Other dilutions: The dilution can also be 1/3, 1/4, 1/5, …, 1/100, etc. 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.

8. Dilution Calculations:  To calculate a dilution factor: - Dilution factor (DF): ratio of final volume/aliquot volume (final volume = aliquot + diluent). - EXAMPLE: What is the dilution factor if you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent? - Dilution factor (DF) = Final volume Aliquot volume - The final volume = the aliquot volume + the diluent volume: 0.1 mL + 9.9 mL = 10 mL - DF = 10 ml = 1 : 100 dilution 0.1 ml

9. Practice What is the dilution factor when 0.2 mL is added to 3.8 mL diluent?

10.  Answer: dilution factor = final volume/aliquot volume 0.2 +3.8 = 4.0 total volume 4.0/0.2 = 1:20 dilution

11. In the previous dilution all successive tubes would have 3.8 mLs of diluent. You would then transfer 0.2 of the initial diluted sample into the next tube, mix transfer 0.2, mix and so on. If you had 4 tubes what would be the final dilution of tube 4? Remember that each successive dilution would be multiplied by the dilution factor.

12.  Calculate DF of tube 4 Tube 1234 Aliquot 0.2 Diluent 3.8 Math *4/0.21/20x1/201/400x1/201/8000x1/20 Dilution 1:201:4001:80001:160,000 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 = 1:160,000

13.  To Calculate the Concentration of a Solution: In the initial and diluted solution, the moles of solute are the same the concentrations and volumes are related by the following equations: For percent concentration C 1 V 1 = C 2 V 2 Concentrated Diluted solution solution V1 x C1 = V2 x C2 C1 = Initial concentration. V1 = Initial volume. C2 = Final concentration. V2 = Final volume. 13

14. Guide to Calculating Dilution Quantities 14

15. Example of Dilution Calculations Using Percent Concentration % What volume of a 2.00% (m/v) HCl solution can be prepared by diluting 25.0 mL of 14.0% (m/v) HCl solution? STEP 1 Prepare a table: C 1 = 14.0% (m/v)V 1 = 25.0 mL C 2 = 2.00% (m/v)V 2 = ? STEP 2 Solve dilution expression for the unknown C 1 V 1 = C 2 V 2 V 2 = V 1 C 1 C 2 STEP 3 Set up the problem using known quantities: V 2 = V 1 C 1 = (25.0 mL)(14.0%) = 175 mL C 2 2.00% 15

16.  Applications of dilution: Why we need to perform a dilution? Serial dilutions are often used in microbiology, biotechnology, toxicology, immunology and in chemistry classes. Serial dilution may also be used to reduce the concentration of microscopic organisms or cells in a sample. * 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, retested, then the result multiplied by the dilution factor.

18. 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. The sample must be diluted.

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

20.  Procedure: Prepare all materials are needed for this experiment. Label tubes for serial dilutions as follows: 1 (1:2); 2 (1:4); 3 (1:8); 4 (1:16); 5 (1:32) Using a micropipettor, pipet 1 mL of distilled water into tubes 1, 2, 3, 4, 5. Be sure cap is firmly closed and mix the dye solution by inverting the tube. Using a micropipettor, pipet 1 mL of blue dye into tube1. Mix gently by drawing the solution up and down 3 times (3X). Transfer 1 mL of solution from tube 1 into tube 2. Mix gently 3X. Transfer 1 mL of solution from tube 2 into tube 3. Mix gently 3X. Continue to transfer and mix through tube 5. Discard the last 1 mL from tube 5. Examine the tube dilutions. Note that the color decreases with increasing tube number.

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