9.2: Part I- Serial Dilutions

Introduction 

A Serial dilution is a series of dilutions made sequentially, using the same dilution factor for each step. The dilution factor is the initial volume divided by the final solution volume.  For example, if you take 1 part of a sample and add 9 parts of water (solvent), then you have made a 1:10 dilution; this has a dilution factor of 10.  These serial dilutions are often used to determine the approximate concentration of an enzyme (or molecule) to be quantified in an assay. Serial dilutions allow for small aliquots to be diluted instead of wasting large quantities of materials, are cost-effective, and are easy to prepare. 

Figure  18.1: Dilution Series

Figure 18.1 shows a ten-fold serial dilution, which can also be called a 1:10 dilution, or a series with dilution factor of 10.  To determine the concentration at each step of the series, you divide the previous concentration by the dilution factor.  

Diagram of 1:2 Serial Dilutions

In your notebook, draw a diagram showing the serial dilutions for the \(\ce{6KMnO4}\) solutions you are preparing. In the diagram, indicate the volume being withdrawn from the concentrated solution, the volume of water added, the concentration of the new solution, and the total volume.  

Practice Calculations

Problem 1.  Assume the original sample used in Figure 1 contained 400 g/L of Reagent X.  

1. Then the first 1:10 dilution tube would have a concentration of  400/10  = __________
2. Then the second 1:10 dilution would have a concentration of ____________

Problem 2. Assume the original sample used in Figure is considered 100% concentration.

1. Then the first 1:10 dilution tube would have a _____ % concentration.
2. The second 1:10 dilution tube would have a _____ % concentration.

Problem 3. To make a serial dilution with a dilution factor of 5, you would need to add 1 part of the reagent plus ___ parts of water to make a total of 5 parts.  This five-fold serial dilution would have concentrations of 100%, ______% in first diluted tube, _____% in second diluted tube, ________% in third diluted tube.

Problem 4.  Suppose the third diluted tube of a two-fold serial dilution has a concentration of 300 g/L.  

1. That means that the second diluted tube has a concentration of _________ 
2. The first diluted tube has a concentration of ________    
3. The original tube has a concentration of _______
4. What formula could you use to calculate the concentration of the original tube from the problem statement?

Activity: Making Serial Dilutions

Materials

Reagents

• Blue food dye
• DI H2O
• 96 well microplate (dry)

Supplies

• P200 Micropipette
• Box of P200 Pipet tips

Note

Use one pipet tip for each serial dilution.

Procedure

Preparing Two-Fold Serial Dilution (Dilution Factor of Two)

1. Obtain a clean, dry 96 well microplate, always touching the edges only.  Use a dry clean paper towel to wipe off any fingerprints on the bottom of the plate.  
2. Hold plate up to the light and check that there are no dirty spots on the three rows that you will use. 
3.  (Optional) You can scan the plate with no liquid, to find out the baseline absorbance of the plastic.
4. Pipet 100 uL of DI-water into the first 5 wells of row A (A1-A5).  
5. Pipet 100 uL of the original blue dye into the first well (A1).  Carefully pipet up and down twice to mix.
6. You do not need to change the pipet tip.  But make sure that you released all liquid into the first well.
7. Transfer 100 uL of the mixture into the next well (A2). Mix carefully and release all liquid. 
8. Transfer 100 uL of the mixture into the next well (A3). Mix carefully and release all liquid. 
9. Transfer 100 uL of the mixture into the next well (A4). Mix carefully and release all liquid. 
10. Transfer 100 uL of the mixture into the next well (A5). Mix carefully and release all liquid. 
11. Transfer 100 uL of the mixture into the next well (A6). Take a photo of the wells on top of a white paper.

Preparing Four-Fold Serial Dilution (Dilution Factor of Four)

1. Using a new pipet tip, pipet 150 uL of DI-water into the first 5 wells of row B (B1-B5).  
2. You do not need to change the pipet tip.  Pipet 50 uL of the original blue dye into the first well (B1).  Carefully pipet up and down twice to mix. Then make sure that you released all liquid into the first well.
3. Transfer 50 uL of the mixture into the next well (B2). Mix carefully and release all liquid. 
4. Transfer 50 uL of the mixture into the next well (B3). Mix carefully and release all liquid. 
5. Transfer 50 uL of the mixture into the next well (B4). Mix carefully and release all liquid. 
6. Transfer 50 uL of the mixture into the next well (B5). Mix carefully and release all liquid. 
7. Transfer 50 uL of the mixture into the next well (B6). This ensures that all wells B1-B5 have the same volume.
8. Take a photo of the wells on top of a white paper.

Preparing Five-Fold Serial Dilution (Dilution Factor of Five)

1. Using a new pipet tip, pipet 160 uL of DI-water into the first 5 wells of row C (C1-C5).  
2. You do not need to change the pipet tip.  Pipet 40 uL of the original blue dye into the first well (C1).  Carefully pipet up and down twice to mix. Then make sure that you released all liquid into the first well.
3. Transfer 40 uL of the mixture into the next well (C2). Mix carefully and release all liquid. 
4. Transfer 40 uL of the mixture into the next well (C3). Mix carefully and release all liquid. 
5. Transfer 40 uL of the mixture into the next well (C4). Mix carefully and release all liquid. 
6. Transfer 40 uL of the mixture into the next well (C5). Mix carefully and release all liquid. 
7. Transfer 40 uL of the mixture into the next well (C6). This ensures that all wells C1-C5 have the same volume.
8. Take a photo of the wells on top of a white paper.

Activity: Measuring Absorbance

A microplate reader is a spectrophotometric instrument that can measure the absorbance of 96 different samples at one time.  Does that save time compared to working with individual cuvettes and a spectrophotometer?  We will use a microplate with 96 wells, so that you can perform all of your serial dilutions onto one plate and scan the entire plate with the microplate reader once.  The microplate has rows marked A-H and columns marked #1-12.  Using blue dye, you will make a 1:2 serial dilution on row A, make a 1:4 serial dilution on row B, and a 1:5 serial dilution on row C. 

Materials

Equipment

• Microplate Reader and cables
• Laptop computer with program installed to run microplate reader

Procedure

1. Attach the cable from laptop computer to microplate reader.  
2. Power “ON” the laptop computer and the microplate reader.  
3. Open the computer program to run the microplate reader. 
4. Push the button to open the microplate reader and expose the microplate loading platform.  
5. Place your microplate securely into the holder area, ensuring that well A1 is at the top left corner.  
6. Push the button to close the microplate reader.
7. On the computer program, start “read new plate” using wavelength 595 nm (for blue dye).  
8. With the computer mouse, highlight the cells corresponding to the microplate wells that you used. Take a photo of the computer screen and note the Absorbance data in the tables below. 
9. Calculate the dye concentration for each well, by dividing the dilution factor for each step of the serial dilution.  The original dye is 100% concentration.

Data Table 18.1.  

Absorbance Measurements of Two-Fold Serial Dilution with Microplate Reader
Well	A1	A2	A3	A4	A5
Absorbance @ 595 nm
Dye Concentration

  

Data Table 18.2.

Absorbance Measurements of Four-Fold Serial Dilution with Microplate Reader
Well	A1	A2	A3	A4	A5
Absorbance @ 595 nm
Dye Concentration

 

Data Table 18.3.  

Absorbance Measurements of Five-Fold Serial Dilution with Microplate Reader
Well	A1	A2	A3	A4	A5
Absorbance @ 595 nm
Dye Concentration