Title: Serial Dilutions
Course: BTEC-1100-301-302-303-F21 > Section: Module 1 - Calculations and Measurements

You will perform serial dilutions of a blue dye solution. The dye solution is in a tube labeled “SmartSpec”.

Sample preparation for the SmartSpec

Obtain 5 microcentrifuge tubes.  Label them “undil”, “2x dil”, “4x dil”, “8x dil”, and “16x dil”.

Tip: To help keep track of your tubes, and what you have done to each one, keep your tubes in a logical order, and place them in a rack in rows for “done” and “not done”.  For instance, empty tubes could be in the top row of the rack.  When you pick up a tube and put something in it, put it into another row.  If you pick it up again to add something else, put it down into yet another row.  Developing this habit can save many mistakes, especially once protocols become more complicated!

- Take 750 µL of blue dye solution from the class stock, and put it in tube “undil”.
- Take 750 µL of blue dye solution from the class stock, and put it in the “2x dil” tube.  Then, add 750 µL of water to the “2x dil” tube.  (Because you mixed 1 part original plus one part water, you did a 2-fold dilution.)  Vortex the tube to mix thoroughly.
- Transfer 750 µL of “2x dil” to the “4x dil”.  Add 750 µL water to the “4x dil”.  (You just did a 2-fold dilution of something that was already 2-fold diluted.  2x2=4.)  Vortex the tube to mix thoroughly.
- Transfer 750 µL of “4x dil” to the “8x dil”.  Add 750 µL water to the “8x dil”.  (You just did a 2-fold dilution of something that was already 4-fold diluted.  2x4=8.)  Vortex the tube to mix thoroughly.
- Transfer 750 µL of “8x dil” to the “16x dil”.  Add 750 µL water to the “16x dil”.  (You just did a 2-fold dilution of something that was already 8-fold diluted.  2x8=16.)  Vortex the tube to mix thoroughly.

To check your work, make sure that tubes “undil”, “2x dil”, “4x dil”, and “8x dil” all have the same volume (750 µL).  Tube “16x dil” should have twice that volume, or 1.5 mL.

Congratulations!  You have just done a serial dilution.  What you did is a 2x serial dilution (because you diluted 2x over and over).  Often, people will do 10x (or 100x) serial dilutions to achieve a drastic change in concentration relatively quickly.

Using the SmartSpec

1. Turn on the SmartSpec, and allow it to warm up and go through its diagnostics.
2. Press the lambda (λ) button.  (The Greek letter λ means wavelength.)
3. The screen should now say “Enter number (1-3) of wavelengths to read.”  Use the keypad to type 1, then press enter.
4. The screen should now say “Enter wavelength:”.  Type 580, then press enter.
5. The screen should now say “Do you want to subtract background reading? NO”.  Press enter to accept that choice.
6. The screen should now say “Ready to read absorbance”.
7. First, you need to zero the spectrophotometer by giving it a cuvette filled with the solvent in which you diluted your sample.  In this case, the sample is diluted in water.  So, put 750 µL of water into a plastic cuvette.  Be sure you touch only the frosted parts of the cuvette, and not the clear portion through which the light beam passes.  If you have any doubts that you may have left fingerprints in the light path, wipe it with a kimwipe.
8. Insert the cuvette into the spec so that the “V” shape on the cuvette is aligned with the arrow next to the cuvette chamber.  (This ensures that the light is going through the proper path.)  Close the lid.
9. Press “read blank”, and wait while it reads.
10. Record the value for the blank.  (A580 = ???)
11. Press the right arrow button to continue.
12. The screen should now say “Ready to read absorbance”.
13. Obtain a new cuvette, and put in one of your samples.  (I suggest starting with the most dilute sample, and working your way up.  Again, it helps to go in some logical order.)
14. Place the cuvette into the spec, making sure orientation is correct.
15. Press “read sample”.  Record the value.
16. Repeat steps 13-15 until you have values for each of your samples.
17. Press the print button to print your results.

Remember: The next step is to come into the lab to practice with the materials, view a demonstration, or to even just ask questions. Also, if you are ready to take the lab final, please inform your instructor. The lab final will be performed by yourself and will be graded with a rubric.