SERIAL DILUTION, PIPETTING PRACTICE
©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
             University of Cincinnati Clermont College,
Batavia OH 45103
23 June 1989, rvsd 18 July 1994, 17 July '95, 18 July '97, 1 July 98, 1 July 99
This page has been accessed times since 18 July 2001.

Serial dilutions are regularly used in microbiology when initial concentrations of bacteria are much too high to perform a plate count, or for producing a series of regular dilutions as in titering serum. It involves a sequential series of dilutions performed as follows: equal measured volumes of diluent are placed in each of a labeled series of test tubes. A small aliquot of the specimen sample is placed in the first tube and mixed. A small aliquot of that dilution is removed with a fresh pipet and added to the second tube. The second tube is then mixed, and an aliquot from it is transferred to the third tube in like manner. The process is continued until the series of dilutions has been completed (a serial dilution). Notice that the concentration decreases exponentially as the dilution series progresses (in the following example, the relative concentrations are 16, 8, 4, 2, and 1.) Dilutions of antibodies or serum for titering are prepared in much the same fashion.

See handout on Dilutions for in-depth explanation of dilutions and sample problems. The handout on sterile delivery with pipets describes pipette use.

Illustrate the following dilution process in your notebook with labeled tubes and volumes involved so that you fully understand what you will be doing before you begin the exercise.

EQUIPMENT AND SUPPLIES per table of two students, each performing expt:
 

eight 16 x 150 mm tubes two test tube racks (larger, fingered) ten 5 mL pipettes in 1000 mL beaker 125 mL flask with 30 mL dH2O two 16 x 150 mm tubes, with 7 mL of:         0.0005 % methylene blue1 each  (A609 = about 1.00)  wax pencil

1 Brinkman Pipetor or pipet bulb one vortex mixer one spectrophotometer, warmed up two cuvettes in plastic test tube rack:     Blank with 3 mL dH2O (marked "B")     Sample (marked "S") wipettes used pipet receptacle (plastic is best)

PREPARATIONS:

Label the test tube with the stock solution of methylene blue "16x." Set up four empty 16 x 150 mL tubes and label 8x, 4x, 2x and 1x to indicate the relative concentration of dye they will contain. Aliquot 3.00 mL of dH₂O into each of these.

1. Add 3.00 mL of the initial methylene blue solution from #16 into tube #8, vortex to mix well. (You should be leaving about 4 mL in tube #16.)  When dilutions are done, 3 mL should remain in each of these tubes, except the last:
2. Using a clean pipet, withdraw 3.00 mL from tube #8, add it to #4. Mix as before.
3. Using a clean pipet, withdraw 3.00 mL from tube #4, add it to #2. Mix as before.
4. Using a clean pipet, withdraw 3.00 mL from tube #2, add it to #1. Mix as before.

Read the A₆₀₉ of each dilution, and plot on a graph with the relative concentration of methylene blue (indicated by the tube number) as the ordinate (X axis) and absorbency being the abscissa (Y axis). Use the blank tube (zero methylene blue and A₆₀₉ = 0.000) as your first (zero) point.