SERIAL DILUTION, PIPETTING PRACTIC

©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 
This page has been accessed Counter times since 18 July 2001. 

3 July 1994, rvsd 26July95, 18Sept95, 23July96, 15Oct96, 21July97, 19July98, 18Sept00, 14July01, 18July04
 

    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 has two advantages:  it allows for achievement of a very high dilution factor, and it requires a relatively small volume of diluent.  It involves a sequential series of dilutions performed as follows:

  1. equal measured volumes of diluent are placed in each of a labeled series of test tubes.
  2. A small aliquot of the specimen sample is placed in the first tube and mixed.
  3. A small aliquot of that dilution is removed with a fresh pipet and added to the second tube.
  4. The second tube is then mixed, and an aliquot from it is transferred to the third tube in like manner.
  5. 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.
 

Per table of two students, each performing his or her own experiment:
 

EQUIPMENT SUPPLIES
eight 16 x 150 mm tubes
two test tube racks (larger, fingered)
ten 5 mL pipettes in 1000 mL beaker
one 125 mL flask with 30 mL dH2O
two 16 x 150 mm tubes, with 7 mL
wax pencil
one 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")
one used pipet receptacle (plastic is best)		 7 mL of 0.0005 % methylene blue1 per student 
(A609 = about 1.00)

distilled water diluent in repipet, set for 3 mL

wipettes

PREPARATIONS:
 

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

SERIAL DILUTIONS:
 

Add 3.00 mL of the 16x (nitial) methylene blue solution from tube #16 into tube #8, vortex #8 tube to mix well. (You should be leaving about 4 mL in tube #16.)  
Using a clean pipet, withdraw 3.00 mL from tube #8, add it to #4. Mix as before.
Using a clean pipet, withdraw 3.00 mL from tube #4, add it to #2. Mix as before.
Using a clean pipet, withdraw 3.00 mL from tube #2, add it to #1. Mix as before.  When dilutions are done, 3 mL should remain in tubes #8, #4 and #2.  Tube #1 should contain 6 mL.

READ AND PLOT THE ABSORBENCY OF THE DILUTION SERIES:
 

Read the A609 of each dilution against a blank of distilled water.  Begin with tube #1, and work your way up.  In this way, you need not wash the cuvette each time, but touch off the last drop before adding the next dilution.
Plot a graph with the relative concentration of methylene blue (indicated by the tube number) as the ordinate (X axis) and absorbency at 609 nm as the abscissa (Y axis). Use the blank tube (zero methylene blue with an A609 = 0.000) as your first (zero) point.

1 Stock solution of methylene blue is 0.3%: Dilute it 0.166 mL into 100 mL in dH2O.