SERIAL DILUTION,
PIPETTING PRACTICE

©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 methylene blue, serially diluted
   16x    8x      4x       2x     1x
Here is the experimental
plan to perform a 
serial dilution
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3 July 1994, rvsd26July95, 18Sept95, 23July96, 15Oct96, 21July97, 19July98, 18Sept00,14July01, 18July04
 Here are the final dilution
tubes of a serial dilution.
Note relative concentration.

 

    Serial dilutions are regularly used in microbiology when, for instance,initial concentrations of bacteria are orders of magnitude too high to perform a plate count, or for producing a series of regular dilutions as intitering serum. It has two advantages:

  1. It allows for rapid achievement ofa very high dilution factor.
  2. It requires a relatively smallvolumeof diluent.
It involves a sequential series ofdilutions performed as follows:
Experimental plan: 

  1. Equal measured volumes of diluentare placed in each of a labeled series of test tubes.
  2. A small aliquot ofthe 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 orserumfor 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 serial 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
eight 5 mL pipettes in 1000 mL beaker
two 16 x 150 mm tubes, with 7 mL MB solution
wax pencil
one Brinkman Pipetoror 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 blue 1 perstudent 
(A609 = about 1.00)

distilled water diluent in a repipet,set for 3 mL

wipettes

PREPARATIONS:
 

Turn on spectrophotometer to warm up.
Set up your work bench with required equipment.  
Label the test tube with theoriginal solutionof methylene blue "16x."  Set up four empty 16 x 150 mL dilutiontubesin a test tube rack.  Label the tubes 8x, 4x, 2x and 1x toindicate therelative concentration of dye which they will contain.
Aliquot 3.00 mL of dH2O into each of these four labeled dilution tubes, using are pipet.  Here is an alternative style repipet.
Brinkman Pipet Bulb   To use the Brinkman Pipet Bulb (click on the image to the left for a labeled version): 
  1. Squeeze air out of bulb to create a partial vacuum
  2. Insert a pipet into the pipet recepticle
  3. Place tip of pipet well into the liquid
  4. Push lever up to draw fluid above desired calibration line.  DO NOT DRAW FLUID INTO BULB!
  5. Press level down to deliver fluid to new container
  6. Press bulb to blow out last of the liquid from the pipet.

SERIAL DILUTIONS:
 

  Using the pipet bulb, transfer3.00 mL of the original 16x  methylene blue solution from tube #16into tube #8, vortex #8 tube to mix well.
(NOTE:  If using a 5 mL pipet, 3 mL are contained when themeniscusis just touching the 2 calibration line.  You should be leavingabout4 mL in tube #16.
After vortexing, use a cleanpipet to withdraw3.00 mL from tube #8, add it to #4. Mix as before, using a vortex.
Using a clean pipet, withdraw3.00 mL fromtube #4, add it to #2. Mix as before.  Here the class isperforming severalof the stages of the serial dilution.
Using a clean pipet, withdraw3.00 mL fromtube #2, add it to #1. Mix as before.  When dilutions are done, 3mLshould 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 yourway up.  In this way, you need not wash the cuvette each time, but touch off the last drop before adding the next dilution.
Graph of A609s of dilutions series Plota graph with the relative concentration of methylene blue(indicated by the tubenumber) as the ordinate (X axis) and absorbency at 609 nm as theabscissa(Y axis). Use the blank tube (zero methylene blue with an A609= 0.000) as your first (zero) point.

[It is puzzling that this curvedoesnot strictly adhere to Beer's law (it should be linear), as ifconcentrated methylene blue absorbs more proportionately thandilute.  Do YOU know why this descrepency?]

1Stock solution of methylene blueis 0.3%: Dilute it 0.166 mL into 100 mL in dH2O to produce ~A609of 1.000.

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