Serial Dilution

Definition
A method used to stepwise dilute substance into solution with constant dilution factor in
each step.

Application
Experimental sciences include biochemistry, pharmacology, and physics as well as in
homeopathy.

Difficulty
Procedure: Medium

Concept: Easy

Concept
Serial dilution is the stepwise dilution of a substance in solution. Usually the dilution
factor at each step is constant, resulting in a geometric progression of the concentration
in a logarithmic fashion.

Serial dilutions are used to accurately create highly diluted solutions as well as solutions
for experiments resulting in concentration curves with a logarithmic scale.
 Serial Dilution

Besides the more conventional uses described above, serial dilution may also be used
to reduce the concentration of microscopic organisms or cells in a sample.

A tenfold dilution for each step is called a logarithmic dilution or log-dilution and a 3.16-
fold (100.5-fold) dilution is called a half-logarithmic dilution or half-log dilution.

Materials
 Buffer used to dissolve the sample
 The sample
 Multiple tubes
 Pipette or graduated cylinder
 Stirring rod

Procedure
  Shake the solution by hand or use the stirring rod to swirl
the solution. Make sure the solution is uniformly mixed.
 Take half of the solution out to a new tube and add equal
amount of buffer into it to dilute the solution concentration
to half of the original solution.
 Take half of the newly made solution to another new tube
and add equal amount of buffer into it to dilute the
solution concentration to one quarter of the original
solution.
 Continue the process until reaching the desired
concentration of the solution.

Analysis
Serial dilution is a process of solution preparation. Therefore, analysis is not necessary.

Title:  Serial Dilutions

Introduction:

A serial dilutions is a technique of proportionally diluting a stock solution.  Each dilution ensures
that there is a differing concentration of the stock solution in the different tubes.  In this lab we
will be preforming a 1/10th serial dilution which means that each proceeding dilution will be
1/10 the concentration of the of the solution in which the sample has come from.
The viable spectrum of colors ranges from violet at around 400nm all the way up to red at
800nm.  The colors that we see in objects are directly related to the amount of light that they
absorb.  As seen in the figure below an absorbency of 440-490nm would relate to an object that
would appear to be blue in color where as something that absorbs 585-620nm of light would be
orange.  In this lab we will be using food coloring dyes in differing concentrations to see what
kind of absorbance that will produce.  The different concentrations will be produced by
preforming a 1/3 serial dilution of the original food coloring dye.

Materials:
  Eppendorf micro pippeter
 Bel-Art products large pippeter
 VWR mini vortexer
 Mini loop
 Screw top 15ml tubes
 E. coli solution
 Water
 Food coloring
 Bunsen burner
 Test tubes
 SLT Spectrometer
 Spreader
 95% ethanol
 NA plates
 Micotiter dish reader
 Precision incubator

Methods:
    Part I:  E. coli 1/10 serial dilution.

1. Make stock solution with 9ml or water and ecoli with GFP.
2. Take 1ml of stock solution and add 9ml of water to new test tube #1.
3. Take 1ml of solution from test tube #1 and add that to 9ml of water into test tube #2.
4. Take 1ml of solution from test tube #2 and add that to 9ml of water into test tube #3.
5. Take 1ml of solution from test tube #3 and add that to 9ml of water into test tube #4.
6. Take 1ml of solution from test tube #4 and add that to 9ml of water into test tube #5.
7. Take 1ml of solution from test tube #5 and add that to 9ml of water into test tube #6.
8. Vortex each dilution tube.
9. Find the absorbance of each tube.
10. Streak out 100 microliters of solution from tubes #4,#5,#6 onto an 3 separate agar plates.
11. Spread out solution on the plates using ethanol and flame sterilized spreader.
12. Let the plates grow over night and then count the number of colonies on each pate.

    Part II:  1/3 dilution of food coloring dyes

1. Make 1/3 dilution of green food coloring dye for 10 wells.  Perform this by taking 1micro
liter of each proceeding dilution and add that to 2 microliters of water.
2. Repeat step 1 for both yellow and blue food coloring.
3. Find the absorbance of each well for the 3 colors used.

Results/Discussions:

Part I:
    
Table 1: 10 Fold Dilution    
Stock Stock
.10 Dilution
#1
.01 Dilution
#2
.001 Dilution
#3
.0001 Dilution
#4
.00001 Dilution
#5
.000001 Dilution
#6

  Table 2: Number of Ecoli Colonies Per Dilution

Stock To many to count
Dilution # 1 To many to count
Dilution # 2 To many to count
Dilution # 3 To many to count
Dilution # 4 93 colonies
Dilution # 5 51 colonies
Dilution # 6 10 colonies

    The results from experiment one did not show a perfect serial dilution, the colony numbers per
plate were not exactly 1/10.  The numbers of colonies per plate however did decrease with each
dilution.  By dilution number 6 there were only 10 colonies on the plate compared to the 51 of
dilution #5 and the uncountable  numbers found on the plates that represented dilutions 1,2,3,
and the stock. 

 Tabel 3: Absorbency of Each E. coli Per Dilution

Stock (7) .125nm
Dilution #1 .016nm
Dilution #2 .007nm
Dilution #3 .005nm
Dilution #4 .002nm
Dilution #5 .001nm
Dilution #6 .003nm
Figure 1:  E. coli absorbance

    The aborbance readings from each dilution also did not reveal a perfect 1/10 serial dilution. 
The only dilution that came close to 1/10 was dilution #1.  This dilution had about 12% the
absorbance of the original stock solution, a perfect 1/10 dilution would have been 10%. The rest
of the dilutions did not have proportional 1/10th absorbance change.  The unproportional
absorbance differences between the dilutions could have been a result of a poor serial dilution
technique or from faulty readings from the spectrometer.  The tubes used for the spectrometer
could have been dirty or had a film on them throwing the absrobance readings off.

Figure 2: Absorbance of Food Coloring Dyes.

The amount of light that each color absorbs decreases with each 1/3 dilution made to the specific
food coloring dyes.  In the normal color spectrum green color will be produced when roughly
490nm of light has been absorbed, yellow is found to be around 570nm, and blue is about
440nm.

References: