Lab 1: Serial Dilution Lab

Background

    Serial dilutions are commonly used in many areas of Science to arrive at a desired concentration in a more accurate way than simply using one large dilution (1).  Serial dilutions can be used to create highly diluted solutions and also to reduce the concentrations of organisms or cells in a sample (2).  The method involves removing a small amount of the original solution and bringing that amount up to the original volume using an appropriate buffer or water (1).


Serial Dilution
Fig. 1)  Example of Serial Dilution Protocol (3)

    In Part 1 of this lab, serial dilutions were used to reduce the concentrations of E. coli containing Green Fluorescent Protein.  The  reduction in concentration was believed to correspond to a reduction in the amount of cells contained in the solution, as would be seen when the various concentrations were plated out and when their absorbances were recorded.

Serial Dilution Plates
Fig. 2) Example of a Serial Dilution involving cells (4)

    In Part 2 of the lab, various food colors underwent serial dilutions.  Again, the absorbances were recorded and it was believed that a reduction in concentration would correspond to a reduction in absorbance.

Part 1) Serial Dilution of E. coli with Green Fluorescent Protein
    
               Materials
                      Bel-Art Products Pi Pump                           Loop                                               Spectrometer
                      95% Ethanol                                                Bunsen Burner                               4 Conical Tubes (15mL)
                      Sterile Water                                                Micropipetter                                  10 Serological Pipettes (1/10mL)
                      6 Tubes                                                        Sterile Sparker                                Scraper
                      Rotating Table                                             Glass Petri Dish                              Nutrient Agar Plates

                Procedure
                   1)  Grow up E. coli containing Green Fluorescent Protein on a Nutrient Agar plate.
                   2)  Innoculate a tube containing 4.5mL of sterile water with E. coli colonies and shake gently.
                   3)  Place 0.5mL of the E. coli dilution in a clean tube and add 4.5mL of sterile water.  Shake gently.
                   4)  Repeat dilution process 4 additional times until a 1/100000 dilution is made.
                   5)  Using 1 Nutrient Agar plate for each dilution, place 100uL of dilution in the middle of the plate and spread cells evenly using a sterilized spreader and rotating table.
                   6)  Allow cells to grow for 1 to 2 days.
                   7)  Using a spectrometer at a wavelength of 600nm, determine the number of bacteria cells on each plate.


Part 2) Serial Dilution of Food Dye

                Materials
                      Pipette                                                               Microtiterdish
                      Micropipette (200uL)                                        Sterile Water

                Procedure
                   1)  Measure out 800uL of food dye into first well of microtiterdish.
                   2)  In the second well of microtiterdish, add 100uL of dye from well #1 and 200uL water.
                   3)  In third well, add 100uL of solution from second well and 200uL of water.
                   4)  Repeat dilution well until the twelth well is reached.
                   5)  Use four different wavelengths (405, 570, 492, and 450nm) to measure the dilutions.


Results
    Part 1) Serial Dilution of E. coli
            A) Absorbance:
Concentration
 Absorbance
Initial
 0.579
1/10
 0.012
1/100
 0.003
1/1000
 0.001
1/10000
 0.001
1/100000
 0

         B) Colonies:
Concentration
 Colonies
Initial
 too numerous to count
1/10
 too numerous to count
1/100
 970
1/1000
 405
1/10000
 250
1/100000
 75

   Table 1)  Effects of Serial Dilution on E. coli----A)  Measure of absorbance at 600nm of the various E. coli concentrations
                            B)  Number of colonies present on plates of various E. coli concentrations

  
   
    Part 2) Serial Dilution of Food Coloring

Well 1
 Well 2
 Well 3
 Well 4
 Well 5
 Well 6
 Well 7
 Well 8
 Well 9
 Well 10
 Well 11
 Well 12
405nm
 3.715
 3.558
 3.917
 3.28
 3.164
 2.203
 0.812
 0.301
 0.133
 0.078
 0.083
 0.052
450nm
 --------
 -------
 --------
 --------
 --------
 1.742
 0.622
 0.230
 0.108
 0.070
 0.063
 0.048
492nm
 --------
 -------
 --------
 2.362
 0.890
 0.350
 0.144
 0.079
 0.054
 0.051
 0.055
 0.041
570nm
 --------
 -------
 --------
 --------
 2.105
 0.760
 0.281
 0.122
 0.068
 0.052
 0.053
 0.044
    Table 2)  Absorbance readings at various wavelengths following Serial Dilutions of Food Coloring (Dashed lines correspond to overflow.)



Discussion

    Serial Dilutions are commonly used to reduce the concentrations of solutions or cells.  In this lab, both purposes were carried out.  A solution of food coloring was diluted in a series of twelve steps and than the absorbance was measured.  A higher absorbance reading corresponds to a higher concentration due to turbidity.  The results of this experiment are seen in Table 2.  As we go from Well 1 to Well 12, the concentration of the food coloring decreases.  As seen in Table 2, as the concentration decreases, so do the absorbance readings.  This follows with what was expected.
    Serial Dilutions involving cells were also performed, using E. coli containing Green Fluorescent Protein.  A series of five dilutions were performed and, again, absorbance readings were taken, this time at only one wavelength.  However, in this part, the different concentrations were plated and, after having time to grow, colonies were counted.  As is expected regarding absorbance, a decrease in concentration is to result in a decrease in the number of colonies that grow.  Results are seen in Table 1.  Part A shows the absorbance readings recorded.  As expected, as the concentration decreases, so do the absorbance readings.  As the amount of cells present in the solution decreases, less turbidity is present.  This is what causes a lower absorbance reading.  Part B shows the amount of colonies that successfully grew on the different plates.  Again, the results followed what was expected.  As the concentration decreased, less colonies grew.  If there are a lower number of cells present in a solution, there is less of a chance of something growing.


References

1) http://www.accessexcellence.org/AE/APEC/WWC/1993/serial.html
2) http://en.wikipedia.org/wiki/Serial_dilution
3) http://www.sciencebuddies.org/mentoring/project_ideas/MicroBio_img019.gif
4) http://www.phys.ksu.edu/gene/photos/lab12a.jpg