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Dilution Tutorial and Problems

The document provides two methods for calculating the concentration or titer of bacterial cultures from dilution plating experiments. Method 1 uses the average number of colonies counted from replicate plates and the dilution factors to calculate the original concentration through stepwise applications of the dilution equation. Method 2 takes a pictorial approach by representing each dilution as a fraction and calculating an overall dilution factor called the plating factor, which is used along with average colonies to determine the original concentration. The document emphasizes that either method can be used and method 2 is commonly applied in microbiology, where the plating factor term is important to know.

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Dilution Tutorial and Problems

The document provides two methods for calculating the concentration or titer of bacterial cultures from dilution plating experiments. Method 1 uses the average number of colonies counted from replicate plates and the dilution factors to calculate the original concentration through stepwise applications of the dilution equation. Method 2 takes a pictorial approach by representing each dilution as a fraction and calculating an overall dilution factor called the plating factor, which is used along with average colonies to determine the original concentration. The document emphasizes that either method can be used and method 2 is commonly applied in microbiology, where the plating factor term is important to know.

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© © All Rights Reserved
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Dilution Tutorial

Method 1:

First, consider that the number of colonies on each plate indicates the number of bacterial cells that were in the final 0.1
mL aliquot that was spread on the plate. Each of the three plates shown above were spread with the same volume of
liquid (0.1 mL) from Bottle 3, so we can average the number of colonies on the three plates:
90 + 106 + 98 = 294 colonies
294 colonies / 3 plates = 98 colonies per plate
Remember this is also the average number of cells per 0.1 mL of fluid taken from Bottle 3. Thus, the concentration, or
titer, of Bottle 3 can be calculated:
98 cells / 0.1 mL = 980 cells/mL in Bottle 3
In order to figure the titer of Bottle 2, we must recall lessons from General Chemistry and the equation
C1V1 = C2V2, where C1 is the concentration of the more concentrated solution, V1 is the volume taken out of that
solution, C2 is the concentration of the more dilute solution, and V2 is the final total volume of that solution.
Lets manipulate this equation to apply to our current situation and allow us to calculate the concentration of Bottle 2:
CBottle 2Vremoved from Bottle 2 = CBottle 3Vfinal in Bottle 3
(CBottle 2)(11 mL) = (980 cells/mL)(110 mL)
CBottle 2 = 9.8 x 10^3 cells/mL
We can use this same equation to determine the titer in Bottle 1 and finally in Culture A:
CBottle 1Vremoved from Bottle 1 = CBottle 2VFinal in Bottle 2
(CBottle 1)(1 mL) = (9.8 x 10^3 cells/mL)(100 mL)
CBottle 1 = 9.8 x 10^5 cells/mL
CCulture AVremoved from Culture A = CBottle 1Vfinal in Bottle 1
(CCulture A)(1 mL) = (9.8 x 10^5 cells/mL)(100 mL)
CCulture A = 9.8x 10^7 cells/mL

Method 2:
This method uses more of a pictorial approach.
Step 1:
The first step of this method is to write each dilution as a fraction with the volume transferred out of the more
concentrated solution over the total volume of the less concentrated solution. In the final transfer, 0.1 mL is transferred
out of the more concentrated solution and put directly on the plate. This is a 1/10 dilution.

Step 2:
In the second step of this method, the fractions are converted to scientific notation.

Step 3:
In the third step, the product of the individual dilution factors is calculated to give the final dilution factor:
(1 x 10-2)(1 x 10-2)(1 x 10-1)(1 x 10-1) = Final dilution factor
Step 4:
In microbiology, the reciprocal of the final dilution factor is called the plating factor. In step 4, the plating factor is
calculated.
Plating Factor (p.f.) = 1/(1 x 10-6)
Plating Factor (p.f.) = 1 x 106
Step 5:
Finally, to determine the concentration of Culture A, the average number of colonies (as calculated in method 1) is
multiplied by the plating factor.

p.f. x average number of colonies = titer of original culture

(1 x 106)(98) = 9.8 x 107 bacteria/mL


Please feel free to use either method to determine titer of original cultures. Method is more concise and more clearly
tracks units. In microbiology, method 2 is commonly used and, even if you select method 1, it will be necessary to be
familiar with the term plating factor.
Significant figures:
Experimentally, the number of significant figures that can be retained is determined by the accuracy of the measuring
devices selected. Consider the 5 mL pipet shown in the figure above. Three significant figures could be retained when
using this device because two figures can be read and the third is allowed to have uncertainty. Practice finding 4.55 mL on
this pipet. In problems that are not encountered in the lab (such as the dilution problems assignment), retain the number
of significant figures you are given.

Name:

_______________________________________________

Lab

Section:

Dilution Problems

____________________

1. (1 point) Use the following dilution scheme to determine the titer (bacteria/mL) of culture A.
1 mL

11 mL

86

11 mL

colonies

94

colonies

Culture A

99
mL

99
mL

99
mL

Bottle 1

Bottle 2

Bottle 3

72

colonies

2. (2 points) Use the following dilution scheme to determine the titer (bacteria/mL) of culture B.
11 mL

1 mL

1 mL

125

1 mL

colonies

156

colonies

Culture B

99
mL

4
mL

4
mL

9
mL

Bottle 1

Bottle 1

Bottle 2

Bottle 3

144

colonies

3. (2 points) Culture C has a titer of 2.3 x 107 bacteria/mL. Draw a dilution scheme showing 1 method in which this
culture could be diluted to give plates with between 30 and 300 colonies.

4. (1 point) Use the following dilution scheme to determine the titer (bacteria/mL) of culture D.
1 mL

2 mL

2 mL

colonies

59

colonies

Culture D

99
mL

99
mL

99
mL

Bottle 1

Bottle 2

Bottle 3

54

colonies

5. (2 points) Use the following dilution scheme to determine the titer (bacteria/mL) of culture E.
1 mL

222

1 mL

colonies

199

colonies

99
mL
Culture E

Bottle 2

99
mL
Bottle 3

169

colonies

6. (2 points) Culture F has a titer of 1,555,233 bacteria/mL. Draw a dilution scheme showing 1 method in which this
culture could be diluted to give plates with between 30 and 300 colonies.

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