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Lactose Influence on
Bacterial Metabolism Aidan Towsley
Grade 11
Pittsburgh Central Catholic High School
Problem
How do lactose, glucose, and galactose
affect the growth rates of Escherichia coli
(E.coli) Staphylococcus epidermidis
(Staph)?
E. Coli Metabolism
Background
• E. Coli obtain carbon based foods from their
environment.
• Glucose and galactose are converted to pyruvate
through the EMP pathway as part of the Central
Metabolism of E.coli.
• The Lac Operon regulates metabolism of lactose
while no other carbohydrates are in the system.
Staph. Metabolism
Background
• Staph. metabolize glucose mainly through
glycolysis.
• Staph. can use glucose and lactose to form acid
products.
• Capable of metabolizing glucose, galactose, and
lactose anaerobically through fermentation.
Gram (-) vs Gram (+) Bacteria
• Cell wall contains an
extra layer of
lipopolysaccharides
for extra protection.
• Outer membrane
protects bacteria
from several
antibiotics.
• Most pathogenic
bacteria in humans are
Gram (+) organisms.
• Simple cell wall.
• Some antibiotics work
against the formation of
the cell wall.
Staphylococcus epidermidis
(Staph)• Common surface symbiont in many mammals
(Human).
• Gram (+) bacteria
• Most strains considered non-pathogenic.
• Pathogenic strains can be life-threatening
Escherichia coli (E.coli)• Large and diverse group of gram (-) bacteria
• Free living, symbionts, or pathogens
• Live in the intestinal tract of many mammals.
• Most strains are not pathogenic
• Serve as a common prokaryotic cell model.
Purpose
• To analyze the growth rates of
Staphylococcus epidermidis and
Escherichia coli under the influence of
lactose, glucose, and galactose.
Hypotheses
• Null Hypothesis: Glucose, galactose, and lactose
will not vary significantly in their effect on
bacterial growth rates.
• Alternate Hypothesis: Glucose, galactose, and
lactose will cause significant variation in their
effect on bacterial growth rates.
Materials • Glucose, Galactose, Lactose
solutions
• Micro and Macro pipettes +
sterile tips
• Escherichia coli (functional
Lac-Z), Escherichia coli
(Non-functional Lac-Z),
Staphylococcus epidermidis
• Burner
• 32 Sidearm Flasks
• 2 Incubators
• Tinfoil
• SDF (Sterile Dilution Fluid)
• Lambda broth (Minimal Media)
• Ethanol
• Klett Spectrophotometer
• Gloves/ Goggles
Procedure 1. Bacteria (E.coli and Staph) were grown overnight in sterile
Lambda Media.
2. SDF and Lambda media were sterilized for use via a
burner.
3. Blanks were created for bacteria growing in flasks of SDF
and lambda.
4. 16 flasks of E.coli (non-functional Lac-Z) containing the
appropriate solutions were created for both the SDF and
Lambda media. (4 control, 4 glucose, 4 galactose, 4
lactose)
Blank Solutions
Lambda
Media9mL
SDF 1mL
Lambda
Media.5mL
SDF 9.5mL
Lambda
BlankSDF Blank
Flask Solutions
Lambda
Media/SDF 8.5mL
Carbohydrate 1.0mL
Microbe .5mL
Total Volume 10mL
Lambda
Media/SDF8.5mL
SDF 1mL
Microbe .5mL
Total Volume 10mL
Carbohydrate Flasks Control Flasks
Procedure5. After creating a total of 32 total flasks of E.coli (non-functional Lac-
Z), the turbidity was measured and recorded using a Klett
spectrophotometer.
6. The flasks were placed into incubators set to 37 degrees Celsius.
7. After 30 minutes the flasks were removed from the incubators and
measured for changes in growth. The flasks were placed back into
the incubator immediately after measuring. This step was repeated
until 360 minutes had passed. Note: The flasks were removed and
measured in the same order each time to avoid lag time.
8. After all data was collected for E.coli (non-functional Lac-Z), the
procedure was repeated for Staph. and E.coli (functional Lac-Z)
Carbohydrate Influence on
E.coli and Staph
0
20
40
60
80
100
120
140
160
180
0 30 60 90 120 150 180 210 240 270 300 330 360
Kle
ttR
ead
ing
(T
urb
idit
y)
TIme
Carbohydrates’ Effect on E.coli and Staph
Control E.Coli Non-Func Lambda Lactose E.coli Non-Func Lambda Galactose E.Coli Non-Func Lambda Glucose E.coli Non-Func Lambda
Control E.coli Non-Func SDF Lactose E.coli Non-Func SDF Galactose E.coli Non-Func SDF Glucose E.coli Non-Func SDF
Control Staph SDF Lactose Staph SDF Galactose Staph SDF Glucose Staph SDF
Control Staph Lambda Lactose Staph Lambda Galactose Staph Lambda Glucose Staph Lambda
Control E.coli Functional Lambda Lactose E.coli Functional Lambda Galactose E.coli Functional Lambda Glucose E.coli Functional Lambda
Control E.coli Functional SDF Lactose E.coli Functional SDF Galactose E.coli Functional SDF Glucose E.coli Functional SDF
Dunnett’s TestE.Coli Non-
Functional Lambda
T-Crit: ~3.29
Lactose 2.71
Galactose 1.87
Glucose .916
E.Coli Non-
Functional
SDF
T-crit ~3.29
Lactose .110
Galactose 9.73
Glucose 14.3
E.Coli Functinal
Lambda
T-crit ~3.29
Lactose 2.37
Galactose 4.88
Glucose 6.17
E.Coli Functional
SDF
T-Crit ~3.29
Lactose 11.31
Galactose 13.79
Glucose 15.96
Dunnett’s TestStaph
SDF
T-crit ~3.29
Lactose 6.09
Galactose 12.27
Glucose 6.17
Staph Lambda
T-crit ~3.29
Lactose 1.90
Galactose 2.25
Glucose 1.70
Conclusions
• Reject the Null Hypothesis for all
solutions expect the staph grown in
lambda media.
• Almost all functional E.coli found
significantly different from control
compared to the couple found in non-
functional E.coli and Staph.
Limitations
• Time of exposure in incubator could
have been slightly different.
• Only 4 flask for each set were tested.
• Accuracy of Klett Spectrophotometers
used.
Future Studies
• Use different sources of
energy/carbohydrates.
• Test various cell models.
• Vary exposure times in incubator.
Works Cited• http://www.ncbi.nlm.nih.gov/pubmed/1311956
• https://www.wikigenes.org/e/mesh/e/149.html
• http://www.ndsu.edu/pubweb/~mcclean/plsc431/prokaryo/prokaryo2.htm
• http://www.phschool.com/science/biology_place/biocoach/lacoperon/intro.html
• Muller-Hill, Benno. The Lac Operon: A Short History of a Genetic Paradigm. Berlin: Walter De Gruyter, 1996. Print.
Anova Non-Func E.coli LambdaAnova: Single
Factor
P-value < 0.05
= Significant
SUMMARY
Groups Count Sum Average Variance
Control 4 530 132.5
251.666666
7
Lactose 4 456 114 22
Galactose 4 581 145.25
86.9166666
7
Glucose 4 505 126.25
11.5833333
3
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 2031.5 3
677.166666
7
7.27810120
9 0.004867377
3.49029
4819
Within Groups 1116.5 12
93.0416666
7
Total 3148 15
Anova Non-Func E.coli SDFAnova: Single Factor
P-value <
0.05 =
Significant
SUMMARY
Groups Count Sum Average Variance
Control 4 87 21.75 4.25
Lactose 4 86 21.5 1.666666667
Galactose 4 175 43.75 16.91666667
Glucose 4 216 54 18
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 3180.5 3 1060.166667 103.8530612 7.50938E-09 3.490294819
Within Groups 122.5 12 10.20833333
Total 3303 15
Anova Func E.coli LambdaAnova: Single Factor
P-value <
0.05 =
Significant
SUMMARY
Groups Count Sum Average Variance
Control 4 354 88.5
4.33333333
3
Lactose 4 411 102.75
60.9166666
7
Galactose 4 451 112.75
40.9166666
7
Glucose 4 502 125.5
181.666666
7
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups
2940.2
5 3
980.083333
3
13.6201505
5
0.00036016
7
3.49029481
9
Within Groups 863.5 12
71.9583333
3
Total
3803.7
5 15
Anova Func E.coli SDFAnova: Single Factor
P-value <
0.05 =
Significant
SUMMARY
Groups Count Sum Average Variance
Control 4 131 32.75
6.91666666
7
Lactose 4 345 86.25
32.9166666
7
Galactose 4 392 98
86.6666666
7
Glucose 4 433 108.25
52.9166666
7
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups
13547.187
5 3
4515.72916
7
100.675801
2
8.98059E-
09
3.49029481
9
Within Groups 538.25 12
44.8541666
7
Total
14085.437
5 15
Anova Staph LambdaAnova: Single
Factor
P-value <
0.05 =
Significant
SUMMARY
Groups Count Sum Average Variance
Control 4 308 77
64.6666666
7
Lactose 4 419 104.75
1103.58333
3
Galactose 4 439 109.75 426.25
Glucose 4 407 101.75
99.5833333
3
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups
2553.187
5 3 851.0625
2.00949382
7
0.16642703
3
3.49029481
9
Within Groups 5082.25 12
423.520833
3
Total
7635.437
5 15
Anova Staph SDFAnova: Single Factor
P-value <
0.05 =
Significant
SUMMARY
Groups Count Sum Average Variance
Control 4 33 8.25 5.583333333
Lactose 4 113 28.25 42.25
Galactose 4 194 48.5 17
Glucose 4 114 28.5 21.66666667
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 3240.25 3 1080.083333 49.9460501 4.71621E-07 3.490294819
Within Groups 259.5 12 21.625
Total 3499.75 15
Data Non-Func E.coliNon-Functional E.Coli Lambda Non-Functional E.Coli SDF
Control Control
Time Flask 1 Flask 2 Flask 3 Flask 4 Time Flask 1 Flask 2 Flask 3 Flask 4
0 10 10 18 13.5 0 20 13 21 16
30 10 10 19 15 30 20 12 21 16
60 21 20 26 28.5 60 21 15 23 17
90 35 33 37 36 90 25 17 25 18
120 49 48 49 51 120 23 17 25 19
150 58 62 60 65 150 21 16 21 19
180 69 73 69 81 180 26 20 23 22
210 80 81 77.5 103 210 25 18 22 20
240 90 89 89 112 240 24 19 23 21
270 103 105 99 121 270 24 21 22 21
300 112 116 108 134 300 24 20 24 22
330 119 121 119 142 330 23 20 25 23
360 124 128 122 156 360 23 20 24 20
Lactose Lactose
Time Flask 1 Flask 2 Flask 3 Flask 4 Time Flask 1 Flask 2 Flask 3 Flask 4
0 12 10 19 9.5 0 14 14 17 23
30 12 12 20 9.5 30 15 15 15 21
60 15 22 32 21 60 17 16 19 23
90 34 29 43 32 90 17 19 21 25
120 45 43 53 43 120 18 19 20 23
150 55 50 63 52 150 18 19 19 22
180 71 61 74 63 180 19 19 20 24
210 80 71 84 68 210 20 20 21 24
240 91 79 89 77 240 20 20 21 26
270 96 90 100 90 270 21 21 21 25
300 102 97 106 96 300 22 22 23 25
330 110 114 114 104 330 20 22 21 25
360 116 108 119 113 360 20 21 22 23
Data Non-Func E.coliGalactose Galactose
Time Flask 1 Flask 2 Flask 3 Flask 4 Time Flask 1 Flask 2 Flask 3 Flask 4
0 18 17 11 18 0 15 17 17 13
30 18 18 13 18 30 13 15 16 14
60 33 29 23 27 60 18 19 20 17
90 42 36 33 40 90 21 24 24 20
120 55 49 48 54 120 27 28 28 25
150 72 68 66 72 150 27 32 30 26
180 91 85 84 86 180 31 36 36 30
210 112 99 98 101 210 31 38 34 31
240 125 109 105 110 240 36 43 38 33
270 135 119 117 119 270 36 44 39 34
300 146 131 127 127 300 40 45 42 36
330 151 138 137 134 330 40 49 41 37
360 159 143 140 139 360 43 49 44 39
Glucose Glucose
Time Flask 1 Flask 2 Flask 3 Flask 4 Time Flask 1 Flask 2 Flask 3 Flask 4
0 12 15 14 14 0 21 17 15 21
30 12 16 15 14 30 21 18 15 21
60 25 20 25 27 60 26 21 21 25
90 39 38 36 37 90 30 26 26 32
120 57 55 53 59 120 31 30 30 39
150 76 77 76 76 150 37 36 34 38
180 92 96 95 93 180 47 44 38 44
210 99 105 102 101 210 51 45 40 43
240 105 112 111 110 240 50 49 43 47
270 111 121 121 113 270 53 49 46 47
300 118 125 125 121 300 58 52 48 50
330 122 126 129 126 330 58 53 48 49
360 122 129 129 125 360 59 56 50 51
Data Func E.coliE.Coli Lamda Functional
E.Coli SDF
Functional
Control Control
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 4 13 14 7 0 5 5 6 6
30 4 11 12 4 30 5 3 4 5
60 6 15 15 8 60 6 5 6 6
90 8 21 17 12 90 11 8 10 10
120 23 35 27 22 120 15 12 12 13
150 30 47 37 32 150 20 16 16 16
180 44 57 48 46 180 23 21 21 22
210 49 66 55 50 210 23 21 20 22
240 63 77 65 63 240 28 25 25 26
270 73 80 72 66 270 31 28 28 30
300 78 85 80 72 300 32 29 29 33
330 84 89 86 79 330 34 29 30 35
360 86 91 88 89 360 35 31 30 35
Lactose Lactose
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 13 12 14 9 0 9 9 11 7
30 13 11 13 8 30 7 10 12 9
60 18 15 18 14 60 7 10 15 13
90 22 20 23 22 90 12 17 20 19
120 33 28 32 32 120 19 23 25 25
150 43 42 40 42 150 26 32 34 39
180 52 61 50 64 180 36 43 48 50
210 67 70 60 72 210 43 49 52 60
240 82 79 77 77 240 53 59 63 74
270 85 81 85 82 270 67 71 73 79
300 99 85 88 90 300 71 81 81 82
330 106 93 93 97 330 75 87 85 84
360 114 101 96 100 360 78 91 87 89
Data Func. E.coliGalactose Galactose
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 7 16 7 8 0 6 6 10 7
30 7 15 6 17 30 9 8 12 8
60 11 18 11 25 60 12 12 17 15
90 17 23 17 38 90 18 18 24 20
120 25 33 28 44 120 21 21 35 29
150 35 41 33 57 150 32 32 44 39
180 55 52 46 71 180 43 41 53 48
210 65 67 62 78 210 48 45 60 55
240 76 79 76 84 240 57 58 70 65
270 85 87 86 88 270 66 68 80 77
300 97 95 94 94 300 76 74 94 87
330 106 104 98 100 330 87 81 100 94
360 120 116 106 109 360 95 87 109 101
Glucose Glucose
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 8 30 20 18 0 7 6 7 7
30 7 29 19 20 30 9 8 10 8
60 13 41 28 30 60 15 12 17 16
90 20 60 42 46 90 20 18 24 22
120 28 71 61 60 120 27 26 33 32
150 36 85 70 73 150 37 40 49 48
180 55 98 84 92 180 48 48 50 60
210 72 98 85 96 210 55 58 70 70
240 84 107 92 108 240 67 67 79 81
270 88 116 101 121 270 77 77 85 89
300 93 127 114 127 300 85 86 94 98
330 98 130 121 131 330 94 97 103 103
360 106 135 127 134 360 101 103 115 114
Data Staph Staph SDF Staph Lambda
Control Control
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 7 4 7 5 0 4 4 6 7
30 7 4 6 5 30 5 5 6 6
60 7 4 6 5 60 5 6 6 6
90 6 4 6 4 90 8 12 14 6
120 4 2 5 4 120 13 19 18 7
150 5 3 5 4 150 18 25 26 20
180 5 2 6 5 180 24 33 35 34
210 6 3 6 5 210 27 43 44 36
240 7 3 7 6 240 37 55 53 44
270 8 3 8 6 270 45 65 59 57
300 8 4 8 7 300 58 76 66 63
330 8 4 9 9 330 67 81 69 71
360 8 5 10 10 360 74 89 73 72
Lactose Lactose
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 5 7 8 12 0 8 6 7 4
30 5 6 6 11 30 8 7 8 5
60 5 6 6 10 60 8 7 8 5
90 5 6 6 10 90 14 13 15 7
120 5 5 5 9 120 22 19 19 11
150 6 6 7 12 150 30 26 24 16
180 6 7 8 14 180 45 35 33 22
210 8 11 10 17 210 56 50 45 29
240 11 13 14 20 240 74 60 53 38
270 16 15 15 26 270 90 78 61 45
300 21 18 17 29 300 125 94 71 53
330 24 24 19 34 330 131 110 80 58
360 29 25 22 37 360 142 120 91 66
Data StaphGalactose Galactose
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 8 9 13 8 0 5 6 2 7
30 7 8 12 7 30 6 6 2 7
60 7 8 12 7 60 7 6 2 6
90 8 8 12 7 90 12 9 9 11
120 9 8 11 7 120 18 13 15 15
150 15 9 12 11 150 28 21 16 21
180 18 11 14 12 180 35 30 18 28
210 26 13 18 17 210 56 45 27 29
240 34 20 22 21 240 67 51 35 34
270 37 26 29 28 270 79 60 50 60
300 46 28 35 31 300 115 77 66 84
330 50 35 39 37 330 123 94 71 91
360 54 44 48 48 360 133 105 84 117
Glucose Glucose
Time Trial 1 Trial 2 Trial 3 Trial 4 Time Trial 1 Trial 2 Trial 3 Trial 4
0 12 13 10 15 0 4 6 3 4
30 12 12 10 14 30 4 7 3 4
60 12 11 10 12 60 4 7 3 4
90 11 11 10 13 90 5 12 5 7
120 11 12 10 13 120 6 16 6 9
150 12 12 22 14 150 14 30 9 25
180 13 12 11 14 180 19 43 16 33
210 16 15 14 16 210 40 60 26 54
240 18 18 16 18 240 51 72 38 69
270 21 18 16 19 270 60 91 54 75
300 21 18 17 21 300 79 101 68 91
330 27 21 20 28 330 80 104 76 104
360 32 25 24 33 360 97 109 90 111
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