Lactose Influence on Bacterial Metabolism science... · E. Coli Metabolism Background • E. Coli obtain carbon based foods from their environment. • Glucose and galactose are converted

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.

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

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


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


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


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


Between Groups

2940.2

5 3

980.083333

3

13.6201505

5

0.00036016

7

3.49029481

9


71.9583333

3

Total

3803.7

5 15

Anova Func E.coli SDFAnova: Single Factor

P-value <

0.05 =

Significant

SUMMARY


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


Between Groups

13547.187

5 3

4515.72916

7

100.675801

2

8.98059E-

09

3.49029481

9


44.8541666

7

Total

14085.437

5 15

Anova Staph LambdaAnova: Single

Factor

P-value <

0.05 =

Significant

SUMMARY


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


Between Groups

2553.187

5 3 851.0625

2.00949382

7

0.16642703

3

3.49029481

9


423.520833

3

Total

7635.437

5 15

Anova Staph SDFAnova: Single Factor

P-value <

0.05 =

Significant

SUMMARY


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


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


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


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


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


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


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


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


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


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


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


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

Documents

Lactose Influence on Bacterial Metabolism science... · E. Coli Metabolism Background • E. Coli obtain carbon based foods from their environment. • Glucose and galactose are converted