1. Lactose Influence on Bacterial Metabolism
Aidan Towsley
Grade 11
Pittsburgh Central Catholic High School

2. Problem How do lactose, glucose, and galactose
affect the growth rates of Escherichia coli
(E.coli) Staphylococcus epidermidis
(Staph)?

3. 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.

4. 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.

5. 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.

6. Staphylococcus epidermidis (Staph)
Common surface symbiont in many mammals (Human).
Gram (+) bacteria
Most strains considered non-pathogenic.
Pathogenic strains can be life-threatening

7. 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.

8. Purpose
To analyze the growth rates of Staphylococcus epidermidis and Escherichia coli under the influence of lactose, glucose, and galactose.

9. 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.

10. Materials 2 Incubators Tinfoil SDF (Sterile Dilution Fluid)
Lambda broth (Minimal Media)
Ethanol
Klett Spectrophotometer
Gloves/ Goggles
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

11. Procedure
Bacteria (E.coli and Staph) were grown overnight in sterile Lambda Media.
SDF and Lambda media were sterilized for use via a burner.
Blanks were created for bacteria growing in flasks of SDF and lambda.
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)

12. Blank Solutions Lambda Blank SDF Blank Media .5mL SDF 9.5mL
Lambda Media
9mL
SDF
1mL

13. Flask Solutions Carbohydrate Flasks Control Flasks Lambda Media/SDF
8.5mL
Carbohydrate
1.0mL
Microbe
.5mL
Total Volume
10mL
Lambda Media/SDF
8.5mL
SDF
1mL
Microbe
.5mL
Total Volume
10mL

14. 5. After creating a total of 32 total flasks of E
5. 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)
Procedure

15. Carbohydrate Influence on E.coli and Staph

16. E.Coli Non-Functional Lambda
Dunnett’s Test
E.Coli Non-Functional Lambda
T-Crit:
~3.29
Lactose
2.71
Galactose
1.87
Glucose
.916
E.Coli Functinal
Lambda
T-crit
~3.29
Lactose
2.37
Galactose
4.88
Glucose
6.17
E.Coli Non-Functional
SDF
T-crit
~3.29
Lactose
.110
Galactose
9.73
Glucose
14.3
E.Coli Functional
SDF
T-Crit
~3.29
Lactose
11.31
Galactose
13.79
Glucose
15.96

17. Dunnett’s Test Staph 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

18. 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.

19. Limitations
Time of exposure in incubator could have been slightly different.
Only 4 flask for each set were tested.
Accuracy of Klett Spectrophotometers used.

20. Future Studies Use different sources of energy/carbohydrates.
Test various cell models.
Vary exposure times in incubator.

21. Works Cited http://www.ncbi.nlm.nih.gov/pubmed/1311956
aryo/prokaryo2.htm
ach/lacoperon/intro.html
Müller-Hill, Benno. The Lac Operon: A Short History of a Genetic Paradigm. Berlin: Walter De Gruyter, Print.

22. Anova Non-Func E.coli Lambda
Anova: Single Factor
P-value < 0.05 = Significant
SUMMARY
Groups
Count
Sum
Average
Variance
Control 4
530
132.5
Lactose
456
114 22
Galactose
581
145.25
Glucose
505
126.25
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
2031.5 3
Within Groups
1116.5 12
Total
3148 15

23. Anova Non-Func E.coli SDF
Anova: Single Factor
P-value < 0.05 = Significant
SUMMARY
Groups
Count
Sum
Average
Variance
Control 4 87
21.75
4.25
Lactose 86
21.5
Galactose
175
43.75
Glucose
216 54 18
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
3180.5 3
E-09
Within Groups
122.5 12
Total
3303 15

24. Anova Func E.coli Lambda
Anova: Single Factor
P-value < 0.05 = Significant
SUMMARY
Groups
Count
Sum
Average
Variance
Control 4
354
88.5
Lactose
411
102.75
Galactose
451
112.75
Glucose
502
125.5
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
Within Groups
863.5 12
Total 15

25. P-value < 0.05 = Significant
Anova Func E.coli SDF
Anova: Single Factor
P-value < 0.05 = Significant
SUMMARY
Groups
Count
Sum
Average
Variance
Control 4
131
32.75
Lactose
345
86.25
Galactose
392 98
Glucose
433
108.25
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
E-09
Within Groups
538.25 12
Total 15

26. P-value < 0.05 = Significant
Anova Staph Lambda
Anova: Single Factor
P-value < 0.05 = Significant
SUMMARY
Groups
Count
Sum
Average
Variance
Control 4
308 77
Lactose
419
104.75
Galactose
439
109.75
426.25
Glucose
407
101.75
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
Within Groups 12
Total 15

27. P-value < 0.05 = Significant
Anova Staph SDF
Anova: Single Factor
P-value < 0.05 = Significant
SUMMARY
Groups
Count
Sum
Average
Variance
Control 4 33
8.25
Lactose
113
28.25
42.25
Galactose
194
48.5 17
Glucose
114
28.5
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
E-07
Within Groups
259.5 12
21.625
Total 15

28. Data Non-Func E.coli Non-Functional E.Coli Lambda
Non-Functional E.Coli SDF
Control
Time
Flask 1
Flask 2
Flask 3
Flask 4 10 18
13.5 20 13 21 16 30 19 15 12 60 26
28.5 23 17 90 35 33 37 36 25
120 49 48 51
150 58 62 65
180 69 73 81 22
210 80
77.5
103
240 89
112 24
270
105 99
121
300
116
108
134
330
119
142
360
124
128
122
156
Lactose
9.5 14 32 34 29 43 45 53 55 50 63 52 71 61 74 84 68 91 79 77 96
100
102 97
106
110
114
104
113

29. Data Non-Func E.coli Galactose Time Flask 1 Flask 2 Flask 3 Flask 4 1818 17 11 15 13 30 16 14 60 33 29 23 27 19 20 90 42 36 40 21 24
120 55 49 48 54 28 25
150 72 68 66 32 26
180 91 85 84 86 31
210
112 99 98
101 38 34
240
125
109
105
110 43
270
135
119
117 44 39
300
146
131
127 45
330
151
138
137
134 41 37
360
159
143
140
139
Glucose 12 57 53 59 76 77 92 96 95 93 47
102 51
111 50
121
113 46
118 58 52
122
126
129 56

30. Data Func E.coli E.Coli Lamda Functional E.Coli SDF Functional Control
Time
Trial 1
Trial 2
Trial 3
Trial 4 4 13 14 7 5 6 30 11 12 3 60 15 8 90 21 17 10
120 23 35 27 22
150 47 37 32 20 16
180 44 57 48 46
210 49 66 55 50
240 63 77 65 28 25 26
270 73 80 72 31
300 78 85 29 33
330 84 89 86 79 34
360 91 88
Lactose 9 18 19 43 42 40 39 52 61 64 36 67 70 82 53 59 74 81 71 99
106 93 97 75 87
114
101 96
100

31. Data Func. E.coli Galactose Time Trial 1 Trial 2 Trial 3 Trial 4 7 167 16 8 6 10 30 15 17 9 12 60 11 18 25 90 23 38 24 20
120 33 28 44 21 35 29
150 41 57 32 39
180 55 52 46 71 43 53 48
210 65 67 62 78 45
240 76 79 84 58 70
270 85 87 86 88 66 68 80 77
300 97 95 94 74
330
106
104 98
100 81
360
116
109
101
Glucose 19 13 42 22 61 27 26 36 73 37 40 49 92 50 72 96
107
108
121 89 93
127
114
130
131
103
135
134
115

32. Data Staph Staph SDF Staph Lambda Control Time Trial 1 Trial 2 Trial 37 4 5 6 30 60 90 8 12 14
120 2 13 19 18
150 3 25 26 20
180 24 33 35 34
210 27 43 44 36
240 37 55 53
270 45 65 59 57
300 58 76 66 63
330 9 67 81 69 71
360 10 74 89 73 72
Lactose 11 15 22 16 17 56 50 29 38 78 61 21
125 94
131
110 80
142 91

33. Data Staph Galactose Time Trial 1 Trial 2 Trial 3 Trial 4 8 9 13 5 6 28 9 13 5 6 2 7 30 12 60 90 11
120 18 15
150 28 21 16
180 14 35
210 26 17 56 45 27 29
240 34 20 22 67 51
270 37 79 50
300 46 31
115 77 66 84
330 39
123 94 71 91
360 54 44 48
133
105
117
Glucose 10 4 3 25 19 43 33 40 72 38 69 75
101 68 80
104 76 32 24 97
109
111