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 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

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)

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

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

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

Carbohydrate Influence on E.coli and Staph

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

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

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/prok aryo/prokaryo2.htm http://www.phschool.com/science/biology_place/bioco ach/lacoperon/intro.html Müller-Hill, Benno. The Lac Operon: A Short History of a Genetic Paradigm. Berlin: Walter De Gruyter, 1996. Print.

Anova Non-Func E.coli Lambda Anova: Single Factor P-value < 0.05 = Significant SUMMARY Groups Count Sum Average Variance Control 4 530 132.5 251.6666667 Lactose 456 114 22 Galactose 581 145.25 86.91666667 Glucose 505 126.25 11.58333333 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 2031.5 3 677.1666667 7.278101209 0.004867377 3.490294819 Within Groups 1116.5 12 93.04166667 Total 3148 15  

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 1.666666667 Galactose 175 43.75 16.91666667 Glucose 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 Lambda Anova: Single Factor P-value < 0.05 = Significant SUMMARY Groups Count Sum Average Variance Control 4 354 88.5 4.333333333 Lactose 411 102.75 60.91666667 Galactose 451 112.75 40.91666667 Glucose 502 125.5 181.6666667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 2940.25 3 980.0833333 13.62015055 0.000360167 3.490294819 Within Groups 863.5 12 71.95833333 Total 3803.75 15  

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 6.916666667 Lactose 345 86.25 32.91666667 Galactose 392 98 86.66666667 Glucose 433 108.25 52.91666667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 13547.1875 3 4515.729167 100.6758012 8.98059E-09 3.490294819 Within Groups 538.25 12 44.85416667 Total 14085.4375 15  

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 64.66666667 Lactose 419 104.75 1103.583333 Galactose 439 109.75 426.25 Glucose 407 101.75 99.58333333 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 2553.1875 3 851.0625 2.009493827 0.166427033 3.490294819 Within Groups 5082.25 12 423.5208333 Total 7635.4375 15  

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 5.583333333 Lactose 113 28.25 42.25 Galactose 194 48.5 17 Glucose 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.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

Data Non-Func E.coli Galactose Time Flask 1 Flask 2 Flask 3 Flask 4 18 18 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

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

Data Func. E.coli Galactose Time Trial 1 Trial 2 Trial 3 Trial 4 7 16 7 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

Data Staph Staph SDF Staph Lambda Control Time Trial 1 Trial 2 Trial 3 7 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

Data Staph Galactose Time Trial 1 Trial 2 Trial 3 Trial 4 8 9 13 5 6 2 8 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