1. HONEY ANTIMICROBIAL EFFECTS Alicia Grabiec Freeport Senior High School Grade 10

2. Purpose  To determine the antimicrobial effects of honey on the Escherichia coli (Gram -) and Staphylococcus epidermidis (Gram +)

3. Background Information  Advent of penicillin and other antibiotic drugs in the twentieth century reduced honey's medicinal use  Makua honey, like that found in the antiseptic lotion above, can destroy strains of antibacterial-resistant bacteria, such as MRSA C 6 H 12 O 6 + H 2 O + O 2 → C 6 H 12 O 7 + H 2 O 2 This chemical reaction occurs when honey is mixed with water and oxygen. Its products are glutonic acid and hydrogen peroxide, both possess antibacterial properties.  Honey has been known for its healing properties for thousands of years  Ancient Greeks used it, and so have many other peoples through the ages  Up to the second world war, honey used for its antibacterial properties in treating wounds

4. Background Information  Escherichia coli is a large and diverse group of gram (-) bacteria.  It is found in the intestinal tract of most animals, including humans.  Estimated to cause infection in more than 70,000 patients a year in the United States.  There are many strains of E. coli, most are non-pathogenic  Pathogenic strains can cause illness and death in humans  Staphylococcus epidermidis is a spherical bacterial that is known to cause infections in wounds.  Gram (+) bacteria  This bacteria is commonly found on the skin.  It is resistant to antibiotics like penicillin and methicillin.

5. Null Hypothesis:  Honey exposure will not significantly reduce the survivorship of both E. coli and S. epidermidis. Alternative Hypothesis:  Honey exposure will significantly reduce the survivorship of both E. coli and S. epidermidis.

6. Variables Independent / Manipulated Concentration of honey being tested 0%(control), 1%, 10%, 30%, 50% Dependent/ Responding Bacterial survivorship Constant/ Controlled Incubated at 37 o C 112 sterile petri dishes containing LB Medium Length of Incubation time (48 hours) Limitations Extended viability of E. coli and S. epidermidis (established) Impurities in the honey

7. Pulse Liquid Exposure Procedure: 6. 100 µL of cell culture was then added to the honey concentrations, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates. 9. The plates were incubated at 37°C for 24 hours. 10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. 1. Bacteria (E. coli and S. epidermidis) were grown overnight in sterile LB media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 0.5-0.8x10 5 cells/mL. 5. Honey was mixed with the appropriate amount of SDF to create honey concentrations of 0%, 1%, 10%, 30%, and 50%.

8. Honey Infused Agar (Prolonged Exposure) Procedure: 1. Honey was infused directly into molten agar plates at 0.1, 1, 10% and allowed to solidify. 2. Bacteria (E. coli and S. epidermidis) was grown overnight in sterile LB media. 3. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 4. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 0.5-0.8x10 8 cells/mL. 5. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 6. The culture was then spread evenly across honey infused agar. 7. The plates were incubated at 37°C for 24 hours.

9. Zone of Inhibition Procedure: 1. Bacteria (E. coli and S. epidermidis) was grown overnight in sterile LB media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 0.5-0.8x10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5. The culture was then spread evenly across petri dishes containing sterile LB Media 6. Plates were then incubated for one hour at 37°C 7. 50 µL honey aliquots (droplets) were carefully placed in 5 positions on each plate and repeated for each concentration 8. The plates were incubated at 37°C for 24 hours. 9. Determine and record the average zone of inhibition

10. Pulse Liquid Exposure Data Pulse Liquid Exposure (S. epidermidis) Plate #Percent of Honey 0%1%10%30%50% 11545320 21630000 31590000 41533100 51570000 61602200 71651000 81433010 Avg.156.751.750.750.380.00 Pulse Liquid Exposure (E. coli) Plate #Percent of Honey 0%1%10%30%50% 123323015412080 220621012014090 321118013910083 422616912711287 523116315011786 620917414312184 723016914713089 821016712912681 Avg.219.50182.75138.63120.7585.00

12. Dunnett Test HONEY CONCENTRATION T-VALUE INTERPERATION 1%5.151Significant 10%11.337Significant 30%13.824Significant 50%18.853Significant HONEY CONCENTRATION T-VALUE INTERPERATION 1%82.843Significant 10%83.379Significant 30%83.578Significant 50%83.779Significant Pulse Liquid Exposure (S. Epidermidis) 0.01 Significance Level T-critical = 4.17 T > T-critical = Significant Variation, Reject Null Hypothesis Pulse Liquid Exposure (E. coli)

13. Honey Infused Agar Data Honey Infused Agar (E.coli) Plate #Percent of Honey 0%0.10%1%10% 125721930 2222 00 323420620 422023010 521321741 624821230 721921500 825422310 923022120 1023322611 1123721650 1224621530 Avg.234.42218.502.080.17 Honey Infused Agar (S. epidermidis) Plate #Percent of Honey 0%0.10%1%10% 121015000 222317000 319016700 418618010 522314900 622115810 720316300 816915101 917315510 1016116800 1118016500 1215517100 Avg.191.17162.250.250.08

15. Dunnett Test HONEY CONCENTRATION T-VALUE INTERPERATION 0.01%2.133Insignificant 1%31.132Significant 10%31.389Significant HONEY CONCENTRATION T-VALUE INTERPERATION 0.01%1.412Insignificant 1%9.321Significant 10%9.331Significant 0.01 Significance Level T-critical = 3.58 T > T-critical = Significant Variation, Reject Null Hypothesis Honey Infused Agar (S. epidermidis) Honey Infused Agar (E. coli)

16. Zone of Inhibition Data Zone of Inhibition (mm, E.coli) Plate #Percent of Honey 0%1%10%30%50% 111.58.573.51.5 212.58652 31276.54.51 4137.554.51.5 512.57.56.541.5 Avg.12.37.76.24.31.5 Zone of Inhibition (mm, S. epidermidis) Plate #Percent of Honey 0%1%10%30%50% 1117.53.51.50.5 213.56.52.511 312.5611.50.5 4137210.5 513.55.51.510.3 Avg.12.76.52.11.20.56

18. Dunnett Test HONEY CONCENTRATION T-VALUE INTERPERATION 1%48.42Significant 10%94.21Significant 30%84.21Significant 50%113.68Significant HONEY CONCENTRATION T-VALUE INTERPERATION 1%39.49Significant 10%97.52Significant 30%73.25Significant 50%77.32Significant 0.01 Significance Level T-critical = 4.17 T > T-critical = Significant Variation, Reject Null Hypothesis Zone of Inhibition (mm, S. epidermidis) Zone of Inhibition (mm, E.coli)

19. Conclusions  In the pulse liquid exposure to honey experiment, the different concentrations of honey had a significant impact in reducing the number of E. coli and S. epidermidis  In the honey infused agar experiment, 1% and 10% honey significantly decreased the number of bacteria while the 0.01% honey had an insignificant impact in reducing the number of both bacteria  In the zone of inhibition experiment, all the concentrations of honey tested had a significant impact in reducing the number of both bacteria In conclusion, my hypothesis was partially supported.

20. Future Improvements/ Changes  Conduct more trials  Test a greater variety of honey concentrations

21. Future Extensions/ Experiments  Use a different method to determine the amount and type of bacteria present  Test different types of honey  Test the effect of honey on its ability to inhibit other types of bacteria  Test the effect of sugar and salt on their abilities to inhibit bacterial growth  Test the effects of honey on the bacteria over a greater period of time