1. T HE E FFECT OF S HIITAKE M USHROOMS ON B ACTERIA G ROWTH Matthew Scotti PJAS Central Catholic High School Grade 9

2. P ROBLEM Bacteria that cause infections are becoming increasingly resistant to antibiotics and other antimicrobial agents. Shiitake mushrooms have been thought to have antimicrobial effects. Do Shiitake mushrooms have a significant negative effect on the growth of gram negative and gram positive bacteria. Question

3. L ENTINULA EDODES (S HIITAKE M USHROOMS ) Native to and considered a delicacy in many Asian countries Have been cultivated for centuries in Asia not only for its uses as a food, but also for its medicinal properties Asian civilizations have for centuries attributed health and longevity to Shiitake mushrooms

4. P REVIOUS S TUDIES Have been shown to have a strong negative effect on various species of bacteria Have been shown to have anticancer and anti- pathogen properties

5. E SCHERICHIA COLI (E. COLI ) Gram-negative One of the most common varieties of bacteria found in the human body Historically utilized as the most studied prokaryote in history Most strains are not considered pathogenic Pathogenic forms can produce fatal illness in humans and other mammals.

6. S TAPHYLOCOCCUS E PIDERMIDIS (S TAPH ) Gram-positive Most strains non-pathogenic Can cause many serious infections in humans Common on human skin Main bacteria associated with skin infections

7. E. COLI : G RAM N EGATIVE B ACTERIA Cell wall is thin extra layer of lipopolysaccharide which adds extra level of protection. If the toxin enters the circulatory system it causes a toxic reaction. This outer membrane protects the bacteria from several antibiotics.

8. S TAPH : G RAM P OSITIVE B ACTERIA Most pathogenic bacteria in humans are gram-positive organisms. Simple cell wall. Antibiotics such as penicillin work against the formation of the cell wall.

9. P URPOSE To examine the effects of Shiitake mushrooms on the growth curve of E.coli and Staph bacteria species

10. H YPOTHESES Null: The Shiitake mushrooms will NOT have a significant effect on the growth curve of staph and E. coli Alternative: The Shiitake mushrooms WILL have a significant effect on the growth curve of staph and E. coli

11. MATERIALS LB plates LB Media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Micropipettes Sterile Pipette tips Vortex Sterile Dilution Fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl) Sidearm Flasks (125mL) Spreading Platform, spreader bar, ethanol Escherichia coli bacteria Staphylococcus epedermidis bacteria 580 micro Liters of 25% stock Shiitake Mushrooms Ethanol Micro tubes Matches 0.22 micron sterile syringe filters

12. P ROCEDURE F OR T ESTING S HIITAKE M USHROOMS O N B ACTERIA (A) 1. Bacteria (E. coli and Staph) 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 10 5 cells/mL. 5. A shiitake mushroom puree was mixed with the appropriate amount of SDF to create shiitake mushroom solutions of 0%,.1%, 1% and 10%.

13. C HART OF C ONCENTRATION ( M L) 0 % stock.1 % stock1 % stock10 % stock Microbe0.1 SDF9.9959.959.55.9 Shiitake Mushroom 0.0040.040.44 Total10

14. P ROCEDURE A C ONTINUED 6. 100 µL of cell culture was then added to the Shiitake Mushroom solutions, 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.

15. P ROCEDURE B (A GAR I NFUSION ) 1. 200 micro Liters of Sterilized Shiitake Mushroom was infused into the LB agar media and the media was used to create the LB agar plates. 2. E. coli and Staph were 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 cultures were 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. 5. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL.

16. P ROCEDURE B (A GAR I NFUSION ) C ONT. 6.100 µL of cell culture was then added to an SDF solution of 9.9mL, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7.After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB plates. 8.The plates were incubated at 37 C for 24 hours. 9.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

17. E. C OLI R ESULTS 0% E. coli.1% E. coli1% E. coli10% E. coliInfusedE. coli 193190 135 187133176127159 197134168141159 192164166128102 18320818077176 188195163159

18. S TAPH R ESULTS 0% Staph.1% Staph1% Staph10% StaphInfused Staph 214166197117228 169162198137252 17220422365207 35221583160174 16016915673222 21313717551

19. S HIITAKE M USHROOM ’ S E FFECT O N E. COLI AND S TAPH B ACTERIA S URVIVORSHIP ShiitakeConcentrationShiitakeConcentration Number of Colonies E. coli P value= 0.01312 Staph P value= 0.00321

20. E. C OLI A NOVA Analysis of Variance (One-Way) Summary GroupsSample sizeSumMeanVariance 0% E. coli61,140.190.24.8.1% E. coli61,024.170.666671,033.46667 1% E. coli61,043.173.83333103.36667 10% E. coli6822.137.1,418. ANOVA Source of VariationSSdfMSFp-levelF crit Between Groups8,916.4583332,972.152784.608640.013128.09838 Within Groups12,898.1666720644.90833 Total21,814.62523

21. S TAPH ANOVA Analysis of Variance (One-Way) Summary GroupsSample sizeSumMeanVariance 0% Staph61,114.185.66667534.66667.1% Staph61,053.175.5833.9 1% Staph61,032.172.2,417.6 10% Staph6603.100.51,922.3 ANOVA Source of VariationSSdfMSFp-levelF crit Between Groups27,439.539,146.56.409080.003218.09838 Within Groups28,542.33333201,427.11667 Total55,981.8333323

22. D UNNETT ’ S T EST A NALYSIS Variable Concentration T valueInterpretation 0.1% Shiitake Mushroom 1.30Not Significant 1% Shiitake Mushroom 1.09Not Significant 10% Shiitake Mushroom 3.61Significant Escherichia Coli T Critical = 3.10 Alpha =.05

23. D UNNETT ’ S T EST A NALYSIS Variable Concentration T valueInterpretation 0.1% Shiitake Mushroom 0.5Not Significant 1% Shiitake Mushroom 0.64Not Significant 10% Shiitake Mushroom 3.92Significant T Critical = 3.10 Alpha =.05 Staphylococcus

24. S HIITAKE M USHROOM I NFUSED A GAR ’ S E FFECT O N E. COLI AND S TAPH S URVIVORSHIP Number of Colonies E. coli P value=0.00484 Staph P value=0.13371

25. I NFUSED E. COLI ANOVA Analysis of Variance (One-Way) Summary GroupsSample sizeSumMeanVariance InfusedE. coli5731.146.2823.7 0% E. coli61,140.190.24.8 ANOVA Source of VariationSSdfMSFp-levelF crit Between Groups5,232.109091 13.773540.0048422.85713 Within Groups3,418.89379.86667 Total8,650.9090910

26. I NFUSED STAPH ANOVA Summary GroupsSample sizeSumMeanVariance Infused Staph51,039.207.8438.2 Control Staph61,114.185.66667534.66667 ANOVA Source of VariationSSdfMSFp-levelF crit Between Groups1,336.048481 2.716690.1337122.85713 Within Groups4,426.133339491.79259 Total5,762.1818210

27. D UNNETT ’ S T EST A NALYSIS T valueInterpretation 3.89Significant Agar Infusion Exposure: E. coli T Critical = 2.45 Alpha = 0.05

28. C ONCLUSIONS The null hypothesis, that the Shiitake mushrooms would NOT have a significant effect on the growth curve of bacteria, was rejected for the 10% Shiitake mushroom concentration for both Staphylococcus and Escherichia coli. It was also rejected for the Shiitake mushroom infused agar with Escherichia coli. The null could not be rejected for the.1% and 1% concentrations of Shiitake mushrooms for both the Escherichia coli and the Staphylococcus.It could also not be rejected for the Shiitake mushroom infused agar with Staphylococcus. The alternative hypothesis was accepted for the 10% concentration for both bacteria and for the infused agar plates with E. coli. The alternative hypothesis could not be rejected for the.1% and 1% concentrations for both bacteria or for the infused agar plates with Staph.

29. L IMITATIONS AND E XTENSIONS It was difficult to have completely synchronized plating. There was only one exposure time There were only four concentrations Add lab hands to further synchronize plating Use multiple exposure times Use more than four concentrations Limitations Extensions

30. R EFERENCES http://en.wikipedia.org/wiki/Shiitake http://www.medicalmushrooms.net/ http://en.wikipedia.org/wiki/Escherichia_coli http://en.wikipedia.org/wiki/Staphylococcus