The Effect of Shiitake Mushrooms on Bacteria Growth

The Effect of Shiitake Mushrooms on Bacteria GrowthMatthew ScottiPJASCentral Catholic High SchoolGrade 9

Problem 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.QuestionLentinula edodes(Shiitake Mushrooms)Native to and considered a delicacy in many Asian countriesHave been cultivated for centuries in Asia not only for its uses as a food, but also for its medicinal propertiesAsian civilizations have for centuries attributed health and longevity to Shiitake mushrooms

Previous StudiesHave been shown to have a strong negative effect on various species of bacteriaHave been shown to have anticancer and anti-pathogen properties

Escherichia coli(E. coli)Gram-negativeOne of the most common varieties of bacteria found in the human bodyHistorically utilized as the most studied prokaryote in historyMost strains are not considered pathogenic Pathogenic forms can produce fatal illness in humans and other mammals.

Staphylococcus Epidermidis (Staph)Gram-positiveMost strains non-pathogenicCan cause many serious infections in humans Common on human skinMain bacteria associated with skin infections

E. coli: Gram Negative Bacteria

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.Staph: Gram Positive Bacteria

Most pathogenic bacteria in humans are gram-positive organisms.

Simple cell wall.

Antibiotics such as penicillin work against the formation of the cell wall. Purpose To examine the effects of Shiitake mushrooms on the growth curve of E.coli and Staph bacteria speciesHypotheses Null: The Shiitake mushrooms will NOT have a significant effect on the growth curve of staph and E. coliAlternative: The Shiitake mushrooms WILL have a significant effect on the growth curve of staph and E. coli materialsLB platesLB Media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)MicropipettesSterile Pipette tipsVortexSterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)Sidearm Flasks (125mL)Spreading Platform, spreader bar, ethanolEscherichia coli bacteria Staphylococcus epedermidis bacteria 580 micro Liters of 25% stock Shiitake Mushrooms EthanolMicro tubes Matches0.22 micron sterile syringe filters

Procedure For Testing Shiitake Mushrooms On Bacteria (A)Bacteria (E. coli and Staph) were grown overnight in sterile LB media.

A sample of the overnight culture was added to fresh media in a sterile sidearm flask.

The culture was placed in an incubator (37C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL.

The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

A shiitake mushroom puree was mixed with the appropriate amount of SDF to create shiitake mushroom solutions of 0%, .1%, 1% and 10%.

Chart of Concentration (mL)

0 % stock.1 % stock1 % stock10 % stockMicrobe0.10.10.10.1SDF9.9959.959.55.9Shiitake Mushroom0.0040.040.44Total10101010Procedure A Continued 100 L of cell culture was then added to the Shiitake Mushroom solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.

The solutions were vortexed and allowed to sit at room temperature for 15 minutes.

After vortexing to evenly suspend the cells, 100 L aliquots were removed from the tubes and spread on LB agar plates.

The plates were incubated at 37C for 24 hours.

The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

Procedure B (Agar Infusion)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.

E. coli and Staph were grown overnight in sterile LB media.

A sample of the overnight culture was added to fresh media in a sterile sidearm flask.

The cultures were placed in an incubator (37C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL.

The cultures were diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

Procedure B (Agar Infusion) Cont.6.100 L 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 103 cells/mL.

7.After vortexing to evenly suspend the cells, 100 L 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.

E. Coli Results0% E. coli.1% E. coli1% E. coli10% E. coliInfusedE. coli19319019019013518713317612715919713416814115919216416612810218320818077176188195163159Staph Results0% Staph.1% Staph1% Staph10% StaphInfused Staph21416619711722816916219813725217220422365207352215831601741601691567322221313717551Shiitake Mushrooms Effect On E.coli and Staph Bacteria SurvivorshipShiitake

ConcentrationNumber of Colonies E. coli P value= 0.01312

Staph P value= 0.00321 E. Coli AnovaAnalysis of Variance (One-Way)SummaryGroupsSample sizeSumMeanVariance0% E. coli61,140.190.24.8.1% E. coli61,024.170.666671,033.466671% E. coli61,043.173.83333103.3666710% E. coli6822.137.1,418.ANOVASource of VariationSSdfMSFp-levelF critBetween Groups8,916.4583332,972.152784.608640.013128.09838Within Groups12,898.1666720644.90833Total21,814.62523 Staph anovaAnalysis of Variance (One-Way)SummaryGroupsSample sizeSumMeanVariance0% Staph61,114.185.66667534.66667.1% Staph61,053.175.5833.91% Staph61,032.172.2,417.610% Staph6603.100.51,922.3ANOVASource of VariationSSdfMSFp-levelF critBetween Groups27,439.539,146.56.409080.003218.09838Within Groups28,542.33333201,427.11667Total55,981.8333323Dunnetts Test Analysis

Variable ConcentrationT valueInterpretation0.1% Shiitake Mushroom1.30Not Significant 1% Shiitake Mushroom 1.09Not Significant10% Shiitake Mushroom3.61SignificantEscherichia Coli T Critical = 3.10 Alpha = .05

Dunnetts Test Analysis

Variable ConcentrationT valueInterpretation0.1% Shiitake Mushroom0.5Not Significant 1% Shiitake Mushroom 0.64Not Significant10% Shiitake Mushroom3.92Significant

T Critical = 3.10Alpha = .05Staphylococcus Shiitake Mushroom Infused Agars Effect On E. coli and Staph SurvivorshipNumber of ColoniesE. coli P value=0.00484

Staph P value=0.13371 Infused e. coli anovaAnalysis of Variance (One-Way)SummaryGroupsSample sizeSumMeanVarianceInfusedE. coli5731.146.2823.70% E. coli61,140.190.24.8ANOVASource of VariationSSdfMSFp-levelF critBetween Groups5,232.1090915,232.1090913.773540.0048422.85713Within Groups3,418.89379.86667Total8,650.9090910 Infused staph anova SummaryGroupsSample sizeSumMeanVarianceInfused Staph51,039.207.8438.2Control Staph61,114.185.66667534.66667ANOVASource of VariationSSdfMSFp-levelF critBetween Groups1,336.0484811,336.048482.716690.1337122.85713Within Groups4,426.133339491.79259Total5,762.1818210Dunnetts Test Analysis

T valueInterpretation3.89Significant

Agar Infusion Exposure: E. coliT Critical = 2.45 Alpha = 0.05 ConclusionsThe 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. Limitations and ExtensionsIt was difficult to have completely synchronized plating.There was only one exposure timeThere were only four concentrations

Add lab hands to further synchronize platingUse multiple exposure times Use more than four concentrationsLimitationsExtensionsReferenceshttp://en.wikipedia.org/wiki/Shiitake

http://www.medicalmushrooms.net/

http://en.wikipedia.org/wiki/Escherichia_coli

http://en.wikipedia.org/wiki/Staphylococcus