Effects of Commercial Protein Powders on Microbial Models Harry Suver Grade 11 Central Catholic High School

Gram Staining Invented by Hans Christian Gram Is a method of differentiating bacterial species into two groups, Gram-positive and Gram-negative Gram-positive bacteria have generally a single membrane (monoderm) surrounded by a thick peptidoglycan Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes When stained, a Gram-positive bacteria results in a blue/violet color while Gram-negative results in light red/pink

Gram(-) vs. Gram(+) Gram-negativeGram-positive  Cell walls 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. species consisting of harmless skin-dwelling micro-organisms Species of Staph. are commonly used in microbiological experiments because of variety of strain types within each species – each strain has different features and toxins Gram-positive bacteria Most strains are not pathogenic

Escherichia coli Major cause of food-borne illness Consumption of undercooked meats Consumption of contaminated vegetables or unpasteurized milk Also resides in fecal matter Most studied bacteria in the world and very commonly used in the lab Resides in the digestive tract of animals Not all strains of E.coli can result in sickness or disease Is a Gram-negative bacteria

Whey Protein GNC Pro Performance 100% Whey Protein Whey protein is a mixture of globular proteins isolated from whey, the liquid material created as a by-product of cheese production. Whey protein is commonly marketed and ingested as a dietary supplement, and various health claims have been attributed to it in the alternative medicine community.

Soy Protein GNC Super Foods 100% Soy Protein protein isolated from the soybean. Made from soybean meal that had been dehulled and defatted

Purpose The purpose of this experiment was to see if protein powder had significant effects on the growth of microbial models

Hypothesis Null Hypothesis- The protein powder will not have a significant effect the growth of the bacteria Alternate Hypothesis- The protein powder will have a significant effect on the bacteria and will cause more colonies to grow than the control groups

Materials E.coli Staph. E. GNC pro performance Whey Protein GNC Soy Protein LB media (.5% yeast extract, 1% tryptone, 1% sodium chloride) Agar plates Spreader bars Bunsen Burner Vortex Incubator Micropipettes Sterile pipette tips Sterile dilution fluid ([SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4,.1mM CaCl2, 100mM NaCl) Sterile test tubes Test tube rack Ethanol Matches

Procedure 1. Bacteria (E.coli and Staph. E.) were grown overnight in sterile LB Media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. 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 10⁸ cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. 5. Solutions were made up in sterile test tubes using sterile fluid and variable mL. Of the cell cultures were added to the solutions which brought them to a total volume of 10mL. 7. Solutions were pipetted onto LB plates using a micro-pipette. 8. Spreader bars were sterilized and used to spread solutions on the plates. 9. Plates were put in an incubator set at 37 degrees Celsius for 24 hours. 10. Colonies were counted, data was recorded, results were analyzed.

Concentrations in Tubes CONTROL5%10% STERILE FLUID 9.9 mL7.9 mL VARIABLE 0.0 mL2.0 mL (of 25% stock) 2.0 mL (of 50% stock) BACTERIA 0.1 mL TOTAL VOLUME 10 mL 5 replicates for each group

Stat Analysis ANOVA Shows if there is any variations between the mean of 2 or more variable groups Dunnetts Test Shows the variation between a control group and an experimental groups Alpha=.05 T crit= 3.03

P-value = = Significant Number of Colonies

Dunnett's Test(Staph w/ Soy) ConcentrationT-valueSignificance 5%1.207Not Significant 10%3.34Significant

Staph with Whey Protein P-value = = Not Significant Number of Colonies

Dunnett’s Test (Staph w/Whey) ConcentrationT-valueSignificance 5% Not Significant 10%1.2264Not Significant

P-value = = Significant Number of Colonies

Dunnetts Test (E.coli w/ Soy) ConcentrationT-valueSignificance 5%3.2442Significant 10%4.4968Significant

P-value = = Significant Number of Colonies

Dunnetts Test (E.coli w/ Whey) ConcentrationT-valueSignificance 5% Not Significant 10% Significant

Conclusion The Null Hypothesis can be partially rejected, and the Alternate Hypothesis can be partially accepted. Soy protein had significant effects on both bacterial models at all concentrations and caused more colonies to grow. Whey protein did not have significant effects on either bacterial model at any concentration. While on some occasions the 5% groups had less colonies than the control groups, they had colonies that were larger in size

Limitations Continuations Did not have access to any other model organisms. Lag time between counting of colonies Experiment could be expanded to test protein powder on stem cells or algae. Could compare protein powder’s effectiveness to growth hormones and natural animal proteins.

ANOVA (Staph w/ soy) SUMMARY GroupsCountSumAverageVariance Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

ANOVA (Staph w/ whey) SUMMARY GroupsCountSumAverageVariance Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

ANOVA (E.coli w/ soy) SUMMARY GroupsCountSumAverageVariance Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

ANOVA (E.coli w/ whey) SUMMARY GroupsCountSumAverageVariance Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

References CentralCatholichs.com/biology