Antimicrobial Effects of Vinegar. Daniel Crawford Grade 9 Central Catholic High School

Transcription

1 Antimicrobial Effects of Vinegar Daniel Crawford Grade 9 Central Catholic High School

2 Antibacterial Agents Humans interest in microbial growth/survivorship for over 100 years Many products/technologies have been developed to alter bacterial population Refrigerators, Antiseptics, Soaps, etc. Environmental concerns with many antibacterial agents Do common household items possess antimicrobial properties?

3 Gram (-) vs. Gram (+) Bacteria Cell was 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.

4 Escherichia coli (E. coli) Large and diverse group of gram (-) bacteria Free living, symbiotes, or pathogens Live in the intestinal tract of many mammals Most strains are non-pathogenic Serve as a common prokaryotic cell model

5 Staphylococcus epidermidis (Staph) Common surface symbiont in many mammals (Human). Gram (+) bacteria Most strains considered non-pathogenic. Pathogenic strains can be lifethreatening.

6 Vinegar, CH 3 COOH Cleaning and cooking. Vinegar Synthesis: alcoholic fermentation acetic fermentation Creates acidic environment Variable- Giant Eagle Brand pure distilled white vinegar

7 Sodium Bicarbonate, NaHCO 3 Baking soda, white, powdery substance. In remedies Mouthwash urinary alkalization. Used to balance out the ph of the solutions Variable: Lab Grade Sodium Bicarbonate

8 ph measure of acidity or basicity of an aqueous solution measured on a scale of 0-14 ph x< 7: Acidic x> 7: Basic x= 7: Neutral

9 Pervious Studies Vinegar has shown: Antimicrobial effects To cause significant lower systolic blood pressure Act against tumors Antiglycemic effects

10 Purpose To determine whether: Vinegar affects the growth of Escherichia coli or Staphylococcus epidermidis Vinegar neutralized with Sodium Bicarbonate affects the growth of Escherichia coli or Staphylococcus epidermidis Sodium Bicarbonate affects the growth of Escherichia coli or Staphylococcus epidermidis

11 Hypotheses Null Hypothesis: The vinegar, the mixture of vinegar and sodium bicarbonate, and the sodium bicarbonate will not significantly affect bacterial survivorship. Alternate Hypothesis: The vinegar, the mixture of vinegar and sodium bicarbonate, and the sodium bicarbonate will significantly alter bacterial survivorship.

12 Materials Vinegar(pure white distilled vinegar) and Sodium Bicarbonate(lab grade) Micro and Macro pipettes + Sterile tips Spreader bars LB agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl) Escherichia coli DH5-alpha (E.Coli) (Obtained from Doonan Lab, CMU) Staphylococcus epidermidis (Staph) (Obtained from Ward s Scientific) Burner Sidearm Flask Vortex Incubator Gloves/goggles Klett Spectrophotometer SDF (Sterile Dilution Fluid)(100mM KH 2 PO, 100mM K 2 HPO 4, 10mM MgSO 4, 1m NaCl) Sterile Test Tubes Ethanol 0.22 micron syringe filters and 10mL syringe

13 Procedure (Liquid Exposure) 1. Bacteria (E.coli and Staph) 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.

14 Procedure (con t) Sterile Dilution Fluid 1. The tubes that received vinegar alone were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.1%, 1%, and 10%. Concentration Chart 0% 0.1% 1% 10% 9.9 ml 9.89 ml 9.8 ml 8.9 ml Microbe 0.1 ml 0.1 ml 0.1 ml 0.1 ml Vinegar/V+SB/ SB 0 ml 0.01 ml 0.1 ml 1 ml Total Volume 10 ml 10 ml 10 ml 10 ml

15 Procedure (con t) 2. The tubes that received both vinegar and sodium bicarbonate were combined in the following process. 1. The tubes received vinegar and were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.1%, 1%, and 10%. 2. Sodium bicarbonate added to the solution to neutralized the acidic properties, based on the table. (only [1%] and [10%] received sodium bicarbonate) ph Neutralizing Measurements 1% 10% Original ph 5 4 Sodium Bicarbonate added 0.02g 0.2g Resulting ph 7 7

16 Procedure (con t) 3. The tubes receiving sodium bicarbonate alone received the ingredients in the given measurements below Concentration Chart 0% 0.1% 1% 10% SDF 9.9 ml 9.9 ml 9.9 ml 9.9 ml Microbe 0.1 ml 0.1 ml 0.1 ml 0.1 ml Sodium Bicarbonate 0g 0g 0.02g 0.2g ph

17 Procedures (con t) 4. The solutions were mixed by vortexing and allowed to sit at room temperature for 30 minutes. 5. After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes at time 30 minutes and spread on LB agar plates. 6. The plates were incubated at 37 C overnight and the resulting colonies were counted. 7. Stat tests were completed.

18 ANOVA & Dunnett s Tests ANOVA (analysis of variance) is a statistical analysis which allows comparison of multiple means. Dunnett s test comparison of each experimental group to the control to determine significant variation.

19 Number of Colonies Escherichia coli growth P-value: <0.01 P-Value: <0.01 P-value: <0.01 Vinegar V + SB SB [0%] [0.1%] [1%] [10%] Concentrations

20 Staphylococcus epidermidis Growth P-value: <0.01 P-value: <0.01 P-value: < Vinegar V + SB SB [0%] [0.1%] [1%] [10%] Concentrations

21 Dunnett s Tests E. coli [0.1%] [1%] [10%] Vinegar Not Significant Vinegar + SB Significant SB Significant Significant Significant Significant Significant Significant Significant Staph [0.1%] [1%] [10%] Vinegar Not Significant Vinegar + SB Not Significant SB Not Significant Not Significant Significant Significant Not Significant Not Significant Not Significant

22 Effects on Colony Morphology (Size) Measured against control E. coli [0.1%] [1%] [10%] Vinegar Average Average Large Vinegar + SB Average Average Average SB Average Average Large Staph [0.1%] [1%] [10%] Vinegar Average Average Average Vinegar + SB Average Average Average SB Average Average Average

23 Results Was there a significant difference between the effect of vinegar and sodium bicarbonate combined and the effect of just vinegar? Escherichia coli --- Yes Staphylococcus epidermidis --- No

24 Conclusions The null hypotheses was rejected for Escherichia coli at all concentrations except [0.1%] vinegar The null hypotheses for Staphylococcus epidermidis was rejected at only the concentrations of [1%] of Vinegar + SB and [1%] of Sodium Bicarbonate alone

25 Limitations and Extensions During the testing, plating could have possibly been unsynchronized. Liquid pulse only technique utilized Different types of acids/bases More concentrations Different exposure method- agar infusion Study Growth Curves

26 References Bicarbonate.htm the_power_of_hydrogen.htm 12-19

27 Number of Colonies Escherichia coli LD Vinegar: 0.3 V+SB: N/A SB: Vinegar V+SB SB Concentration

28 Number of Colonies Staphylococcus epidermidis LD Vinegar: N/A V+SB: N/A SB: N/A Vinegar V+SB SB Concentration