Thyme Extract Antimicrobial Assessment

Jonathan MaffeiPittsburgh Central Catholic

Grade 9

Research Question

● How do thyme extract concentrations affect the survivorship of E. coli?

Natural and Artificial Antimicrobials● Antimicrobials are agents that can be used to destroy microorganisms or to

prevent their development● Natural antimicrobials can be used to preserve food from spoilage and kill

pathogenic microorganisms(garlic, honey, thyme, ginger)● Artificial antimicrobial agents are any variety of chemical compounds and

physical agents that are used to destroy microorganisms or to prevent their development(Penicillin, Aminoglycosides, Ofloxacin)

Thyme Extract● Thyme extract is obtained from Thyme leaves ● Used as a natural cough remedy or natural antibiotic● PH between 4.0 - 6.5 at 25 degrees Celsius ● Molecular structure is C50H82O4● Packed with minerals and vitamins

that are essential for optimum health.● Contains 20–54% thymol and compounds,

such as p-cymene, myrcene, borneol, and linalool

Escherichia coli (E. coli)

● Rod-shaped, 2 micrometers diameter● Gram negative bacteria● Survival, growth, and replication require only a single carbon

source and ammonium salts● Most common prokaryote model● Common mammalian intestinal symbiont● Some strains can cause diseases(food poisoning)

Gram Negative and PositiveGram Negative

● Cell wall has a thin extra layer of

lipopolysaccharide which adds

extra level of protection

● This outer membrane protects

the bacteria from several antibiotics

Gram positive

● Simple cell wall

Purpose

● Determine the effect of thyme extract concentrations on E. coli survivorship.

Hypothesis

● Alternative Hypothesis: Thyme extract will significantly reduce the survivorship of the E. coli.

● Null Hypothesis: There will be no effect of thyme extract on the survivorship of E.coli.

Materials● Spreader bars● Escherichia coli(DHS5

Alpha) ● Burners● Sidearm flask● Turn-table● Vortex● Incubation● Thyme Extract● SDF(sterile dilution Fluid)● Ethanol● Pipettes

● 25 liquid pulse plates( with concentrations of 0%, 0.01%, 0.1%, and 1%)

● 12 Agar infusion plates( with concentrations of 0%, 0.01%, 0.1%, and 1%)

● LB Agar plates(0.5% yeast extract, 1% tryptone, 1% sodium chloride)

Procedure(Liquid Pulse)

1. Bacteria (E.coli) 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

4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL

5. The thyme extract was sterilized

6. The following ingredients were combined to create the desired concentrations of 0%, 0.01%, 0.1%, and 1%

Procedure Continued(Liquid Pulse)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

Concentrations

Concentrations 0% 0.01% 0.1% 1%

Sterile fluid 9.9mL 9.89mL 9.8mL 8.9mL

Microbe 0.1mL 0.1mL 0.1mL 0.1mL

Sterile Thyme extract

0.0mL 0.01mL 0.1mL 1mL

Total 10mL 10mL 10mL 10mL

Effects of thyme concentrations on E. coli(liquid Pulse)

Dunnett's Test (liquid Pulse)

Concentration T-value Results

0.01% 7.30 Significant

0.1% 7.06 Significant

1% 12.03 Significant

T-crit:2.35

Procedure(Agar Infusion)1. 0.1ml of thyme concentrations, 0%, low (20μL thyme, 180μL SDF) and high

(200μL thyme) were spread on LB agar plates and incubated for 1 hour, allowing the agar to absorb the chemicals(low~0.1% thyme high~1% thyme)

2. 100 uL aliquots of bacterial suspensions from the original control tube were spread onto the infused plates

3. The plates were incubated at 37 C for 24 hours4. The resulting colonies were counted visually, each; colony being assumed to

have arisen from one cell

Effects of thyme concentrations on E. coli(Agar infusion)

Dunnett's Test (Agar infusion)

Concentration T-value Results

High 7.54 Significant

Low 1.111 Not significant

T-crit:2.35

Conclusion● The null hypothesis was rejected for all concentrations of thyme extract

● The alternative hypothesis was supported for all concentrations in liquid pulse and for high exposure in the Agar infusion

● 1%, 0.1%, and 0.01% all had a significant effect of the survivorship of E. coli

limitations● Lag time between plating● Only 3 concentrations of variable tested● Only one exposure time utilized● Only one species tested● Only survivorship assessment

Extensions● Test more than one exposure time● Test other concentrations of the variable● Use other microbial models and human cells● Test synergistic effects of chemicals

Bibliography https://pubchem.ncbi.nlm.nih.gov/compound/Thyme-oil

http://herbgardening.com/growingthyme.htm

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507285/

https://www.healthline.com/health/health-benefits-of-thyme

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/13%3A_Antimicrobial_Drugs/13.3%3A_Commonly_Used_Antimicrobial_Drugs/13.3A%3A_Synthetic_Antimicrobial_Drugs

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074903/=

https://byjus.com/chemistry/antimicrobial-agents/

https://www.medicalnewstoday.com/articles/321108.php

Anova(liquid pulse)P-value:

0.000000005

Anova(Agar Infusion)P-value:

0.0000008

Statistical TestsANOVA Statistical

Analysis of Variance- Multiple mean comparison, allows you to compare different groups of statistical data

P-value is smaller than the Alpha Value (0.05)= variation Dunnett's Test

• This is a follow up stats test,which examines the individual variation between each of the concentrations compared back to the control

Data Table(Agar Infusion)

Control High Low

392 296 412

416 312 380

371 286 404

386 310 414

382 278 396

Data Table(Liquid pulse)

Concentrations 0% 0.01% 0.1% 1%

1 392 260 286 210

2 416 250 248 136

3 371 248 260 104

4 386 270 286 196

5 382 294 258 220