The Effects Of Temperature and

Antibiotic on E.coli and Staph epidermidis

James ShaverGrade 9

Central Catholic High School

AmpicillinAntibiotic belonging to the amino penicillin group of penicillin

• Used to treat urinary tract infections, Salmonella, some Influenza strains, and Meningitis.• Commonly used in the lab and the medical world.• Has Gram-positive coverage but limited Gram-negative coverage.• The Amino group in the antibiotic helps it penetrate the membrane of Gram-negative bacteria.

- Beta-lactam antibiotic which is part of the aminopenicillin family.

- Roughly equivalent to its successor, amoxicillin in terms of spectrum and level of activity.

The most common side effects include:

-rash

-fever

-diarrhea

-nausea

The Effects of Ampicillin

Bacterial Heat Stress

- Bacterial heat-shock response allows for bacteria to change (stress) in the environment.

- Bacteria use various response mechanisms for coping with the stress and for adapting to the new conditions.

-Most pathogenic bacteria in humans are Gram (+) organisms.

-Simple cell wall.

-Some antibiotics work against the formation of the cell wall.

-Cell was contains an extra layer of lipopolysaccharides for extra protection.-Outer membrane protects bacteria from several antibiotics.

Gram (-) Vs. Gram (+) Bacteria

- Large and diverse group of gram (-) bacteria

-Free living, symbiotes, or pathogens

-Live in the intestinal tract of many mammals.

-Most strains are not pathogenic

-Serve as a common prokaryotic cell model.

Escherichia coli

Staphylococcus epidermidis

-Common surface symbiont in many mammals (Human).

-Gram (+) bacteria

-Most strains considered non-pathogenic.

-Pathogenic strains can be life-threatening.

Purpose

● To determine if Ampicillin and temperature have a synergistic effect on Staphylococcus epidermidis and Escherichia coli survivorship.

HypothesesNull: 1. Ampicillin and Heat individually will not reduce bacterial survivorship.

2. Ampicillin and Heat will not have synergistic effects at reducing bacterial survivorship.

Alternative: 1. The effects of Ampicillin and Heat individually will significantly reduce bacterial survivorship.

2. Heat and Ampicillin in synergy will significantly reduce bacterial survivorship.

Materials

- Escherichia coli DH5-alpha(E.Coli) (Obtained from Doonan Lab, CMU)- Staphylococcus epidermidis (Staph) (Obtained from Carolina Science Supply)- Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)- Thermometer - Latex Gloves- Micro pipettes- Micro tubes- Ampicillin- 72 LB agar plates- 72 infused LB agar plates- Spreader Bar- Incubator- Ethanol- Matches- Bunsen Burner- Vortex- Sharpie - hot plate- Luria Broth(LB) Agar Plates ( 0.5% yeast extract,1% tryptone, 1% NaCl)

Procedure for Liquid Pulse1.Bacteria (E.coli/Staph epidermidis) 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. Ampicillin Stock of AMP 200 (this stock represents 10x or 10 times the recommended working concentration) was sterile filtered.6.The Ampicillin Stock was mixed with the appropriate amounts of SDF to create concentrations of 0x, 0.01x,0.1x and 1x.

Chart of Concentration

Control 0.01x 0.1x x

E. coli 0.1 ml 0.1 ml 0.1ml 0.1ml

Ampicillin 0 ml 0.01ml 0.1ml 1ml

Sterile Water 9.9 ml 9.89 9.8 8.9

Total Volume 10ml 10ml 10 ml 10ml

Procedures Continued

7. After addition of 100 microliters of bacterial suspension, the tubes were incubated at the selected temperatures; 40,50,60 degrees F.8. 100 microliter aliquots from each tube were pipetted on LB agar plates.9. The LB agar plates were placed in the incubator overnight and the number of colonies were then counted.

Procedures for Infused Plates

1.The following volumes of ampicillin were spread onto Lb agar plates to create the following concentrations; 60 microliters(X), 6 microliters of sub stock ( 0.01x).2. The plates were placed in the incubator for one hour to be infused.3.Each E. coli population subjected to the various temperatures was then pipetted onto the infused plates.4.The plates were then incubated for 24 hours at 37 C.

Anova

-Abbreviation for analysis of variance-Statistical test comparing variation within and between experimental groups- If the P- value is lower than the alpha value (.05), then the result is significant (a result of the variable influence)

Dunnett’s Test

- A procedure for comparing the mean of each experimental group individually against the mean of the control group.- A Dunnett’s Test allows you to see if there was a significant difference from the control.- If the T Value is greater than the T Critical value than the difference is significant.

Anova

P- Value

Staph- 0.01289 E coli- 4E-07

Dunnett’s Test T- Crit (3.25)

T- Value 50 C

1.2265Not Sig

2.259Not Sig

T- Value 60 C

2.313Not Sig

14.407Sig

The Effects of Temperature on Bacterial Survivorship

The Effects of Ampicillin on Bacterial Survivorship 40 C

Anova

P- Value

Staph- 0.203322 E coli- 8.52E-08

Dunnett’s Test T-Crit (3.25)

T Value of 0.01x

0.50861Not Sig

3.88418Sig

T- Value of 1x

2.14252Not Sig

17.2157Sig

210

178.8

Interaction P- Value

0.000414

Synergistic Effects on Staph Survivorship(Infusion)

Concentration of Ampicillin

Num

ber

of

Bac

teria

Col

onie

s

0x 0.1x 1x

P-Value Interaction

3.75E-11

Synergistic Effects on E coli Survivorship(Infusion)N

umb

er

of B

acte

ria C

olo

nies

Concentration of Ampicillin 0x 0.1x 1x

Effects of Temperature on E coli Survivorship

Anova

P-Value 5.29E-05

Dunnett’s Test T-Crit-2.86

50 C 1.14947(Not Sig)

60 C 3.31763(Sig)

Dunnett’s Test T- Crit(3.10)

0.01x 3.680174 Sig

0.1x 11.771 Sig

1x 11.8156 Sig

Anova

P-Value 4E-06

Effects of Ampicillin on E coli Survivorship

Ampicillin Effects on E coli (Liquid Pulse)N

umb

er o

f B

acte

ria C

olon

ies

Concentration of Ampicillin

P-Value Interaction

5.59E-09

0x 0.01x 0.1x 1x

0.1x 1x

E coli 40 C 17% 77%

50 C 32% 85%

Staph Epidermidis

40 C 36% 71%

50 C 18% 63%

Percent Decrease In Survivorship Compared to Control

ConclusionExperiment 1(Liquid Pulse)

- 1st null hypothesis is rejected because temperature and ampicillin individually had effects on bacterial survivorship.- 2nd null hypothesis is rejected because temperature and ampicillin had synergistic effects on bacterial survivorship.

Experiment 2(LB Agar Infusion)

- 1st null hypothesis is rejected because temperature and ampicillin individually had effects on bacterial survivorship.- 2nd null hypothesis is rejected because the temperature and ampicillin had synergistic effects on bacterial survivorship.

Limitations And Future Studies

- Plating could have been unsynchronized.

- Only three concentrations of the variable were tested.

- Only survivorship examined.

- Use more Antibiotics

- Use a greater range of temperatures including those that are closer to eachother

-Perform a Trypan blue exclusion essay to test for dead cells

- Explore growth curve effects

References

-http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html

-http://www.mayoclinic.org/diseases-conditions/e-coli/basics/definition/con-20032105

-http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0000105/

Data Infused Plates

Infused Plates

E coli

Control Low Concentration High Concentration

40 degrees 224,228,236,204 168,176,204,189 54,44,39,68

50 degrees 144,156,168,224 104,110,123,131 24,16,31,29

60 degrees 0,0,0,0 0,0,0,0 0,0,0,0

Data Infused Plates

Infused Plates Staph epidermidis

Control Low Concentration High Concentration

40 degrees ,256,360,224 ,236,256,220 96,88,92,99

50 degrees 252,234,208,204 156,176,193,207 81,96,78,76

60 degrees 0,0,0,0 0,0,0,0 0,0,0,0

Data Liquid Pulse

Regular Plates E coli

0mg Stock 0.01x 0.1x 1x

40 degrees 208,352,284,333,305,252

421,532,327,554,366,496

276,175,180,189,201,222

184,184,304,209,258,196

50 degrees 176,208,304,222,258,202

240,288,311,225,233,316

240,220,176,220,228,248

211,144,156,202,178,198

60 degrees 0,0,0,0,0,0 0,0,0,0,0,0 0,0,0,0,0,0 0,0,0,0,0,0

Data Liquid Pulse

Regular Plates E coli

0mg Stock 0.01x 0.1x 1x

40 degrees ,352,284,333,305,252

421,327,366, ,175,180,189,201,222

184,184,209,196

50 degrees 176,208,304,222,258,202

240,288,311,225,233,316

240,220,176,220,228,248

,144,156,202,178,198

60 degrees 0,0,0,0,0,0 0,0,0,0,0,0 0,0,0,0,0,0 0,0,0,0,0,0