effects of diphenhydramine on e. coli science/pjas/pjas 14 final... · effects of diphenhydramine...
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Effects of Diphenhydramine on E. coliJoseph Connolly
Grade 9 Central Catholic High School
PJAS 2014
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Human Microbial Flora- About 10 % of the biomass on the body is symbionts - Comprised of all the microorganisms that reside in or on
the human body - Most species are non-pathogenic, commensals or
mutualists - Almost 1000 species of bacteria live in the gut of an
average human body. - Vast majority of drugs tested on human cells- Microflora?
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Diphenhydramine - Antihistamine - Used to relieve allergies - Hinders the assembly of
histamines that are produced during an allergic reaction
- Found in medicines like Genahist, Sominex, Unisom and Benadryl
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Benadryl - Over-the-counter drug used to
relieve allergy symptoms such as watery eyes and runny nose
- Diphenhydramine concentration of 12.5 mg./5mL
- Contains sodium benzoate, sodium chloride, sucrose, and glycerin
- Targets acetylcholine (histamine) to prevent allergic reactions
- Taken orally in 5mL to 10 mL doses
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Escherichia coli (E.coli) - Ubiquitous gram-negative
bacteria - Inhabits intestines of most
mammals - Most strains harmless and
often beneficial - Some pathogenic - Lab strain DH Alpha used as
model
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Problem● Does diphenhydramine significantly affect E. coli
survivorship?
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Recent Study● 2008 study- examined effects of diphenhydramine
on bacterial survivorship in propofol ● Significant inhibitory effect found on growth of
bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicansin) at concentrations greater than 1%
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Purpose- To determine if diphenhydramine has a significant
effect on E. coli survivorship
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Hypotheses Null Hypothesis- Diphenhydramine WILL NOT
significantly reduce E. Coli survivorship Alternative Hypothesis- Diphenhydramine WILL
significantly reduce E. coli survivorship
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Materials- Benadryl - E. Coli DHS Alpha - LB Agar Plates + Media (1% Tryptone, 0.5% Yeast
extract, 1% NaCl) - Test tubes - Sterile fluid (100mM KH2PO4,
100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
- Petri dishes - Pipettes - 50 Klett Spectrophotometer
- Side arm flask - Pipette tips - Spreader bars - Ethanol - Bunsen Burner - Vortex - Incubator
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Procedure1. E. Coli was grown overnight in sterile LB media 2. A sample of the overnight culture was added to fresh
media in a sterile sidearm flask 3. The culture was incubated at 37 degrees Celsius until a
density of 50 Klett spectrophotometer units was reached. (Cell density approximately 10^8 cells/mL
4. The culture was diluted in a sterile dilution fluid to a concentration of approximately 10^5 cells/mL
5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL.
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Procedure (continued)6. 0.1 mL of cell culture was then added to the test tubes,
yielding a final volume of 10 mL. and a cell density of approximately 10^3 cells/mL
7. The solution was mixed by vortexing 8. After vortexing to evenly suspend cells, 0.1 aliquots were
removed from the tubes and spread on LB agar plates 9. The plates were incubated at 37 degrees Celsius for 24
hours 10. The resulting colonies were counted. Each colony is
assumed to have risen from one cell.
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Concentration of Benadryl in test tubes
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0% 0.1% 1% 10%
E. Coli 0.1 mL 0.1 mL 0.1 mL 0.1 mL
Sterile Fluid
9.9 mL 9.89 mL 9.8 mL 8.9 mL
Benadryl 0 mL 0.01 mL 0.1 mL 1 mL
Total (mL)
10 mL 10 mL 10 mL 10 mL
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Data0% 0.1% 1% 10%
Trial 1 358 297 341 311Trial 2 374 313 349 344Trial 3 402 336 368 347Trial 4 319 352 309 385Trial 5 394 372 348 303Trial 6 348 313 311 342Trial 7 327 381 331 364Trial 8 424 385 370 296
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Benadryl effects on E. Coli
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Col
ony
Cou
nt
0
100
200
300
400
Benadryl Concentration
0% 0.1% 1% 10%
P-value= 0.456053
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ANOVAAnova: Single Factor
SUMMARY
Groups Count Sum Average Variance
Column 1 8 2946 368.25 1369.35714285714
Column 2 8 2749 343.625 3555.98214285714
Column 3 8 2827 353.375 868.839285714286
Column 4 8 2692 336.5 959.714285714286
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 4532.625 3 1510.875 0.8948172746934310.4560535540589472.94668526878167
Within Groups 47277.25 28 1688.47321428571
Total 51809.875 31
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Conclusion● The data supports the acceptance of the null
hypothesis that diphenhydramine would not significantly reduce E. coli survivorship
● The ANOVA statistical analysis yielded a p-value of 0.456053, which is significantly greater than the 0.05 cutoff for accepting the null hypothesis
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Limitations- Unknown effects of secondary compounds in Benadryl - E. coli colonies could have been too small to count - E. coli colonies could have been too close together to
count - Only one type of test (liquid pulse exposure) used to
assess effect - Slight deviations in plating times - Only one exposure time
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Extensions- Different medications containing diphenhydramine - Pure diphenhydramine could have been tested - Other bacterial models - Synergistic effects - Different exposure
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References● "Diphenhydramine HCl Oral : Uses, Side Effects, Interactions, Pictures,
Warnings & Dosing - WebMD." WebMD. WebMD, n.d. Web. 31 Dec. 2013.
● "Diphenhydramine: MedlinePlus Drug Information." U.S National Library of Medicine. U.S. National Library of Medicine, n.d. Web. 31 Dec. 2013.
● "Drugs and Human Performance FACT SHEETS - Diphenhydramine." Drugs and Human Performance FACT SHEETS - Diphenhydramine. N.p., n.d. Web. 04 Jan. 2014.
● "E. Coli." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 03 Aug. 2012. Web. 31 Dec. 2013.
● "The Preventive Effect of Diphenhydramine on Bacterial Growth in Propofol: A Laboratory Study." NCBI. U.S. National Library of Medicine, n.d. Web. 04 Jan. 2014.
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