By : Phillip DeRenzo 9th Grade Central Catholic High School.

The Effects of Secondhand Smoke on Microbial Flora

By : Phillip DeRenzo 9th Grade

Central Catholic High School

Introduction People all over the world smoke. Most of them know that

smoking is harmful to their bodies, but do they know the damaging effects that smoke has on their cells?

In addition, what effects does secondhand smoke have on flora in the body? (Ecology)

Two experiments were performed to explore smoke toxicity.

Cellular Model – Yeast Smoke Products- Cigarettes (Garcia y Vega: English

Coronas)/Cigars (Marlboro: Filtered Cigarettes)

Cigarette/Cigar Smoke Cigarette/Cigar smoke has

harmful effects- releases toxins, irritants, etc.

Main pollutants are acetone, carbon monoxide, arsenic, benzene, butane, cyanide, nicotine, lead, formaldehyde, and many others

Background of Cigar vs. Cigarette Smoke

Past studies focus on adverse effects on human body. Cigars have shown to be more damaging than cigarettes.

Higher levels of carcinogensThese carcinogens can induce abnormal cell cycling

(cancer). Cigarettes appear to be more addictive. Cigarette smokers inhale more deeply, creating more

respiratory tree problems (lungs). Upon inhalation, nicotine can reach the brain 10 seconds.

Secondhand Smoke Smoke that is inhaled

involuntarily, or passively, by someone who is not smoking.

A child of a smoker is more likely to have asthma and other breathing problems because of secondhand smoke inhalation.

Yeast Eukaryotic, unicellular

fungi. Has been widely used in

genetics and cell biology, largely because the cell cycle in a yeast cell is very similar to the cell cycle in higher organisms (human).

Experimental Cell ModelSaccharomyces cerevisiae- a

common yeastGreat ease of manipulation in the

laboratory (single-celled microbe)Similar biochemistry, genetics, cell

cycle to other eukaryotes, including human cells

MOST STUDIED CELL IN THE WORLD

ColoniesPetri Dish

Agar

Purpose

Do cigarettes or cigars affect the survival of microbial flora?

Can secondhand smoke persist on a surface and harm microbial flora?

Does increased exposure increase the damaging effects of smoke on microbial flora?

HypothesesNull Hypothesis :

Smoking product exposure will not reduce survivorship of yeast.

Alternate Hypothesis : The smoking products will reduce survivorship of yeast.

Materials YEPD agar plates (1% yeast

extract, 2% glucose, 1.5% agar)

YEPD media (1% yeast extract, 2% peptone, 2% glucose)

Sterile capped test tube with sterile dilution fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

Klett Spectrophotometer

Incubator Saccharomyces cerevisiae- a

common yeastMicropipettes Permanent MarkerSpreader BarEthanol VortexLighterBoxCigars (Garcia y Vega: English

Coronas)Cigarettes (Marlboro: Filtered

Cigarettes)

Procedure (1st Experiment)1. Saccharomyces cerevisiae was

grown overnight in sterile YEPD media.

2. A sample of the overnight culture was added to fresh YEPD media in a sterile sidearm flask.

3. The culture was placed in an incubator (30oC) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of 107 cells/mL.

4. The cell suspension was serially diluted to a density of approximately 103 cells/mL

5. The tubes were vortexed and then 0.1 mL aliquots were spread onto YEPD plates.

6. Sets of 8 plates were taped to the lid of a box, facing down toward the bottom of the box, and the lids removed .

7. A cigarette or cigar was lit and set it at the bottom of the box. For a time exposure of 1 and 3 minutes, the plates were exposed to the smoke rising from the cigarette lying at the bottom of the box.

8. After exposure, the plates were removed from the box and their lids were put back on.

9. The plates were incubated for 48 hours at 32C.

10. The resulting colonies were counted. Each colony assumed to have arisen from one cell.

Procedure (Infused Smoke)1. 16 YEPD agar plates were

selected. 2. 8 of them at a time were taped

to the lid of a box, facing down toward the bottom of the box.

3. The lids of the plates were removed, allowing the plates to be exposed to smoke.

4. A cigarette or cigar was lit and set on the bottom of the box.

5. For 10 minutes, the agar plates were exposed to the smoke rising from the cigarette lying at the bottom of the box.

6. When the 10 minutes were over, the plates were removed and covered.

7. 0.1mL aliquots from the 103 yeast cell suspension were spread onto the plates

8. The 16 plates were placed in a 32C incubator for 48 hours.

9. The resulting colonies were counted. Each colony assumed to have arisen from one cell.

Cigarette Smoke Effects

Exposure Time

Num

ber O

f Col

onie

s

0 Seconds 60 Seconds 180 Seconds0

20

40

60

80

100

120

Cigar

p-value = 1.7E-09

Cigar Smoke Effects

Exposure Time

Num

ber O

f Col

onie

s

0 Seconds 60 Seconds 180 Seconds0

20

40

60

80

100

120

Cigarette

p-value = 0.000406

Smoke Infused Agar Effects

Exposure Time

Num

ber O

f Col

onie

s

0 Seconds Infused 0

20

40

60

80

100

120

Cigar Ciagrette

p-value = 2.68E-06

Anova (Cigarette)Anova: Single Factor for Cigarette

SUMMARY

Groups Count Sum Average Variance

Column 1 8 895 111.875 228.125

Column 2 8 720 90 42.85714

Column 3 8 400 50 114.2857

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 15752.08 2 7876.042 61.32908 1.7E-09 3.4668

Within Groups 2696.875 21 128.4226

Total 18448.96 23

Anova (Cigar)Anova: Single Factor for Cigar

SUMMARY

Groups Count Sum Average Variance

Column 1 8 895 111.875 228.125

Column 2 8 570 71.25 469.6429

Column 3 8 490 61.25 791.0714

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 11502.08 2 5751.042 11.58831 0.000406 3.4668

Within Groups 10421.88 21 496.2798

Total 21923.96 23

Anova (Infused Agar)Anova: Single Factor for Infused

SUMMARY

Groups Count Sum Average Variance

Column 1 8 895 111.875 228.125

Column 2 8 575 71.875 349.5536

Column 3 8 405 50.625 345.9821

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 15475 2 7737.5 25.13098 2.68E-06 3.4668

Within Groups 6465.625 21 307.8869

Total 21940.63 23

Dunnetts’ Test (Cigarette)

Variable T Value Interpretation

60 Seconds 3.2 Insignificant (Accept Null)

180 Seconds 9.1 Significant (Reject Null)

T Crit = 4.0

Dunnetts’ Test (Cigar)

Variable T Value Interpretation

60 Seconds 3.8 Insignificant (Accept Null)

180 Seconds 4.7 Significant (Reject Null)

T Crit = 4.0

Dunnetts’ Test (Infused Agar)

Variable T Value Interpretation

Cigarette Smoke Infused Agar Plates

4.5 Insignificant (Reject Null)

Cigar Smoke Infused Agar Plates

7.0 Significant (Reject Null)

T Crit = 3.77

ConclusionThe null hypothesis that the exposure and time of

exposure to cigar or cigarette smoke will not effect the growth of yeast was accepted for 60 seconds of exposure.

The null hypothesis was rejected for the exposure times of 180 seconds cigarette and cigar.

The null hypothesis was rejected for the cigar and cigarette smoke infused agar experiment.

Limitations and ExtensionsSmoke might not have

gone directly to the cellsTrials could have gone

over/under the time of exposure

Have a team of people synchronize the spread plating

More controlled smoke chamber for exposure

More variations of exposure times

SourcesScientific American Magazine

http://ezinearticles.com/?Which-Is-Worse-For-You,-Cigarettes-Or-Cigars?&id=1181914

http://quitsmoking.about.com/od/tobaccostatistics/a/CigaretteSmoke.htm

http://www.quitsmokingsupport.com/cigars.htm