Effects of Road Salt Deicers on Yeast Survivorship

James BrunnerPittsburgh Central Catholic High School

Grade 9

Introduction

In this experiment, a yeast culture was tested under the effects of various chloride salts

Model- yeast, Variable- chloride salts, Interaction- yeast survivorship

Idea arose from wondering what effects salting roads has on local ecosystems

Road Salt Runoff

In PA, PENNDOT uses 96,367 tons of salt on roads in winter

Several salts used as deicers

These salts have harmful effects on everything from plants and animals to concrete and metals

Sodium Chloride

Standard salt used to de-ice roads

Known damages- harmful to water, plants, metals, and even the roads it’s used on

One of the cheapest salts to melt ice

Chemical Symbol: NaCl

Magnesium Chloride

Also used as road salt de-icer

Less harmful then Sodium Chloride

Known to damage metal and concrete, but still known as best for metal and concrete

Contains 17-56% more chloride ion than other salts

Melts ice at slower rate than other chloride salts

Chemical Symbol: MgCl2

Potassium Chloride

Another salt used as de-icer

Thought to be one of the “safer” salts to melt ice with

Used as sodium-free substance for table salt

Known as best for use with vegetation

Chemical Symbol: KCl

Yeast

Eukaryotic organism used in foods and drinks

Most commonly studied organism in labs

Easy to use- grows quickly, easy to count colonies, and nonpathogenic (safe to use)

Less tolerable to extreme conditions- achieves better data

Past Studies

Several studies of the nature of this experiment have been conducted in the past, such as: Effects of NaCl on yeast Effects of NaCl on macro-invertebrates

In both studies, it was found that NaCl had a negative effect on the organisms’ survivorship

Purpose & Hypotheses

Purpose- to determine the effects of various chloride salts on a yeast culture

Null Hypothesis- The survivorship of the yeast cells will not be significantly affected by any of the concentrations of the three chloride salts

Alternative Hypothesis- all three chloride salts will reduce survivorship compared to the control

Materials

YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar)

YEPD Media (1% yeast extract, 2% peptone, 2% glucose (dextrose))

Sidearm flask Spreading platform, spreader

bar, ethanol 20 mL Sterile capped test

tubes with Sterile Dilution Fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

Saccharomyces cerevisiae (Yeast)

0.22 micron syringe filters + 10 mL syringe

Weigh boat Scale Incubator Vortex Genie Stir Plate NaCl (10%) KCl (10%) MgCl2 (10%) Pipettes Sterile test tubes Water Ethanol (95%) Spreader Bar Micro Burner

Procedure 1. Yeast was grown overnight in sterile YEPD media.2. A sample of the overnight culture was added to fresh media in a sterile

sidearm flask.3. The culture was placed in an incubator(30 Degrees) until a density of 50

Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/ml.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.

5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations for a total of 9.9 ml. For Example:1 ml of 10% variable ‘z’ solution + 8.9 ml of SDF= final concentration of almost 1% ‘z’ (the addition of 0.1 ml of cell culture will result in a total of 10 ml and a 1% concentration).

6. 103 100 ml of cell culture was then added to the test tubes, yielding a final volume of 10 ml and a cell density of approximately 103 cells/ml

7. The solution was mixed by vortexing and allowed to sit at room temperature.

8. After vortexing to evenly suspend cells, 0/1 ml aliquots were removed from the tubes and spread on YEPD+LB plates.

9. The plates were incubated at 30 degrees for 48 hours.10. The resulting colonies were counted. Each colony is assumed to have

arisen from one cell.

Test Tube Contents

0% 0.1% 0.5% 1%

SDF (ml)

9.9 9.8 9.4 8.9

10% Salt Stock (ml)

0 0.1 0.5 1

Yeast (ml)

0.1 0.1 0.1 0.1

Total Volume (ml)

10 10 10 10

Results

All salts affected the yeast; yeast survivorship declined as salt concentration rose

NaCl was the least harmful, while MgCl2 was the most harmful

All plates grew yeast well; except for the plates containing .5% KCl, which was contaminated and did not grow any yeast

The results in this experiment pertained to those of past studies of this nature

Effects of Salt on Yeast Survivorship

0

20

40

60

80

100

120

140

NaCl KCl MgCl2 No Salt

0.10%0.50%1%0%

#

o

f

colonies

Type of Salt

Salt Concentration

0.02

0.04

0.03

= P values lower then 0.05

Pictures

NaCl 1% KCl .1%

MgCl2 .5% No Salt Added

ANOVA ChartAnova: Single FactorAll DataSUMMARY

Groups Count Sum Average VarianceColumn 1 6 743 123.8333 290.1667Column 2 6 689 114.8333 120.1667Column 3 6 644 107.3333 340.6667Column 4 6 733 122.1667 142.5667Column 5 6 584 97.33333 196.6667Column 6 6 636 106 161.6Column 7 6 511 85.16667 2066.967Column 8 6 573 95.5 44.3Column 9 6 728 121.3333 599.0667

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

Between Groups 8828.667 8 1103.583 2.506772 0.024113 2.152134Within Groups 19810.83 45 440.2407

Total 28639.5 53

Conclusions

After the data was collected, the following concentrations of salt rejected the null hypothesis: 1%KCl, and 1% MgCl2

All colonies affected negatively by the salts

Alternative Hypothesis correct- all salts reduced yeast survivorship

According to data, road salt de-icers inhibit the growth of yeast colonies

Conclusions (continued)

After ANOVA and Dunnett tests, null hypothesis rejected

Most ANOVA tests showed null hypothesis accepted, with no significant difference from control (most p values over .05), but Dunnett test showed that it was rejected, with significant difference from control (most t values less than t critical)

Dunnett test more powerful than ANOVA- compares better over two groups of data

ANOVA tests may have shown that null was accepted as decreases in yeast survival not very large, but clearly exist

Limitations and Extensions

All .5% KCl contaminated-data could not be obtained

Difficult to ensure exact exposure times to salt variables due to time variation in spread plating. This could be more easily overcome by using a team of people

More concentrations and various salts could be tested

Other cellular models could be tested Another model, such as bacteria, added to

achieve better data

References

“Rock Salt (NaCl) Sodium Chloride.” Peterschemical.com Dec. 21, 2007. <http://www.peterschemical.com/sodium-chloride/

“Potassium Chloride and Urea.” Peterschemical.com Dec. 21, 2007.

<http://www.peterschemical.com/potassium-chloride/ “Magnesium Chloride.” Peterschemical.com Dec 21, 2007.

<http://www.peterschemical.com/magnesium-chloride/ “Yeast.” Wikipedia.org Dec. 21, 2007.

<http://en.wikipedia.org/wiki/Yeast “Sodium Chloride.” Wikipedia.org Dec. 21, 2007.

<http://en.wikipedia.org/wiki/Sodium_chloride “Magnesium Chloride.” Wikipedia.org Dec. 21, 2007

<http://en.wikipedia.org/wiki/Magnesium_chloride

References (continued)

“Potassium Chloride.” Wikipedia.org Dec. 21, 2007 <http://en.wikipedia.org/wiki/Potassium_chloride

“Road Salt and Water Quality.” Des.state.nh.us Dec. 21, 2007<http://www.des.state.nh.us/factsheets/wmb/wmb-4.htm

“Field and laboratory investigations on the effects of road salt (NaCl) on stream macroinvertebrate communities.” Sciencedirect.com Dec. 21, 2007 <http://www.sciencedirect.com/science?_ob=ArticleURL&_u di=B6VB5-

455VM763&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=c9d9b919f172d211bbad1839fd5d0afe

“Effect of Sodium Chloride on Bakers' Yeast Growing in Gelatin.”Aem.asm.org Dec. 21, 2007<http://aem.asm.org/cgi/content/abstract/43/4/757

“Winter Maintenance- Notes and Other Interesting Information” Dot.state.pa.us Jan. 28, 2008.

<http://www.dot.state.pa.us/Penndot/Districts/district6.nsf/winter_maint_notes?ReadForm