Alcohol Effects on Microbial Survivorship Brian Long Central Catholic High School Grade

Yeast Saccharomyces cerevisiae. Easy to manipulate in the laboratory. Unicellular, 3-4 µm diameter. Most commonly studied cell. Very similar in structure and biochemistry to more complex eukaryotes, including human cells. Buds in single colonies, can be easily counted.

Ethyl Alcohol Flammable, colorless liquid. Also known as ethanol. Produced by fermentation. In alcoholic beverages. Molecular composition – C 2 H 5 OH

Ethyl Alcohol’s Effects on Humans Negative effects – Causes physical and behavior abnormalities when exposed to fetus. – Enlarges kidneys and alters hormones. Positive effects – Recent studies show moderate use of alcohol may be beneficial to coronary system.

Past Studies Gerhard Hoppe and Geoffrey Hansford studied ethanol inhibition of continuous anaerobic yeast growth. – Found that ethanol inhibited cell growth rate and cell viability. S.W. Brown and S.G. Oliver studied the difference in complexity and magnitude of ethanol’s effects on yeast growth. – Results showed that ethanol stunted yeast growth and that increased alcohol concentrations negatively affected the yeast.

Purpose and Hypothesis Purpose – Determine the effects of varying concentrations of ethyl alcohol on: yeast survivorship, cell proliferation, and cell respiration. Null – The ethyl alcohol will have no significant effect on any of these parameters. Alternative – The ethyl alcohol will significantly effect these parameters.

Materials YEPD agar plates (1% yeast extract, 2% glucose, 2% peptone, 1.5% agar) YEPD media (1% yeast extract, 2% peptone, 2% glucose) Yeast (Saccharomyces cerevisiae) Sterile capped test tube Sterile dilution fluid (SDF) (10 mm KH2PO4, 10 mm K2HPO4, 1 mm MgSO4, 0.1 mm CaCl2, 100 mm NaCl) 100% Ethyl Alcohol 15 ml sterile conical tubes Plate spreader Ethanol (for sterilization) Bunsen Burner Klett Spectrophotometer Incubator Sidearm flask Test tube rack Permanent marker

Procedure 1 (Growth Experiment) 1.Saccharomyces cerevisiae was grown overnight in a sterile YEPD media. 2.A sample of the overnight cultures was added to fresh media in a sterile sidearm flask. 3.The culture was incubated at 30 o C until a density of 150 Klett Spectrophotometer units was reached. 4.A 100% ethyl alcohol solution, yeast stock, YEPD media, and sterile water were added to sidearm flasks. 5.The flasks were swirled to evenly dissolve the solution. 6.Klett spectrophotometer readings were taken every 30 mins for 4.5 hours.

Dunnett's Test (Growth Exp.) If t value is greater than 2.68 (t crit), significant variation. [ ] of alcoholResults.1%T value(2.81)>2.68 Significant 1%T value(2.19)<2.68 Not significant 10%T value(19.34)>2.68 Significant [ ] of alcoholResults.1%T value(2.71)>2.68 Significant 1%T value(3.72)>2.68 Significant 10%T value(26.64)>2.68 Significant At 240 minutesAt 390 minutes (final reading)

Procedure 2 (Toxicity Experiment) 1.Saccharomyces cerevisiae was grown overnight in a sterile YEPD media. 2.A sample of the overnight cultures was added to fresh media in a sterile sidearm flask. 3.The culture was placed in a shaking water bath. 4.The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/ml. 5.The selected concentrations of ethyl alcohol were added to the variable tubes and allowed to sit for 30 minutes (0%, 0.1%, 1%, and 10% alcohol). 6.After vortexing to evenly suspend yeast cells, 0.1 ml. aliquots were removed from the tubes and spread on 32 plates. (8 plates per concentration). 7.The plates were incubated at 30 0 C for 48 hours. 8.The resulting colonies were counted. Each colony is assumed to have arisen from one cell. Flask AFlask BFlask CFlask D Yeast0.1 ml Sterile H 2 O9.9 ml9.89 ml9.8 ml8.9 ml Alcohol0 ml.01 ml0.1 ml1 ml Total10 ml [ ] alcohol0%0.1%1%10%

t= 9.52 t= t= P value X

Dunnett’s Test (Toxicity Exp) If t value is greater than 2.54 (t crit), significant variation. [ ] of AlcoholResults 0.1%T value(9.52)>2.54 Significant 1%T value(10.93)>2.54 Significant 10%T value(28.26)>2.54 Significant

Procedure 3 (Respiration Experiment) 1.Sugar, water, and a 100% alcohol solution were added to 16 side arm flasks in the ratios as indicated in the table. 2.7 grams of Red Star rapid rise Cooking Yeast was added to each flask and balloons were immediately affixed to each flask. 3.The flasks were transferred to a 30 o C water bath. 4.After 90 minutes of incubation, each balloon was removed from the flask carefully to prevent any leakage of gas. (Each balloon was pinched at the neck and twisted off). 5.The volume of gas was quantified by displacement of water. – The cylinder was inverted and immersed into the water and the plastic wrap was removed. – The balloon was placed into the water with the opening placed into the cylinder. – The opening was slowly released and the air in the balloon was released into the graduated cylinder. The volume of gas was then recorded.

Flask AFlask BFlask CFlask D Yeast7g Glucose8 ml Sterile H 2 O32 ml31.96 ml31.6 ml28 ml Alcohol0 ml.04 ml.4 ml4 ml Total40 ml [ ] Alcohol0%.1%1.0%10% Concentration Table of Respiration Experiment

t= 2.38 t= 4.99 t= 6.92 P value X 10 -5

Dunnett’s Test (Respiration Exp) If t value is greater than 2.68 (t crit), significant variation. [ ] of AlcoholResults 0.1%T value(2.38)<2.68 Not significant 1%T value(4.99)>2.68 Significant 10%T value(6.92)>2.68 Significant

Conclusions Growth Experiment – Null hypothesis was rejected for all concentrations (There appeared to be a dosage effect.) Toxicity Experiment – Null hypothesis was rejected for all concentrations. Respiration Experiment – The null hypothesis was rejected for the concentrations 1% and 10%.

Limits and Extensions Limits – Using balloons to measure respiration causes some CO 2 to be lost when removing balloon. – Plating could not be exactly synchronized possibly resulting in varying colony counts. – The Respiration experiment had a lag time of about 5 minutes which could have allowed for more cellular respiration in some flasks than in others. Extensions – Incorporate different temperatures to eliminate temperature as a variable. – Use wider range of concentrations of alcohol. – Trypan blue exclusion assay

References alcoholism.com/statistics-on-alcoholics.html / htm