Analysis of Lunch Meat Microbial Contamination Experiment and Analysis by Donald Kline, Jr

Problem of providing safe food Throughout history, people have been faced with the challenge of not just providing food, but keeping it safe from contamination

Problems of having safe meat Meat is especially susceptible to disease because it came from a living animal with natural flora like E. coli in addition to diseases the animal could have contracted during its life The nutrients in meats provide can provide an excellent growth source for microbial life

Pathogens  Pathogens in food are the leading cause of death in some undeveloped countries, with a death toll of around two million people annually  Medical care for pathogens costs over a billion dollars per year worldwide

Common contaminants in meat  E. Coli  Salmonella  Clostridium botulinum  Campylobacter jejuni  Listeria monocytogenes  Clostridium perfringens  Staphylococcus aureus

Historical ways people kept meat safe  Until 1860, it was thought that food became spoiled through spontaneous generation  Drying using the sun is the oldest form of food preservation  Salting can be an effective means of preserving meat  Smoking and heating were other common ways to keep food safe  Meats usually could not be kept safe from contamination very long

Common ways to keep meat safe  Keep the meat in a hypertonic environment  Sterile packaging  Cooking  Refrigeration  Salting  Freeze-Drying  Canning and Bottling

Preservatives  A preservative is used to prevent spoilage in foods from microbial growth and contamination  Common chemical preservatives are calcium propionate, sodium nitrate, sodium nitrite, sulphites, disodium EDTA, formaldehyde, glutaraldehyde, and methylchloroisothiazolinone  Natural preservatives include salt, sugar, vinegar, and diatomaceous earth. Citric and ascorbic acids can also assist in the preservation of certain foods.

Preservatives in this experiment  Virginia Brand ham contained water, sugar, salt, dextrose, potassium chloride, sodium phosphate, sodium erythorbate, sodium nitrite, and sugar  Giant Eagle contained the following preservatives: water, salt, sugar, dextrose, sodium phosphates, sodium erythorbate, and sodium nitrite.  Russer ham contained water, salt, dextrose, sodium nitrate, sodium phosphate, and sodium erythorbate.

E. Coli  E.Coli is a pathogen that is found in the lower intestines of warm blooded animals  Represents prokaryotic cell model in this experiment  There are almost 73,000 cases of infection and 61 deaths per year in the United States

Yeast  Type of yeast used in this experiment was Saccharomyces cerevisiae  Yeasts also contribute to food spoiling by producing waste products when they metabolize food  Yeasts contribute to making many foods we use today  They represent the eukaryotic cell model in this experiment

Purpose and Hypothesis  Determine how different brands of sterilized lunchmeat’s preservatives and ingredients would affect the survivorship of yeast and E. coli cells  The null hypothesis was that the lunch meats would not significantly affect survivorship of S. cerevisiae and E. coli cells

Materials 96 LB agar plates( 1.5 % tryptone,.5 % yeast extract, 1% NaCl, 1.5 % agar) LB media (1 % tryptone, 5 % yeast extract, 1% NaCl) 72 YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar) Sterile dilution fluid (10mM KH 2 PO 4, 10mM K 2 HPO 4, 1mM MgSO 4,.1mM CaCl 2, 100mM NaCl) Klett spectrophotometer Sterile pipette tips and Micropipettors Vortex Incubator Sidearm flask Spreader bar Ethanol 20 mL Sterile capped test tubes E.coli B Saccharomyces cerevisiae Giant Eagle Old Fashioned Ham, Russer Cooked Ham, and Dietz and Watson Virginia Brand Ham Hole puncher Metric Scale and weigh boat Micro burner

Procedure 1. E. coli B and Saccharomyces cerevisiae were grown over night in a sterilized media 2. A sample of the overnight cultures were added to separate fresh LB (bacteria) and YEPD (yeast) in a sterile sidearm flask. 3. The cultures were incubated at 37°C (bacteria) and 30°C (yeast) until a density of 50 Klett spectrophotometer units was reached. These represent cell densities of approximately 10 8 (bacteria) and 10 7 (yeast) cells per mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells per mL. 5. The hams were sliced, massed to.4 grams for each brand of ham and sterilized separately by soaking in 95% ethanol. After the ethanol was evaporated, the hams were placed in 15mL sterile polystyrene conical tubes uL of the 10 5 cells/mL cell suspensions were pipetted directly onto the surface of the ham pieces.

Procedure 2 7. The tubes were allowed to incubate at room temperature for the following time periods: 0, 45, 90, and 135 minutes (bacteria) and 0, 30, and 60 minutes (yeast). 8. One mL of SDF was pipetted onto the ham/cell mixture and the tubes were gently vortexed. 9. After vortexing to evenly suspend cells, 100uL of the cell suspension was transferred to 9.9mL of SDF and then the SDF was vortexed and 100 uL aliquots were then spread onto either LB agar or YEPD agar. 10. The plates were incubated for 24 hours at 37°C (bacteria), or 48 hours at 30°C (yeast). 11. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

10 2 cells10 3 cells/mL 10 5 cells/mL with ham 10 8 cells/mL (bacteria) or 10 7 cells/mL (yeast) 100 uL 10 5 cells/mL for both yeast and bacteria 1mL of SDF 100 uL

p Values for Bacteria ControlVirginiaG. EagleRusser 0 min Bac p ›.05 45min Bac1.35E-10 p ›.05p ‹.01 90min Bac1.1E-12 p ‹.01 p › min Bac6.83E-13 p ›.05p ‹.01p ›.05

Conclusions for Bacteria  Only Giant Eagle Ham had a significant negative impact on E. coli survivorship.  Russer Cooked Ham appeared to have the least negative impact on E. coli survivorship. The variation was not statistically significant.  Giant Eagle Old Fashioned Ham appeared to have the most negative impact on bacterial survivorship

Data Chart of p values for yeast ControlVirginiaG. EagleRusser 0 min Yeast p ›.05 30min Yeast p ›.05 60min Yeast p ›.05 p ‹.05p ›.05

Yeast Conclusions  Giant Eagle ham had a statistically significant negative impact on yeast survivorship, while Russer cooked ham and Virginia Brand Ham did not  Russer Cooked Ham appeared to have the least negative impact on yeast survivorship. The variation was not statistically significant.

Limitations and Extensions In the process of sterilizing the hams, it might have affected the composition of the preservatives The moisture content of the hams might have varied due to the sterilizing and drying processes Some hams might retain the bacteria or yeast on the surface longer than others Infuse the meats directly into agar to allow longer exposure times for the cells Sterilize ham with gamma irradiation Use different models of bacteria and eukaryotic cell models

Sources Centers for Disease Control and Prevention (CDC) Food and Drug Administration (FDA) Partnership for Food Safety Education (PFSE) World Health Organization (WHO) Acknowledgement to Professor John Wilson University of Pittsburgh Biostatistician for his advice and statistical analysis