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D value

D and Z values determination. D value The time requires to inactivate 1 log scale (90%) of bacterial population at a particular temperature. Z value The difference of temperature between 1 log scale of D-values. Materials and Methods. Bacteria: Escherichia coli Medium: TSA

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D value

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  1. D and Z values determination • D value • The time requires to inactivate 1 log scale (90%) of bacterial population at a particular temperature. • Z value • The difference of temperature between 1 log scale of D-values.

  2. Materials and Methods • Bacteria: Escherichia coli • Medium: TSA • Dilution buffer: Butterfield’s phosphate buffer (BPB, pH 7.2) • Water bath at 60, 70, 80°C • Pipette • Petri-dish • incubator

  3. Procedures • Incubate two flasks of E. coli culture. • Turn on water bathes before class begins. Make sure water bathes have enough water. • Each group has one tube containing phosphate buffer saline (PBS) into water baths. • Place the tube into the water bath. • Aliquot the E. coli culture into tubes. Each table has one tube. (teaching assistant) • Take the PBS tube from water bath. • Transfer 1 mL E. coli culture into a tube containing 9 mL PBS. Place the culture into one tube immediately. • Bacterial density in each tube should be at 108 CFU/mL.

  4. Start timing. Gently shake the tubes during heating. • When heating time is achieved, place the tubes under running tap water. Gently rotating the tube during cooling for one minute. • Decimally serial dilution • For 0 dilution using pour plate :transfer one mL of dilutant into a petri-dish. Gently mixed agar and bacterial suspension. • For other dilutions using spread plate: transfer 0.1 mL onto the agar surface. • Spread the plates, DO NOT pack the plates vertically. • Place the plates into 37C incubator. Incubate at 37C for 24 hour. Count colonies.

  5. 1 mL 9 mL 9 mL Bacterial culture Heating After cooling 1 mL 0.1 mL 0.1 mL 0, pour plate -1 -2 -3 -4 -5 -6 -7 Duplicate plates/dilution

  6. Control 1 mL 9 mL Bacterial culture -1 -2 -3 -4 -5 -6 -7 0 0.1 mL -6 -7 -8 Duplicate plates/dilution

  7. Record colony number: 25-250 • Calculate average number of the duplicate plates • Average number x dilution = bacterial population • Example: 42 x 103= 4.2 x 104=4.62 log10 CFU/mL • Calculate bacterial population of control • Example: 42 x 108= 4.2 x 109=9.62 log10 CFU/mL • Therefore, bacterial population in the first dilution tube (the same dilution used for heating process) should be = 4.2 x 108=8.62 log10 CFU/mL • The control number is time 0 number (the number on Y axie)

  8. Record bacterial population. • Draw a figure. Use regression function. • Y axle: bacterial population (log CFU/mL) • X axle: heating time. • Obtain an equation. • Obtain D value.

  9. y = -1.0155x + 8.3753 • when y= 6, x=2.339 • When y=5, x= 3.234 • When y=4, x=4.308 • D value = 0.98 minute • Small D values mean more sensitive to heat • Time to reduce 90% (log scale) population • Different temperature different D values • Higher temperature, smaller D values • 12D • Reduce 12 log scale

  10. Population (log CFU/mL) 60℃ 70℃ 75℃ min

  11. Z values • Combine data from all groups. • Draw a figure. Use regression function. • Y axle: D values(log scale) • X axle: temperatuare • Obtain an equation. • Obtain Z value.

  12. Z values (℃ or ℉) • log D values vs. temperature • The time between 1 log scale D values log D values Z value Z value temperatures

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