D and Z values determination D value The

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D and Z values determination • D value – The time requires to inactivate

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 • Dilution buffer: Butterfield’s

Materials and Methods • Bacteria: Escherichia coli • Medium: TSA • Dilution buffer: Butterfield’s phosphate buffer (BPB, p. H 7. 2) • Water bath at 60, 70, 80°C • Pipette • Petri-dish • incubator

Procedures 1. Incubate two flasks of E. coli culture. 2. Turn on water bathes

Procedures 1. Incubate two flasks of E. coli culture. 2. Turn on water bathes before class begins. Make sure water bathes have enough water. 3. Each group has one tube containing phosphate buffer saline (PBS) into water baths. 4. Place the tube into the water bath. 5. Aliquot the E. coli culture into tubes. Each table has one tube. (teaching assistant) 6. Take the PBS tube from water bath. 7. Transfer 1 m. L E. coli culture into a tube containing 9 m. L PBS. Place the culture into one tube immediately. 8. Bacterial density in each tube should be at 108 CFU/m. L.

7. Start timing. Gently shake the tubes during heating. 8. When heating time is

7. Start timing. Gently shake the tubes during heating. 8. When heating time is achieved, place the tubes under running tap water. Gently rotating the tube during cooling for one minute. 9. Decimally serial dilution 10. For 0 dilution using pour plate : transfer one m. L of dilutant into a petri-dish. Gently mixed agar and bacterial suspension. 11. For other dilutions using spread plate: transfer 0. 1 m. L onto the agar surface. 12. Spread the plates, DO NOT pack the plates vertically. 13. Place the plates into 37 C incubator. Incubate at 37 C for 24 hour. Count colonies.

group temperature time dilutions 1 60°C 12 min 0 -1 -2 -3 2 60°C

group temperature time dilutions 1 60°C 12 min 0 -1 -2 -3 2 60°C 8 min -1 -2 -3 -4 3 60°C 6 min -2 -3 -4 -5 4 60°C 4 min -3 -4 -5 -6 5 70°C 6 min 0 -1 -2 6 70°C 6 min 0 -1 -2 7 70°C 4 min 0 -1 -2 -3 8 70°C 2 min -1 -2 -3 -4 9 75°C 4 min 0 -1 -2 10 75°C 2 min 0 -1 -2 11 75°C 1 min 0 -1 -2 值日組(1) No heating control -6 -7 -8

1 m. L 9 m. L Bacterial culture 9 m. L Heating 1 m.

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

Control 1 m. L 9 m. L Bacterial culture 0 -1 -2 -3 -4

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

 • Record colony number: 25 -250 • Calculate average number of the duplicate

• 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 log 10 CFU/m. L • Calculate bacterial population of control • Example: 42 x 108= 4. 2 x 109=9. 62 log 10 CFU/m. L – Therefore, bacterial population in the first dilution tube (the same dilution used for heating process) should be = 4. 2 x 108=8. 62 log 10 CFU/m. L – The control number is time 0 number (the number on Y axie)

 • • • Record bacterial population. Draw a figure. Use regression function. Y

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

 • • y = -1. 0155 x + 8. 3753 when y= 6,

• • y = -1. 0155 x + 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 • 12 D – Reduce 12 log scale

Population (log CFU/m. L) 60℃ 75℃ min

Population (log CFU/m. L) 60℃ 75℃ min

Z values • • • Combine data from all groups. Draw a figure. Use

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.

Z values (℃ or ℉) • log D values vs. temperature • The time

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