1. Each microorganism has an optimum growth temperature, which below it growth decreases reversibly and above it decreases irreversibly due to degradation of vital molecules. Lethal effect of heat

2. Sensitivity of microorganisms to heat Principle Non spore forming bacteria are sensitive to heat at temperature exceeding 55 o C while spore forming bacteria are only heat sensitive to higher temperature.

3. In order to investigate microbial resistance (sensitivity) to heat, the two following parameters are determined: 1) Thermal death point (TDP) The lowest temp required to kill all m.o. in suspension within certain time (usually 10 min). 2) Thermal death time (TDT) (extinction time) Shortest time required to kill all m.o. in a suspension at certain temp.

4. Materials 1- sterile Petri dishes. 2- nutrient agar tubes. 3- two sets of five test tubes containing broth cultures of Staphylococcus aureus, Escherichia coli, or Bacillus subtilis. 4- A thermometer. 5- A water bath. 6- Inoculating loop.

5. Procedure 1- On the back of each plate, draw equal six sectors & mark them as follows: C: for control U: for uninoculated 5: for 5 minutes exposure 10: for 10 minutes exposure 20: for 20 minutes exposure 30: for 30 minutes exposure 2- On the top of plate write the name of each of the test m.o.

6. C 5’ 10’ 20’ 30’U

7. 3- Aseptically pour the nutrient agar in the plates & allow to solidify. 4- The sectors marked “U” in all plates are left uninoculated i.e. –ve control for sterility of the medium. 5- Inoculate sector marked “C” with the appropriate organism as a check for its viability by making a single straight line for a fixed distance on the agar surface.

8. 6- Maintain the water bath at the specified temp. (60, 80, 90, or 100 o C) & immerse the culture tube in the water bath at the specified temp. 7- At different time intervals (5, 10, 20 & 30 min.) take one loopful from the culture tube after shaking & streak on the agar surface in the respective sector. 8- Invert the plates & incubate at 37 o C for 24 h.

9. 9- Examine the plates for the presence (+) or absence (-) of growth & record the results in the table. 10- From the data presented in the table, determine i) TDP for each m.o. (after 10 min). ii)TDT for each m.o. at different temperatures.

10. Bacillus S. aureus 100907060100907060 temp. 0 c Time min ++++---+ 5 -++ +---+ 10 -+++---- 20 -+++---- 30 ≤ 10 > 5 >30 ≤ 5 ≤ 20 > 10 TDT TDP for S. aureus = ≤ 70 & > 60 TDP for Bacillus = > 90 & ≤ 100