Moist Heat

It is possible to quantitate the response of microorganisms to heat by measuring the time required to kill them at different temperatures. The lowest temperature required to sterilize a standardized pure culture of bacteria within a given time (usually 10 minutes) can be called the thermal death point of that species, and, conversely, the time required to sterilize the culture at a stated temperature can be established as the thermal death time.

Purpose	To demonstrate destruction of microorganisms by moist heat applied under controlled conditions of time and temperature
Materials	Tubed nutrient broths (5-ml aliquots) Nutrient agar plates Sterile 1.0-ml pipettes 24-hour broth culture of Staphylococcus epidermidis Six-day-old broth culture of Bacillus subtilis

Procedures

1. Set up a beaker water bath and heat to boiling.
2. Divide one nutrient agar plate in half by marking the bottom of the plate with a wax pencil or ink marker.
3. Streak a loopful of the S. epidermidis culture onto one-half of the nutrient agar plate. Label the section of the plate with the name of the organism and the word Control.
4. Repeat step 3 with the culture of B. subtilis, inoculating the second half of the plate.
5. Place the “control” plate in the 35°C incubator for 24 hours.
6. Divide two nutrient agar plates into 4 quadrants by marking the bottom of the plates with a wax pencil or ink marker. Label one plate S. epidermidis and the other B. subtilis. Label the 4 quadrants on each plate as follows: 5, 10, 15, 30 minutes.
7. Take a pair of broth tubes and inoculate each, respectively, with 0.1 ml of S. epidermidis and B. subtilis. Place these tubes in the boiling water bath. Note the time.
8. Leave the pair of broth cultures in boiling water for 5 minutes. Remove the tubes and cool them quickly under running cold tap water. Streak a loopful of each boiled culture onto the quadrant of nutrient agar labeled 5 minutes.
9. Return the tubes to the boiling water bath for an additional 5 minutes. Begin timing when the water comes to a full boil. Cool the tubes as in step 8 then streak a loopful of each culture onto the quadrant of nutrient agar labeled 10 minutes.
10. Repeat step 9 twice more, streaking loopfuls of culture onto the quadrants of the plates labeled 15 and 30 minutes, respectively.
11. Incubate subcultures from boiled tubes at 35°C for 24 hours.

Results

1. Read all plates for growth (+) or no growth (−). Record your results in the chart.
   
   


2. State your interpretation of these results for each organism:
   • S. epidermidis:
   • B. subtilis: