The Lethal Effects of High Temperature on Bacteria
Each bacterial species possesses the ability to grow over a range of temperatures, but if the maximum temperature for growth is exceeded, a killing effect will be observed. The susceptibility of different organisms to high temperature can be measured as the Thermal Death Point or the Thermal Death Time. The former is the lowest temperature at which a suspension of bacteria is killed in 10 minutes; while the later is the time required to kill a finite number of cells or spores at a given temperature.
Wet heat is often used as a means of killing bacteria. How effective this is as a means of destroying organisms depends on the type of bacteria. Generally, Gram negative organisms, which are usually found inside other organisms (such as people) are the most sensitive to temperature. Gram positive organisms, which are often found free in the environment, are a little less sensitive to temperature. Endospore-formers (Bacillus and Clostridium) are the most resistant, due to the highly resistant endospore. It is important to note that the vegetative cell of a spore former is only as resistant as other Gram positive organisms.
In this exercise, students will expose a bacterial suspension to temperatures of 60°C, 80°C, and boiling water. At intervals of 5, 10 and 20 minutes (total time) a sample of the suspension will be removed and plated out to test for viability. We will compare results from a gram positive organism, a gram negative organism and a spore-forming organism.
Materials:
1. Three broth cultures of the organism to be studied
2. Three Tryptic Soy Agar plates
3. Water baths set for 60°C, 80°C, and boiling water
4. Inoculating loop
Procedure:
1. Follow group descriptions provided by instructor. Students at a table will work with the same organism, but each set of lab partners will expose the organism to a different temperature. Different tables will work with different organisms.
2. Obtain three Tryptic Soy Agar plates, invert them, and use a marking pen to label them 60, 80, and 100. Each set of lab partners will also label with their exposure time, i.e., 0 min, 5 min, 10 min, and 20 min.
3. Inoculate the 60, 80, and 100 plate with a loopful of your test culture prior to exposure time. Streak the loop in a "Z" pattern. Zero time will serve as a control to demonstrate organism viability prior to exposure to high temperature.
4. Place the culture in the appropriate water bath. After 5, 10, and 20 minutes (total time), mix your heated specimen by gently tapping the tube and then obtain a loopful of inoculum. Streak the appropriate plate. Work quickly so that cooling does not occur while the sample is being obtained, and return your culture to the bath after the sample has been collected.
5. Incubate plates as directed until the next laboratory period.
7. At the next laboratory period, examine each plate for growth and attempt to determine the Thermal Death Time of your test specimen for each temperature studied.
8. Compare your results with those obtained by the other groups in the class.