The thermal death point of bacteria

Heat can be used to kill microbes and different organisms have different susceptibilities to heat. Exposing bacteria to a range of temperatures and then subculturing these cultures will show if bacteria have survived or not.

This is how to determine the thermal death point of a culture. You are provided with three cultures and four sterile tubes for each. In addition one sample for each culture has already been autoclaved for fifteen minutes. This tube is marked with tape showing dark brown stripes, indicating a successful autoclave treatment. Autoclave tape appears uniformly buff until it has been exposed to the appropriate temperature in the autoclave, when brown stripes develop.

Using aseptic technique, dispense a small volume of the first untreated sample into its four sterile tubes.

Repeat the procedure for all three bacterial cultures.

Remember to use a new pipette for each sample.

Three of the four sets of tubes should be placed in pre-set waterbaths and exposed to temperatures of fifty-five, sixty-five and seventy-five degrees Celsius.

To expose cultures to a temperature of one-hundred degrees Celsius use a boiling water bath. Place some water in the bottom of an enamel mug and bring it to the boil on a tripod placed over a Bunsen burner. Do not place much water in the mug, since it will take too long to reach boiling point. Once it is boiling, carefully place your test cultures in the mug. Tubes should be left in the boiling water - or in the relevant waterbath - for fifteen minutes.

Having exposed your cultures to a range of temperatures, you should now test for survival of bacteria within the culture by plating them onto nutrient agar.

For each test organism, divide a nutrient agar plate into six sections. Label each section: "C" for the control culture, followed by the five different temperatures to which the cultures have been exposed.

The first section is inoculated with the original culture, which has not been exposed to elevated temperatures. Each of the remaining cultures will be inoculated onto the relevant section of the plate.

Remember this is an aseptic procedure. Flame the loop between samples - do not put the cap on the bench - and flame the top of the tube before and after sampling.

Repeat the procedure for all three cultures.

The plates are incubated at thirty-seven degrees Celsius overnight.

The bacteria on this plate indicate survival of the unexposed control, and of bacteria exposed to fifty-five and sixty-five, but not to higher temperature. This indicates that the thermal death point for this organism is between sixty-five and seventy-five degrees Celsius.

On this plate, the bacterium has survived exposure to elevated temperatures less well. There is only growth of the control bacterium and of cells exposed to fifty-five degrees Celsius. This bacterium, therefore, has a thermal death point that lies between fifty-five and sixty-five degrees Celsius.

This plate shows some growth in all divisions except that inoculated with the autoclaved culture. There is a luxuriant growth of bacteria exposed to temperatures of fifty-five and sixty-five degrees Celsius. At senventy-five there is a slight drop in the number of colonies. This is because vegetative cells in the culture are susceptible to exposure at this temperature. Even after boiling for fifteen minutes, a few colonies can be seen. These result from the germination of heat-resistant spores that have survived the boiling. Exposure to moist heat in an autoclave will kill even these spores.

You have not been told which culture is which. You should work this out by examining a Gram film prepared from the bacteria from each set of cultures.

Remember E. coli is a rod-shaped bacterium that is Gram-negative. It cannot retain the crystal violet-iodine complex of the Gram stain and so it appears as small red rods when viewed under the microscope.Both Enterococcus faecalis and Bacillus subtilis are Gram positive, and retain the crystal violet-iodine stain complex. Their shape can differentiate them. Members of the genus Bacillus, as their names suggest, are rod-shaped; some cells (this shot indicates an enlarged cell with its spore) contain spores, seen as an absence of staining. Enterococci have round or round-ish cells.