Effect of temperature on growth of microorganisms- TDT and TDP

 Aim

To determine the thermal death time and thermal death point of the given test organism

Principle

Temperature is one of the most important physical factors influencing the growth of microorganisms. Bacteria unlike eukaryotes lack homeostatic mechanism and they do not regulate the heat generated by metabolism thus are affected readily by changes in temperature. Enzymatic reactions have maximum efficiency at optimum temperature which varies with organisms. For every 10⁰C rise in temperature, there is 2 fold increase in the rate of enzyme catalyzed reactions for a limited range of temperature. At high temperatures, proteins are irreversibly denatured and there is a total enzyme destruction. At low temperature, the enzyme reactions are merely inactivated and are thus less harmful.

Bacteria are divided into three major groups with respect to their temperature requirements:

1)     Psychrophiles with optimum temperature between 0 and 20⁰C

2)     Mesophiles with optimum temperature between 20 and 40⁰C

3)     Thermophiles with optimum temperature between 40 and 60⁰C

Normally, the lethal range of temperature for bacteria is between 50 and 100⁰C. Time of exposure is a vital factor in assessing the lethal effect of high temperature on bacterial cells. Determination of thermal death time (TDT) and thermal death point (TDP) are done for this purpose.

1)     Thermal death point (TDP) – Temperature at which an organism is killed in 10 minutes of exposure. Lethal action of heat has a temperature-time relationship. Thermal death point is done to determine the degree of heat tolerance of the organism. Some factors such as pH, moisture, composition of media and age of cells influence TDP.

     2)     Thermal death time (TDT) – The time required to kill cells/spores at a given temperature. The length of time that the microbes are exposed to heat contributes to lethal effect. This is assessed by exposing cells to fixed temperature which is determined as thermal death time for increasing periods of time.

 

Materials required

Cultures of S. aureus, E. coli, nutrient tubes, nutrient agar plates, water bath, incubator etc

Methodology

1)     Thermal death point (TDP)

 Into each of the sterile test tubes, 5 ml of sterile nutrient broth was dispensed and tubes were marked 40, 50, 60, 70, 80, 90 and 100 ⁰C for different organisms. A loopful of culture was inoculated into the respective tubes and incubated for ten minutes at each temperature. They were then plated on nutrient agar plates and incubated at 37⁰C for 24 hours. The temperature above which the organism was completely killed and did not grow was noted and this was determined as the thermal death point of the organism.

2)     Thermal death time (TDT)

Each sterile tube containing 5 ml nutrient broth was inoculated with loopful of cultures and incubated at their thermal death point (TDP). A loopful of cultures from the tubes were taken at regular intervals of 3 minutes each, starting from 0 minutes till 15 minutes and plated on to nutrient agar followed by incubation at  37⁰C for 24 hours. The time period above which the organisms were completely killed and did not grow was determined as thermal death time (TDT).

Result

The thermal death point (TDP) and thermal death time (TDT) for S. aureus was --------- and for E. coli was ----------.