Experiment no.

10
Determination of Thermal Death Time (TDT)
Aim: To determine the Thermal Death Time (TDT) of the given bacterial culture
Principle: Bacteria have varying heat sensitivities. Some of them are killed by even a brief
period of exposure to a particular high temperature, while others require longer period. The lethal
time is of practical importance as it is constant for given bacterium. The minimum time taken to
kill the given cell suspension when exposed to a fixed elevated temperature is known as
Thermal Death Time. Its required for the culture characterization, for deciding the time of
exposure to kill that organism, in fermentation industries for maintaining the temperature and
time below lethal levels. When a series of tubes containing washed cells of 24hr culture are
exposed to constant temperatures at increasing time intervals and inoculated into nutrient media,
only the cells that survived the high temperature will grow and produce turbidity after
incubation. The first tube that shows no growth in the series is the TDT value for the culture at
that temperature.
Requirements:
1.
2.
3.
4.
5.

Sterile test tubes

Culture suspension
Water bath set at 800C
Sterile nutrient broth
Miscellaneous: micropipettes, tips, incubator set at 370C

Procedure:
1. Take a set of 8 sterile test tubes and label them as 5, 10, 15, 20, 30 mins, PC, NC and MC
2. Dispense 0.1ml of the given culture suspension into each of the first 7 tubes. Do not
dispense any culture in the tube labeled MC.
3. Set the constant temperature water bath to 800C and switch on the equipment.
4. As soon as the temperature reaches 800C, arrange the first five tubes in the metal rack and
remove each tube after its required treatment time.
5. Dispense 5ml of sterile nutrient broth to all these tubes except NC (Negative control).
6. The tube labeled PC (Positive control) will have unheated culture and medium. And tube
labeled MC will have only the medium and no culture.
7. Place the tube labeled NC (containing 0.1ml culture) in a boiling water bath at 1000C for
20 mins and dispense 5ml nutrient broth in it.
8. Incubate all the tubes at 370C for 24 hours.
9. Entire procedure has to be carried out in total aseptic conditions.
Observation: After incubation, the tubes were observed for turbidity by shaking gently. It was
seen that NC and MC tubes remained sterile and no turbidity was seen. PC tube showed uniform

Practical Handout - Microbiology

Sem III (BE Biotech)

Sruthi Pillai
TSEC

turbidity of pure culture. Among the rest of the tubes, 5,10,15,20 tubes showed turbidity but the
30mins tube had no cell growth.
Result & Discussion: Thermal Death Time (TDT) of the given bacterial culture was found to be
30 minutes at 800C.
Draw this table on LHS:
Table no. 1: observation of cell growth after 24hrs at 370C
Time of exposure
at 800C (mins)

Growth after
24hrs at 370C

10

15

20

30

PC

NC

MC

Practical Handout - Microbiology

Sem III (BE Biotech)

Sruthi Pillai
TSEC