Lab 22: Physical Methods of Heat Control

temperature: optimal growth ranges

1. psychrophilic 2. psychrotrophic 3. mesophilic 4. thermophilic 5. hyperthermophilic

pasteurization

1. temperature is maintained at 63 degrees Celsius for 30 min or 72 degrees Celsius for 15 seconds 2. kill designated organisms that are pathogens

Effectiveness of Heat: Measurements

1. thermal death time (TDT) 2. thermal death point (TDP) 3. decimal reduction time (DRT or value D)

Assume that a DRT value for autoclaving a culture is 1.5 minutes. How long would it take to kill all the cells if 10x6 cells were present. What would happen if you stopped the heating process at 9 minutes?

10^6 X (0.1) ^7=0.1 You need 10.5 minutes of killing to kill one million cells. 7x 1.5 minutes= 10.5 minutes

Give an example of an application (use) of thermal death time.

In order to make sure our food is sterile to eat, we must know the thermal death time of whatever microbes might be living in the food (e.g. food canning).

decimal reduction time (DRT or value D)

the time, in minutes, in which 90% of bacteria at a given temperature will be killed

thermal death point (TDP)

temperature required to kill all bacterial in a liquid culture in 10 minutes

autoclaving

1. most effective moist heat method 2. steam under pressure 3. increased pressure raises the boiling point of water 4. standard conditions are 15 psi, at 121 degrees Celsius, 15 minutes 5. kill endospores and render materials sterile

moist heat

1. moisture transfers heat energy to the microbial cell more efficiently than dry air, results in denaturation of enzymes 2. methods include pasteurization, boiling, and autoclaving

Results of E. coli 63 degrees Celsius

0 time =1 growth 1 min=1 growth 2 min=.5 growth 5 min= 0 growth 15 min=0 growth

Results of B. subtilis 63 degrees Celsius

0 time =1 growth 1 min=2 growth 2 min=4 growth 5 min= 3 growth 15 min=4 growth

Results of E. coli 80 degrees Celsius

0 time =3 growth 1 min=0 growth 2 min=0 growth 5 min= 0 growth 15 min=0 growth

Results of S. epi 80 degrees Celsius

0 time =3 growth 1 min=1 growth 2 min=0 growth 5 min= 1 growth 15 min=.5 growth

Results of S. epi 63 degrees Celsius

0 time =4 growth 1 min=1 growth 2 min=2 growth 5 min= 1 growth 15 min=1 growth

Results of B. subtilis 80 degrees Celsius

0 time =4 growth 1 min=2 growth 2 min=1 growth 5 min= 3 growth 15 min=3 growth

psychrophilic

0-20 degrees Celsius

Vegetative cells will be killed at __________ degrees Celsius in _________ minutes

1. 100 2. ten

boiling

1. 100 degrees Celsius 2. 10 minutes 3. will kill vegetative bacterial cells 4. doesn't inactive endospores

Give an example of a non-laboratory use of the following methods to control microbial growth.

1. Incineration: cremation of the dead 2. Pasteurization: milk 3. Autoclaving: metals to be inserted into surgical tools

Types of Heating

1. dry heat 2. moist heat

dry heat

1. hot-air ovens or incineration, denatures enzymes, dehydrates microbes, and kills by oxidation effects 2. standard application is a 170 degree Celsius hot-air oven for 2 hours 3. heat of hot air is not readily transferred to a cooler body such as a microbial cell

Procedure

1. inoculate 0 time section, place in bath. 2. inoculate 1 min section, place in bath, 1 min. 3. inoculate 2 min section, place in bath, 2 min 4. inoculate 5 min section, place in bath, 5 min 5. inoculate 15 min section, place in bath, 15 min 6. incubate 7. record your results for growth and the type of growth (minimum, moderate, heavy, maximum growth

psychrotrophic

20-30 degrees Celsius

mesophile

25-40 degrees Celsius

thermophilic

45 to 65 degrees Celsius

hyperthermophilic

80 degrees Celsius +

Compare the heat sensitivity of fungal spores to that of bacterial endospores.

Fungal spores are more sensitive to heat. Fungal spores can be killed at 60 degrees Celsius. Endospores can stand temperatures up to 100 degrees Celsius

Explain why fungi and Bacillus sometimes grow better after heat treatment.

Fungi and Bacillus are producing endospores. Heat activates these endospores meaning the Bacillus and fungi are able to divide and replicate because the heat has activated the endospores

Compare the effectiveness of autoclaving and dry heat.

Sterilizing in autoclaving is most effective when the organisms are contacted by steam directly or are contained in H20. Dry heat can sterilize items that can't be sterilized in steam.

Indicators are used in autoclaving to ensure that sterilization is complete. One type of chemical indicator turns color when it has reached a specific temperature; the other type turns color when it has reached a specified temperature and been exposed to steam. Which type of indicator should be used?

Tape indicators will change color when temperatures are over 120C. This indicates that the required temperature was attained and you are unlikely to have heat-resistant spores. This is the best indicator to use.

A biological indication used in autoclaving is a vial containing 10^9 Geobacillus ste. cells that is placed in the autoclave with the material to be sterilized. After autoclaving, the vial is incubated and examined for growth. Why is this species used rather than E. coli or Bac. sub.?

The substance is used because it can withstand high temperatures which are needed to be sterilized without the bacteria drying. It is a thermophilic bacteria, which has the presence of heat resistant endospores.

thermal death time (TDT)

length of time required to kill all bacteria in a liquid culture at a given temperature