Physical Methods of Control - Heat
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
1.
2.
3.
4.
5.
6.
Explain what is meant by moist heat sterilization.
Explain what is meant by dry heat sterilization.
Explain what is meant by an autoclave.
Explain what is meant by psychrophile, mesophile, and thermophile.
Explain what is meant by thermal death time.
Determine thermal death time.
One of the most critical aspects of aseptic processing is the control and decontamination of
microorganisms in a clean room environment. This includes the sterility (absence of viable
microorganisms) of materials used in aseptic processing, which may include equipment,
instrumentation, packaging, water, gloves, and the product itself. In a given environment,
microorganisms' growth is affected by two categories of factors: 1) physical, and 2)
biochemical.
These factors affect the rate of and the potential for the growth of the microorganism. In
this, these factors can be used to control microorganisms. As we have seen,
microorganisms exhibit great diversity as to their oxygen requirements, this is also true in
regard to their growth temperature range requirements. Many bacteria will grow at room
temperature (20°C to 25°C) and at body temperature (37°C). Still many others will grow
below and above these temperatures. The majority of microorganisms are categorized into
three growth temperature ranges:
1. Psychrophiles: Thrive in environments with temperatures ranging from -5°C to 20°C.
2. Mesophiles: Thrive in environments with temperatures ranging from 15°C to 45°C.
3. Thermophiles: Thrive in environments with temperatures ranging from 42°C to 110°C.
Recall that certain microorganisms can form spores that will allow them to survive in
extreme conditions until more favorable conditions return. Such is the case with spore
formers as the spore is able to with stand certain temperature extremes. Antarctic bacteria
can remain dormant for at least 10,000 years at a temperature of -14°C in ice 430 meters
deep. And some bacteria can withstand hours of boiling. Even so, temperature can be used
as an effective means to control microorganisms.
In this laboratory exercise we will be investigating the temperature ranges in which
microorganisms thrive and the use of heat as a physical means of microbial control. The
two most effective means for sterilization are: 1) dry heat sterilization and 2) moist heat
sterilization. See Table 1.
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Table 1. Recommended use of heat to control bacterial growth.
Treatment
Incineration (dry heat)
Temperature
>500°C
Dry heat (hot air oven)
160°C
Dry heat (hot air oven)
170°C
Autoclave and pressure cooker
(steam under pressure, moist heat)
121°C
Boiling (moist heat)
100°C
Intermittent boiling (moist heat)
100°C
Pasteurization (batch method,
moist heat)
63°C
Pasteurization (flash method,
moist heat)
72°C
Ultrapasteurization (direct
method, moist heat)
140°C
Effectiveness
Vaporizes organic material on
nonflammable surfaces but may
destroy many substances in the
process.
2 hours for materials that must
remain dry. Good for glassware
and metal but not for plastic or
rubber items.
1 hour. Same as above. Note
increasing temperature by 10
degrees shortens the sterilizing
time by 50 percent.
15 minutes at 15psi pressure kills
all forms of life including
bacterial endospores. The
substance being sterilized must be
maintained at the effective
temperature for the full time.
30 minutes of boiling kills
microbial pathogens and
vegetative forms of bacteria but
may not kill bacterial endospores.
Three 30 minute intervals of
boiling followed by periods of
cooling kills bacterial endospores.
30 minutes kills most vegetative
bacterial cells including
pathogens such as streptococci,
staphylococci and Mycobacterium
tuberculosis.
15 seconds effective on bacterial
cells similar to batch method; for
milk, this method is more
conducive to industry and has
fewer undesirable effects on
quality or taste
2 seconds effective on most
bacterial cells is lethal. For milk,
this method creates a product with
relatively long shelf life at
refrigeration temperatures.
Dry heat methods of sterilization include hot air ovens and incineration where as moist heat
methods include boiling, pasteurization, and autoclaving. Exposures to boiling water for
30 minutes will result in disinfection only, as all vegetative cells will be killed, but the
heat-resistant spore will not. Autoclaving, on the other hand, is a common application of
using free-flow steam under pressure as a means of sterilization, see Figure 1. Standard
conditions for autoclaving are 15 psi, at 121°C, for 15 minutes. This is usually sufficient to
kill spores and sterilize materials.
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Figure 1. Autoclave. (http://bioinfo.bact.wisc.edu)
Applying a method for heat disinfection or sterilization requires a method of measuring
heat effectiveness. The two methods used for measuring heat effectiveness are: 1) thermal
death point (TDP), and 2) thermal death time (TDT). The thermal death point is the
temperature at which an organism in liquid culture is killed in 10 minutes. This method has
been replaced with the more common thermal death time which is the time required to kill
all organisms and spores in a liquid culture at a given temperature.
In this laboratory exercise you will be comparing the heat effectiveness of a dry heat
method with a moist heat method by determining the thermal death time for each method.
You will also determine the optimal temperature for growth and pigment production. Your
instructor may also demonstrate the autoclave.
Materials (per group)
1 Pure Culture Broth of Serratia marcescens
2 Pure Culture Broth of 48 to 72 hour Escherichia coli
3 TSA or Nutrient Agar Slants
10 TSA or Nutrient Broth Tubes
2 Sterile 0.1 mL or 1 mL Pipettes
China Marker or Sharpie
Hot Plate
Dry Oven Set to 70°C
1 400 mL Beaker
1 Glass Thermometer
Inoculating/Transfer Loop
Bunsen Burner
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Procedure 1
Dry Heat Method
Note: Procedure 1 and Procedure 2 should be performed simultaneously to ensure
that both procedures are completed within the lab period.
Acquire a pure culture broth tube of 48 hour Escherichia coli.
Note: Use aseptic technique throughout all transfers.
1. Label five of the uninoculated culture broth tubes: "Dry - Control", "Dry - 10",
"Dry - 20", "Dry - 30", and "Dry - 40" and with your group initials and the date.
2. From the pure culture broth tube of 48 hour Escherichia coli, pipette 0.1 mL into the
tube labeled "Dry - Control".
3. Place the pure culture broth tube of 48 hour Escherichia coli into an oven that has be
preheated to 70°C (use either a metal tube rack or glass beaker to hold the tube in the
upright position).
4. Heat the culture for 10 min. then pipette 0.1 mL into the tube labeled "Dry - 10".
5. Every 10 min. pipette 0.1 mL into the appropriate tube.
6. Incubate the inoculated tubes at 37°C for 24 to 48 hours.
7. Observe the tubes for growth and record your results.
Procedure 2
Moist Heat Method
Acquire a pure culture broth tube of 48 hour Escherichia coli.
1. Set up a hot plate with 200 mL water in a 400 mL beaker and heat to 70°C. Note: Be sure
to maintain this temperature throughout the entire procedure.
2. Label five of the uninoculated culture broth tubes: "Moist - Control", "Moist - 10",
"Moist - 20", "Moist - 30", and "Moist - 40" and with your group initials and the date.
2. From the pure culture broth tube of 48 hour Escherichia coli, pipette 0.1 mL into the
tube labeled "Moist - Control".
3. Place the pure culture broth tube of 48 hour Escherichia coli into the hot water bath that
has be preheated to 70°C.
4. Heat the culture for 10 min. then pipette 0.1 mL into the tube labeled "Moist - 10".
5. Every 10 min. pipette 0.1 mL into the appropriate tube.
6. Incubate the inoculated tubes at 37°C for 24 to 48 hours.
7. Observe the tubes for growth and record your results.
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Procedure 3
Acquire the pure culture broth tube of Serratia marcescens.
Note: Incubation temperatures may range from 5°C to 10°C (refrigerator), 20°C to
25°C (room temp.), and 36°C to 38°C (incubator).
1. Setup and light a Bunsen burner.
2. Label the three uninoculated agar slants, "5°C", "25°C", and "37°C", and with the
name of the organism, your group initials and the date.
3. Inoculate all three agar slants, separately, by transferring one loop-full of inoculum from
the pure culture broth tube using aseptic technique.
4. Incubate the inoculated tubes as marked at 5°C, 25°C, and 37°C for 24 to 48 hours.
5. Observe the tubes for growth and pigment production and record your results.
Results and Observations
For the Dry Heat tubes record your observations below:
Tube
Dry - Control
Dry - 10
Dry - 20
Dry - 30
Dry - 40
Growth (+) or (-)
Thermal Death Time (Yes) or (No)
For the Moist Heat tubes record your observations below:
Tube
Moist - Control
Moist - 10
Moist - 20
Moist - 30
Moist - 40
Growth (+) or (-)
Thermal Death Time (Yes) or (No)
For the inoculated agar slants record your observations below:
Tube
5°C
25°C
37°C
Growth (+) or (-)
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Pigment Production (Yes) or (No)
Laboratory Review
1. Explain the differences between moist heat sterilization and dry heat sterilization:
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2. Explain the difference between boiling moist heat and autoclave moist heat:
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3. Explain the differences between psychrophiles, mesophiles, and thermophiles:
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4. What is the limitation imposed by using dry heat sterilization:
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5. In your own words, define thermal death time:
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6. Temperature is one of the most important factors that influence the activity of bacterial
enzymes. Enzymes have minimal, optimal, and maximal temperatures which relates
directly to the speed at which an enzymatic reaction progresses. At minimal temperature
enzyme activity is very slow. At optimal temperature enzymatic reaction progresses at
maximum speed. At temperatures greater than the maximal temperature, enzymes tend to
denature and become inactive. With this in mind, try to relate enzymatic activity and
pigment production to temperature:
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