- Learn how temperature, moisture and UV light affect microbial growth.
- Be able to determine the thermal death point (TDP) and thermal death time (TDT).
- Understand the uses of dry heat and moist heat and the advantages and disadvantages of each.
Just as there is a great deal of diversity in the metabolic properties of bacteria, there is also a great deal of diversity in the types of environments in which different species of bacteria can survive. Microbes are affected in different ways by their physical environment. In today’s lab you will perform experiments that will determine the effects of some of these physical factors on microbial growth.
Effect of temperature
Prokaryotes are found in all types of environments. Some microbes are adapted to live in very cold temperatures, whereas others can survive only in very hot temperatures. Each species has a minimum temperature (lowest temperature for growth), maximum temperature (highest temperature for growth) and optimal temperature (temperature at which it grows best). The ranges of temperatures that different types of microbes grow at are as follows:
Figure 7.1.1: Growth Rates for Different Microorganisms in Response to Temperature
Use the chart to determine the range of growth temperatures for the following:
Thermophiles_________________________________________________________________________ Hyperthermophiles ___________________________________________________________________.
In addition to the categories listed here, some bacteria are classified as thermoduric—these species can survive short bursts of heat but will only grow at lower temperatures.
Temperature Control Methods
Because microbial growth can be affected by temperature, both high and low temperatures can be used to control the growth of microorganisms.
One of the most important means of controlling bacterial growth is through the use of temperature. Low temperatures are primarily bacteriostatic—they inhibit bacterial growth and/or reduce the total number of bacteria. Low temperatures inhibit enzyme activity, so biochemical reactions are slowed or cease, thus reducing the rate at which the bacteria can metabolize and reproduce. This inhibition of enzymatic activity is usually not permanent—if the temperature increases, the enzymes can function at their normal rate, and the bacteria will resume metabolizing and reproducing. Low temperatures are commonly used to prevent food from “spoiling”—i.e., to inhibit bacterial growth, but low temperatures cannot be used to sterilize materials.
High temperatures can be bactericidal—they can kill bacteria. When you heat your inoculating loop in the Bunsen burner flame this results in sterilization, since the bacteria are incinerated. Of course not all materials can be sterilized by incineration. Both dry heat methods and moist heat methods can be used to kill bacteria. Dry heat kills by causing oxidation of cellular molecules and by desiccating (drying) the bacteria. Dry heat is commonly used to sterilize materials that could be damaged by moisture (corrosive metal surgical instruments, dry powders). Dry heat methods generally require longer times and higher temperatures, and are less penetrating than moist heat methods. For example, the hot air oven requires 2 hours at 160 – 180°C to sterilize materials.
Moist heat works by denaturing nucleic acids and enzymes in the bacteria; once these molecules are denatured they are no longer capable of functioning, so even if the temperatures are reduced, the bacteria are incapable of metabolizing or reproducing. Moist heat methods include boiling, pasteurization and autoclaving.
1. Boiling: The temperature of boiling water at sea level is 100°C. This temperature is high enough to kill many vegetative cells, but the exact time required varies depending on the bacterial species. Boiling does not guarantee sterilization, because some bacteria can produce spores that are resistant to high temperatures.
2. Pasteurization: Pasteurization is a heating method that is used to control the growth of microbes in food materials such as milk and fruit juices. Regular pasteurization (the holding or batch method) is a low heat treatment (63°C for 30 min.) that is used to reduce the number of bacteria to what are considered to be acceptable levels. It is primarily focused on eliminating Mycobacterium tuberculosis, Escherichia coli and Salmonella sp. from milk. More recently two other pasteurization methods have been developed: flash pasteurization (71.6°C for 15 sec) and the ultra-high temperature (UHT) method (140°C for 3 sec.) The UHT method can sterilize if it is done under proper aseptic conditions.
3. Autoclave: The autoclave is an instrument that uses steam under pressure to destroy microbes. An autoclave is generally set to apply 15 lbs/in2 of pressure, which allows liquids inside the autoclave to reach a temperature of 121.5°C, a higher temperature than can be achieved by a liquid at normal atmospheric pressures. Autoclaves are capable of achieving sterilization—killing all forms of life, including viruses and spores. They are commonly used to in the laboratory to sterilize growth media, glassware and other solutions. They are used in hospital settings to sterilize bedding, IV solutions, instruments and other heat-resistant objects.
Figure 7.1.2: Effect of temperature on bacterial growth
Determining the Effectiveness of Heating Methods
Since heating methods are commonly used to control microbial growth, it is important to be able to define the effectiveness of a heating method for a particular bacterial species. One way to do this is to determine the thermal death point (TDP) and the TDT (thermal death time).
Thermal Death Time (TDT)
The TDT is the minimum time it takes to kill a population of microbes at a specific temperature.
Figure 7.1.3: Death curves for three species at 70°C
Figure 7.1.3 shows death curves for 3 bacterial species (A, B, C), each treated at 70°C for the given time period. Based on the definition of TDT, provide the following information:
What is the TDT for Bacterial species A at 70°C? _______________________
Which bacterial species is more likely to be a thermophile? _________________
Fifteen minutes; C
Thermal Death Point (TDP)
The TDP is the lowest temperature that is required to kill a population of microbes when applied for a specific time.
Figure 7.1.4: Death curves for three species treated for ten minutes
Figure 7.1.4 shows death curves for bacterial species A, B and C, each treated for 10 min. over a range of temperatures. Based on the definition of TDP, provide the following information:
What is the TDP for Bacterial species C at 10 min.? ________________________
Which bacterial species is more likely to be a psychrophile? __________________
As you learned in above, different bacterial species have different temperature requirements for growth. It is therefore not surprising that it would require higher temperatures or longer heating times to kill some bacteria than others.
Effect of moisture
All living organisms, including bacteria, require water to survive. However, bacterial species vary in their ability to survive in a dry environment. Although some species will die very quickly under dry conditions, others can persist for varying amounts of time. The bacterial endospore is not affected by dry conditions, and can germinate to form vegetative cells when it finds itself in a moist environment. Other nonspore formers can also persist in the environment for varying amounts of time based on their cell wall properties and amount of glycocalyx they produce.
Effect of UV radiation
Ultraviolet (UV) radiation is electromagnetic radiation or light having a wavelength longer than that of x-rays but shorter than that of visible light (between 100 nm - 400 nm). UV radiation damages the DNA molecule by causing the formation of pyrimidine dimers, which are unnatural bonds between adjacent thymine or cytosine nucleotides. These bonds distort the DNA molecule (see Figure 5.), which can interfere with the processes of DNA replication and transcription. Although cells do have repair mechanisms to fix the damage, DNA damage caused by UV radiation exposure (particularly in the range of 240-260 nm) can result in mutations, and long-term exposure can overwhelm the repair mechanisms and cause cell death. UV radiation is used to control microbial growth in such locations as sterile hoods, operating rooms, and other areas when they are not in use. Humans need to use these methods with caution, as the UV radiation can damage skin cells as well.
Figure 7.1.5: Effect of UV light on DNA
psychrophile, mesophile, thermophile, hyperthermophile, thermoduric, UV radiation, pyrimidine dimers, bacteriostatic, bactericidal, dry heat, moist heat, thermal death point, thermal death time, autoclave, pasteurization