Sterilization by heat is the most reliable and relatively inexpensive method to sterilize
Physical Control of Microorganisms: Heat Sterilization
Introduction and Importance:
The killing effect of heat on microorganisms has long been known. Heat is fast, reliable, and relatively inexpensive, and it does not introduce chemicals to a substance, as disinfectants sometimes do. A visit to any supermarket will demonstrate that heat preserved canned goods represent one of the most common methods of food preservation. Laboratory media and glassware, and hospital instruments, are also usually sterilized by heat.
Mode of Action:
Above maximum growth temperatures, biochemical changes in the cell's organic molecules result in its death. These changes arise from alterations on enzyme molecules and the resultant changes to the three dimensional shape of proteins inactivate proteins or chemical breakdown of structural molecules, especially in cell membranes. Heat also droves off water, and since all organisms depend on water, this loss may be fatal. Heat appears to kill microorganisms by denaturing their enzymes.
Principles and Applications of Heat Killing:
Heat—Preferred Agent of Sterilization:
Heat is preferred agent of sterilization for all material not damaged by it. It rapidly penetrates thick materials not easily penetrated by chemical agents.
Measurements to Determine Killing Power:
The killing rate of heat may be expressed as a function of time and temperature. For example, tubercle bacilli are destroyed in 30 minutes at 58oC, but in only 2 minutes at 65oC, and in a few seconds at 72oC. Several measurements have been defined to quantify and killing power of heat. The thermal death point is the temperature that kills all the bacteria in a 24-hour-old broth culture at neutral pH in 10 minutes. Factor to be considered in sterilization is the length of time required.The thermal death time is the time required to kill all the bacteria in a particular culture at a specified temperature. Both TDP and TDT are useful guidelines that indicate the severity of treatment required to kill a given population of bacteria.
Decimal reduction time (DRT or D value) is a third concept related to bacterial heat resistance. DRT is the time, in minutes, in which 90% of a population of bacteria at a given temperature will be killed (in table 1, DRT is 1 minute). (The temperature is indicated by a subscript: D80°C, for example.)
Table 1
Microbial Death Rate: An Example
Time (mm)
Death per Minute
Number of Survivors
0
0
1,000,000
1
900,000
100,000
2
90,000
10,000
3
9000
1000
4
900
100
5
90
10
6
9
1
Significance of Measurements:
These measurements have practical significance in industry as well as in the laboratory. For example, a food-processing technician wanting to sterilize a food as quickly as possible would determine the thermal death point of the most resistant organisms that might be present in the food and would employ that temperature. In another situation it might be preferable to make the food safe for human consumption by processing foods containing proteins that would be denatured, thereby altering their flavor or consistency. The processor would then need to know the thermal death time at the desired temperature for the most resistant organism likely to be in the food.
Factors Important for Determination of Time and Temperature:
When determining the time and temperature for microbial destruction with heat, certain factors bear consideration.
1) Type of Organism:
One factor is the type of organism to be killed. For example, if materials are to be sterilized, the physical method must be directed at bacterial spores. Milk, however, need not be sterile for consumption, and heat is therefore aimed at the most resistant vegetative cells.
2) Type of Material:
Second factor is the type of material to be treated. Powder is subjected to dry heat rather than moist heat, because moist heat will leave it soggy. Saline solutions, by contrast, can be sterilized with moist heat but are not easily treated with dry heat.
3) Presence of Organic, Acidic or Basic Material:
Third important factor is the presence of organic matter and the acidic or basic nature of the material. Organic matter may prevent heat from reaching microorganisms, while acidity or alkalinity may encourage the lethal action of heat.
Incineration
Dry Heat
Moist Heat
Live Steam
Fractional Sterilization
Steam Under Pressure
Hot Water
Hot Oil
Desiccation
Physical Methods
1. Incineration
2. Dry Heat
3. Moist Heat
4. Boiling
5. Hot Oil
6. Pasteurization
7. Pressure
8. Filtration
9. Ultrasonic Vibrations
10. Radiation
Incineration
-most common method of treating infectious waste
-toxic air emissions and the presence of heavy metals in ash have limited the use of incineration in most large cities
Moist heat (steam under pressure)
-fastest and simplest physical method of sterilization
-used to sterilize biohazardous trash and heat-stable objects; (e.g., autoclave)
-121 degrees celsius and 132 degrees celsius most common
-media, liquids, instruments autoclaved 15 minutes at 121 deg. celsius
-infectious medical waste is often sterilized at 132 deg cel for 30-60 minutes for steam penetration through waste and displace trapped air inside autoclave bag
Dry heat
-requires longer exposure 1.5-3 hrs and higher temps 160-180 deg cel
-sterilize glassware, oil, petrolatum, or powders
Filtration
-method of choice for antibiotic solutions, toxic chemicals, radioisotopes, vaccines, and carbohydrates which are all heat sensitive
-using high-efficiency particulate air (HEPA) filters designed to remove organisms larger than 3.0 um from isolation rooms, op rooms, and biological safety cabinets (BSCs)
Ionizing (gamma) radiation
used for sterilizing disposables such as plastic syringes, catheters, or gloves before use.
Physical methods of microbial control
Control of microbial growth means the reduction in numbers and activity of the total microbial flora, is effected in two basic ways i.e., either by killing microorganisms or by inhibiting the growth of microorganisms. Controlling of microorganisms is done to prevent transmission of disease and infection, to prevent contamination by the growth of undesirable microorganisms and to prevent deterioration and spoilage of materials by microorganisms. Control of microorganisms usually involves the use of physical agents and chemical agents.
Species of microorganisms differ in their susceptibility to physical and chemical agents. In Spore forming species, the growing vegetative cells are much more susceptible than the spore forms. Bacterial spores are the most resistant of all living organisms in their capacity to survive under adverse physical and chemical conditions. The major physical agents used for the control of microorganisms are temperature, desiccation, osmotic pressure, radiation and filtration.
Temperature
Microorganisms can grow over a wide range of temperature, from very low temperature characteristic of psychrophiles to the very high growth temperatures characteristic of thermophiles. Temperatures above the maximum generally kill, while those below the minimum usually produce stasis and may even considered preservative.
High temperature
High temperatures combined with high moisture is one of the most effective methods of killing microorganisms. Dry heat is used to sterilize surfaces, and materials which are not likely to break down in high heat and which do not contain any liquids, e.g., glass Petri dishes and culture vessels, and metal surgical instruments. Dry heat penetrates more slowly than moist heat which destroys microorganisms by coagulating their proteins and also destroys microorganisms by oxidizing their chemical constituents. Moist heat penetrates more quickly than dry heat, and is used to sterilize culture solutions and agar preparations, and to sterilize surgical instruments etc. Pressurized steam heat is needed to kill bacterial endospores, which can withstand boiling. Typically a pressure of 15 psi (pounds per square inch) is needed to create steam at a high enough temperature (121°C) to kill endospores. Spores of Clostridium botulinum are killed in within 20 minutes by moist heat at 120°C, whereas a 2-h exposure to dry heat at the same temperature is required.
The thermal death time refers to the shortest period of time to kill a suspension of bacteria or bacterial spores at a prescribed temperature and under specific conditions. The thermal death point is the lowest temperature at which all microorganisms in a particular liquid will be killed in ten minutes. The decimal reduction time is the time in minutes that it takes for 90% of a given population of microorganisms to be killed at a given temperature. It is the time in minutes for the thermal death-time curve to pass through one log cycle. A disinfectant has been applied to a contaminated surface and the result is shown in the graph-1 (figure-1).
Figure 1: Graph showing the decimal reduction time
Figure 1: Graph showing the decimal reduction time
The graph is showing the concept of decimal reduction time, the time in minutes to reduce the microbial population by 90 percent. Here, the cells are dying at a constant rate of 90% each minute. The D value is independent of time when the response is logarithmic, that is when the same length of time is required to accomplish any given log decrease in survivors.
The second graph (figure-2), shows the thermal-death-time curve for spores of a bacterial species encountered in a type of canned-food spoilage. All these values express a time-temperature relationship to killing. In thermal death time, the temperature is selected as the fixed point and the time varied. Decimal reduction time is a modification of thermal death time which measures a 90 percent rather than 100 percent kill rate.
Figure 2: Graph showing the thermal-death-time curve
Figure 2: Graph showing the thermal-death-time curve
Low temperature
Low temperature retards the growth of microorganisms by slowing their metabolism, but it does not always kill them and some bacteria (like Listeria) and fungi do grow at near freezing temperatures. Low temperatures are useful for preservation of cultures, since microorganisms have a unique capacity for surviving extreme cold. Refrigeration at 5° C retards the growth of many bacteria and fungi, freezing at – 10° to - 20° C (typical home freezer) is also an effective but not perfect means to retard microbial growth. Thus from a practical standpoint, high temperatures may be considered as microbicidal and low temperatures as microbistatic.
Moist heat
Steam under pressure
Heat in the form of saturated steam under pressure is the most practical and dependable agent for sterilization. Pressurized steam heat has the advantages of rapid heating, penetration, and moisture in abundance, which facilitates the coagulation of proteins. The prime example which provides steam under pressure is an autoclave, which is a double-jacketed steam chamber equipped with devices which permit the chamber to be filled with saturated steam and maintained at a designated temperature and pressure for any period of time. It is not the pressure that kills the organisms but the temperature of the steam. The autoclave (figure-3) is used to routinely sterilize many media, solutions, discarded cultures, and contaminated materials inside laboratory.
Figure 3: Cross sectional view of an autoclave
Figure 3: Cross sectional view of an autoclave
Fractional sterilization
Some microbiological media, chemical solutions, and biological materials are sterilized by fractional sterilization which involves heating the material at 100°C on three successive days with incubation periods in between. Resistant spores germinate during the incubation periods; on subsequent exposure to heat, the vegetative cells will be destroyed. If spores are present which do not germinate during the incubation periods, the material will not be sterilized.
Boiling water
Boiling water can't thoroughly sterilize the contaminated materials. Though all vegetative cells will be destroyed within minutes by exposure to boiling water but some bacterial spores can withstand this for many hours. Boiling water can be used as a method of disinfection but not as a method of sterilization.
Pasteurization
The process of pasteurization was discovered and named after Louis Pasteur, who discovered that controlled heat treatment was effective in preventing the spoilage of beer and wine. The idea behind pasteurization is to use enough heat to reduce the number of microbes without negatively affecting the taste or quality of the food product. Now milk, cream and certain alcoholic beverages are subjected to pasteurization, which reduces the microbial load and kills many pathogens but does not kill all bacterial pathogens and does not kill endospores.
Dry heat
Hot-air sterilization
Hot-air sterilization is used when it is undesirable to make a direct contact of the materials to be sterilized, e.g., certain laboratory glassware(petridishes and pipettes), oils, powders and similar substances with the autoclave. Hot air sterilization is done in a special type of apparatus e.g., an electric or a gas oven. For laboratory glassware, a 2-hour exposure to a temperature of 160°C is sufficient for sterilization.
Incineration and flaming
Destruction of microorganisms by burning is practised routinely in the laboratory when the transfer needle is introduced into the flame of the Bunsen burner. When the transfer needle is sterilized, care should be taken to prevent spattering, because the droplets which fly off are likely to carry viable organisms. Spattering can be prevented by using a Bunsen burner which is so modified that the transfer needle is exposed to a flame within a tubular space.
Incineration is used for the destruction of animal carcasses, bags and wipes, contaminated dressings, and infected laboratory materials to be disposed of. Care should be taken that the exhaust fumes do not carry particulate matter containing viable microorganisms into the atmosphere.
Desiccation and lyophilization
Desiccation, or drying, has been used for thousands of years to preserve such foods as fruits. It inhibits microbial growth because the evaporation of water inhibits metabolism. Desiccation of the microbial cell causes a cessation of metabolic activity, followed by a decline in the total viable population. The time of survival of microorganisms after desiccation varies, depending upon several factors: the kind of microorganisms, the material on which the organisms are dried, the completeness of the drying process and the physical conditions to which the dried organisms are exposed.
Bacterial species of Gram-negative cocci such as gonococci and meningococci are very sensitive to desiccation; die within hours, whereas Streptococci are much more resistant; some survive weeks after being dried. Dried spores of microorganisms are known to remain viable indefinitely.
Lyophilization, or freeze-drying, preserves microbes and other cells for many years by freezing a culture in liquid nitrogen and removing residual water via a vacuum. Lyophilization prevents the formation of large damaging ice crystals, leaving enough viable cells to enable the culture to be reconstituted many years later. This is useful when storing a bacterial culture for future use in a laboratory.
Filtration
Filtration is used to sterilize heat labile liquids and gases. Filtration (figure-4) is the passage of air or a liquid through a material that traps and removes microbes. These filters are made of different materials. The mean pore diameter in these biological filters are available in several grades, based on the average size of pores. Apart from porosity, other factors such as the electric charge of the filter, the electric charge carried by the organism, and the nature of the fluid being filtered, can influence the efficiency of filtration.
Figure 4: The process of filtration
Figure 4: The process of filtration
Now-a days, a new type of filter called membrane or molecular filter has been developed whose pores are of a uniform and specific predetermined size. These membrane filters are composed of biologically inert cellulose esters and contain millions of microscopic pores of very uniform diameter. Some membrane filters manufactured of nitrocellulose or plastic have pores small enough to trap the smallest viruses and even some large protein molecules. Membrane filters are used extensively in the laboratory and in the industry to sterilize fluid materials. The fluid is normally forced through the filter by applying a negative pressure to the filter flask by use of vacuum or a water pump to impose a positive pressure above the fluid in the filter chamber, thus forcing it through. After completion of filtration, when the filtered material is transferred to other containers, care must be taken to prevent contamination.
The development of high-efficiency particulate air (HEPA) filters has made it possible to remove microbes and particles from air and to deliver clean air. This type of air filtration together with a system of laminar airflow is now used to produce dust and bacteria free air.
Osmotic pressure
If cells are exposed to solutions with higher solute concentration, water will be drawn out of the cell and the process is called plsmolysis and the reverse process, which is the passage of water from a low solute concentration into the cell, is known as plasmoptysis. The pressure built up within the cell as a result of this water intake is termed osmotic pressure. Plasmolysis results in dehydration of the cell and as a consequence metabolic processes are retarded partially or completely. Due to the great rigidity of the microbial cell, the cell wall doesn't exhibit distortions as a result of plasmolysis, but shrinkage of protoplast and changes in the cytoplasmic membrane can be observed during plasmolysis.
High concentrations of salt or sugar inhibit microbial growth by osmotic pressure. Hyperosmotic conditions can preserve foods, because they cause water to be drawn out of bacteria and fungi so that they cannot thrive. Jam and pickles are classic examples because of their high solute loading – this makes jams and pickles highly hyperosmotic to the cytoplasm of bacteria and fungi which forces water to leave the cells by osmosis, but some microbes (some yeasts in brine pickles, or surface molds in jam) do grow in hyperosmotic conditions.
Radiation
Energy transmitted through space in a variety of forms is generally called radiation. The most significant type of radiation is probably electromagnetic radiation, which has the dual properties of a continuous wave phenomenon and a discontinuous particle phenomenon; the particles are quanta of energy called photons, which vibrate at different frequencies.
Gamma rays and x-rays, which have energies of more than about 10eV, are called ionizing radiations, because they have enough energy to knock electrons away from molecules and ionize them. When such radiations pass through cells, they create free hydrogen radicals, hydroxyl radicals, and some peroxides which in turn can cause different kinds of intracellular damage.
X-rays and Gamma rays
Ionizing radiation using X rays or gamma rays is an effective means for killing microbes. X-rays have considerable energy and penetration ability. But they are impractical for purposes of controlling microbial populations as they are very expensive to produce in quantity and they are difficult to utilize efficiently.
Gamma rays are high-energy radiations emitted from certain radioactive isotopes such as 60Co. Gamma rays are similar to x-rays but are of shorter wavelength and higher energy. These rays have greater penetrating power into the matter and they are lethal to all form of life. X-rays and gamma rays in particular are used to sterilize foods such as those used by astronauts or in packaged foods for the armed forces. There is a lot of contention about irradiation of food, it has become a public and political issue.
Results of quantitative studies on the effect of ionizing radiations on the cells have resulted in the establishment of the ''target'' theory of action which says that the radiant energy particle makes a direct heat on some essential substance such as DNA within the bacterial cell, causing ionization which results in the death of the cell.
Cathode rays
In an evacuated tube, when a high-voltage potential is established between a cathode and an anode, the cathode emits a beam of electrons known as cathode rays. The electron accelerator, a type of equipment which produces the high-voltage cathode rays, is used today for the sterilization of surgical supplies, drugs and other materials. In this process, the material can be sterilized after it has been packaged at room temperature. Electron-beam radiation has limited power of penetration and within this limited power, sterilization is accomplished.
Effective sterilisation techniques are essential for working with isolated cell lines for obvious reasons you don’t want bugs from the environment growing in your nice culture medium, and equally, cultures must be sterilised before disposal.
So what are the most common methods of sterilisation, and how do they work? Unsure? Read on…
WET HEAT (Autoclaving)
The method of choice for sterilisation in most labs is autoclaving; using pressurised steam to heat the material to be sterilised. This is a very effective method that kills all microbes, spores and viruses, although for some specific bugs, especially high temperatures or incubation times are required.
Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is efficiently achieved by intense heat in the presence of water.
The intense heat comes from the steam. Pressurised steam has a high latent heat; at 100degC it holds 7 times more heat than water at the same temperature. This heat is liberated upon contact with the cooler surface of the material to be sterilised, allowing rapid delivery of heat and good penetration of dense materials.
At these temperatures, water does a great job of hydrolysing proteins… so those bugs don’t stand a chance.
DRY HEAT (Flaming, baking)
Dry heating has one crucial difference from autoclaving. You’ve guessed it – there’s no water, so protein hydrolysis can’t take place.
Instead, dry heat tends to kill microbes by oxidation of cellular components. This requires more energy than protein hydrolysis so higher temperatures are required for efficient sterilization by dry heat.
For example sterilisation can normally be achieved in 15 minutes by autoclaving at 121degC, whereas dry heating would generally need a temperature of 160degC to sterilize in a similar amount of time.
FILTRATION
Filtration is a great way of quickly sterilizing solutions without heating. Filters, of course, work by passing the solution through a filter with a pore diameter that is too small for microbes to pass through.
Filters can be scintered glass funnels made from heat-fused glass particles or, more commonly these days, membrane filters made from cellulose esters. For removal of bacteria, filters with an average pore diameter of 0.2um is normally used.
But remember, viruses and phage can pass through these filters so filtration is not a good option if these are a concern.
SOLVENTS
Ethanol is commonly used as a disinfectant, although since isopropanol is a better solvent for fat it is probably a better option.
Both work by denaturing proteins through a process that requires water, so they must be diluted to 60-90% in water to be effective.
Again, a it’s important to remember that although ethanol and IPA are good at killing microbial cells, they have no effect on spores.
RADIATION
UV, x-rays and gamma rays are all types of electromagnetic radiation that have profoundly damaging effects on DNA, so make excellent tools for sterilization.
The main difference between them, in terms of their effectiveness, is their penetration.
UV has limited penetration in air so sterilisation only occurs in a fairly small area around the lamp. However, it is relatively safe and is quite useful for sterilising small areas, like laminar flow hoods.
X-rays and gamma rays are far more penetrating, which makes them more dangerous but very effective for large scale cold sterilization of plastic items (e.g. syringes) during manufacturing.
So those are some of the main methods for sterilization I can think of. If I’ve missed any, please feel free to let me know in the comments section.