Physical Antimicrobial Control

Heat

Heat is one of the most common and easily available methods for controlling bacterial growth.

Learning Objectives

Evaluate heat as an agent of microbrial control

Key Takeaways

Key Points

  • Different methods are used to achieve sterilization. One of the most common is applying moist heat which includes autoclaving (pressure cooking), boiling, and Tyndallisation.
  • Dry heat sterilization is accomplished by conduction and is used widely for instruments.
  • Other heat methods include flaming and incineration. Flaming is commonly used to sterilize small equipment used to manipulate bacteria aseptically.

Key Terms

  • sterilization: Any process that eliminates or kills all forms of microbial life present on a surface, solution, or solid compound.
  • Tyndallisation: Tyndallisation is the process of three successive steam treatments to achieve sterilization over the course of three days. This works by killing vegetative cells, allowing germination of surviving spores, and killing the resulting vegetative cells before they have time to form further spores.

Applying heat to bacterial media and utensils in research and the medical field as well as to sterilize food is one of the most common methods for control of bacterial growth. To achieve sterilization, different techniques and tools are used.

Moist Heat Sterilization

Moist heat causes destruction of micro- organisms by denaturation of macromolecules, primarily proteins. Autoclaving (pressure cooking) is a very common method for moist sterilization. It is effective in killing fungi, bacteria, spores, and viruses but does not necessarily eliminate prions. When sterilizing in this way, samples are placed into a steam chamber. The chamber is closed and heated so that steam forces air out of the vents or exhausts. Pressure is then applied so that the interior temperature reaches 121°C. This temperature is maintained for between 15 and 30 minutes. This elevated temperature and pressure is sufficient to sterilize samples of any commonly encountered microbes or spores. The chamber is then allowed to cool slowly or by passive heat dissipation. Pressure sterilization is the prevailing method used for medical sterilization of heat-resistant tools. It is also used for sterilization of materials for microbiology and other fields calling for aseptic technique. To facilitate efficient sterilization by steam and pressure, there are several methods of verification and indication used; these include color-changing indicator tapes and biological indicators. For any method of moist heat sterilization, it is common to use biological indicators as a means of validation and confirmation. When using biological indicators, samples containing spores of heat-resistant microbes such as Geobacillus stearothermophilis are sterilized alongside a standard load, and are then incubated in sterile media (often contained within the sample in a glass ampoule to be broken after sterilization). A color change in the media (indicating acid production by bacteria; requires the medium to be formulated for this purpose) or the appearance of turbidity (cloudiness indicating light scattering by bacterial cells) indicates that sterilization was not achieved and the sterilization cycle may need revision or improvement. Other moist methods are boiling samples for certain period of time and Tyndallisation. Boiling is not efficient in eliminating spores. Tyndallisation inactivates spores as well, but is a more lengthy process.

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Autoclave: Large autoclave used for moist sterilization of media and equipment

Dry Heat Sterilization

Dry heat destroys microorganisms by causing coagulation of proteins. The dry heat sterilization process is accomplished by conduction; that is where heat is absorbed by the exterior surface of an item and then passed inward to the next layer. Eventually, the entire item reaches the proper temperature needed to achieve sterilization. The time and temperature for dry heat sterilization is 160°C for 2 hours or 170°C for 1 hour. Instruments should be dry before sterilization since water will interfere with the process. Other heat sterilization methods include flaming and incineration. Flaming is commonly used to sterilize small equipment used to manipulate bacteria aseptically. Leaving transfer loops in the flame of a Bunsen burner or alcohol lamp until it glows red ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating infectious material may be “sprayed” from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area. Another problem is that gas flames may leave residues on the object, e.g. carbon, if the object is not heated enough. A variation on flaming is to dip the object in 70% ethanol (or a higher concentration) and merely touch the object briefly to the Bunsen burner flame, but not hold it in the gas flame. The ethanol will ignite and burn off in a few seconds. 70% ethanol kills many, but not all, bacteria and viruses. It has the advantage that it leaves less residue than a gas flame. This method works well for the glass “hockey stick”-shaped bacteria spreaders. Incineration will also burn any organism to ash. It is used to sanitize medical and other bio hazardous waste before it is discarded with non-hazardous waste.

Radiation

Both non-ionizing and ionizing radiation methods are applied for sterilization.

Learning Objectives

Compare non-ionizing and ionizing radiation in terms of microbe inhibition

Key Takeaways

Key Points

  • Ultraviolet light irradiation is a non-ionizing method useful only for sterilization of surfaces and some transparent objects.
  • Common methods of ionizing radiation are gamma rays, electron beams, X-rays, and subatomic particles.
  • However, ionizing radiation could be a lethal health hazard if used inappropriately. The proper use of these methods is regulated and monitored by world and national safety organizations.

Key Terms

  • Graft-versus-host disease: A complication after tissue or organ transplant or blood transfusion if the blood was not irradiated. White blood cells of the transplanted tissue or organ (the graft) attack cells in the recipients body (the host).
  • NRC: Nuclear Regulatory Commission

Both non-ionizing and ionizing radiation methods are applied for sterilization.

Non-ionizing radiation sterilization

Ultraviolet light irradiation (UV, from a germicidal lamp) is useful only for sterilization of surfaces and some transparent objects. Many objects that are transparent to visible light such as glass, absorb UV. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses, but is ineffective in shaded areas. The drawback of UV radiation is that it damages some plastics, such as polystyrene foam, if they are exposed for prolonged periods of time.

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UV light used in the laboratory: UV light is commonly used to irradiate and sterilize laminar flow cabinets between uses.

Ionizing radiation sterilization

Ionizing radiation could be a lethal health hazard if used inappropriately. The proper use of these methods is regulated and monitored by world and national safety organizations. Any incidents that have occurred in the past are documented and thoroughly analyzed to determine root cause and improvement potential.

  • Gamma rays

Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets, and food. The gamma radiation is emitted from a radioisotope (usually cobalt-60 or cesium-137). Cesium-137 is used in small hospital units to treat blood before transfusion in order to prevent Graft-versus-host disease. Use of a radioisotope requires shielding to ensure the safety of the operators while in use and in storage as these radioisotopes continuously emits gamma rays (cannot be turned off). An incident in Decatur, Georgia where water soluble cesium-137 leaked into the source storage pool requiring NRC intervention has led to near elimination of this radioisotope; it has been replaced by the more costly, non-water soluble cobalt-60. Sterilization by irradiation with gamma rays may, in some cases affect material properties.

  • Electron beams

Electron beam processing is also commonly used for sterilization. Electron beams use an on-off technology and provide a much higher dosing rate than gamma or x-rays. Due to the higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is reduced. A limitation is that electron beams are less penetrating than either gamma or x-rays. Facilities rely on substantial concrete shields to protect workers and the environment from radiation exposure.

  • X-rays

High-energy X-rays are a form of ionizing energy allowing to irradiate large packages and pallet loads of medical devices. X-ray sterilization is an electricity based process that does not require chemical or radioactive material. High energy and high power X-rays are generated by an X-ray machine that can be turned off when not in use, and therefore does not require any shielding when in storage. Irradiation with X-rays or gamma rays does not make materials radioactive.

  • Subatomic particles

Subatomic particles may be more or less penetrating, and may be generated by a radioisotope or a device, depending on the type of particle. Irradiation with particles may make materials radioactive, depending on the type of particles, their energy, and the type of target material: neutrons and very high-energy particles can make materials radioactive but have good penetration, whereas lower energy particles (other than neutrons) cannot make materials radioactive, but have poorer penetration.

Irradiation is used by the United States Postal Service to sterilize mail in the Washington, DC area. Some foods (e.g. spices, ground meats) are irradiated for sterilization.

Low Temperatures

Low temperatures usually inhibit or stop microbial growth and proliferation but often do not kill bacteria.

Learning Objectives

Identify how low temperatures are used for microbial control

Key Takeaways

Key Points

  • Refrigeration (4ºC) and freezing (-20ºC or less) are commonly used in food, pharmaceutical and biotechnology industries.
  • Refrigeration preserves food by slowing down the growth and reproduction of microorganisms as well as the action of enzymes which cause food to rot.
  • Freezing food slows down decomposition by turning residual moisture into ice, inhibiting the growth of most bacterial species. Freezing kills some microorganisms by physical trauma, while sublethally injuring others which may recover to become infectious.

Key Terms

  • proliferation: The process by which an organism produces others of its kind; breeding, propagation, procreation, reproduction.

Temperature is an important factor for microbial growth. Each species has its own optimal growth temperature at which it flourishes. Human microbial pathogens usually thrive at body temperature, 37ºC. Low temperatures usually inhibit or stop microbial growth and proliferation but often do not kill bacteria. Refrigeration (4ºC) and freezing (-20ºC or less) are commonly used in the food, pharmaceuticals and biotechnology industry.

Refrigeration preserves food by slowing down the growth and reproduction of microorganisms and the action of enzymes which cause food to rot. The introduction of commercial and domestic refrigerators drastically improved the diets of many in the 1930s by allowing foods such as fresh fruit, salads and dairy products to be stored safely for longer periods, particularly during warm weather. It also facilitated transportation of fresh food on long distances.

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The Dunedin: This ship executed the first completely successful refrigerated shipment of food.

Refrigeration is also used to facilitate the preservation of liquid medicines or other substances used for research where microbial growth is undesirable, often combined with added preservatives. Fridge temperatures inhibit the proliferation of bacteria better than molds and fungi.

For longer periods of preservation, freezing temperatures are preferred to refrigeration. Since early times, farmers, fishermen, and trappers have preserved their game and produce in unheated buildings during the winter season. Freezing food slows down decomposition by turning residual moisture into ice, inhibiting the growth of most bacterial species.

Freezing temperatures curb the spoiling effect of microorganisms in food, but can also preserve some pathogens unharmed for long periods of time. While it kills some microorganisms by physical trauma, others are sublethally injured by freezing, and may recover to become infectious.

Frozen products do not require any added preservatives because microorganisms do not grow when the temperature of the food is below -9.5°C, which is sufficient in itself to prevent food spoilage. Long-term preservation of food may call for food storage at even lower temperatures.

High Pressure

Under very high hydrostatic pressure(HHP) of up to 700 MPa, water inactivates pathogens such as E. coli and Salmonella.

Learning Objectives

Explain high pressure as a antimicrobial control

Key Takeaways

Key Points

  • High pressure processing (HPP), pascalization or bridgmanization, is a method of preserving and sterilizing food, in which a product is processed under very high pressure, leading to the inactivation of certain microorganisms and enzymes in the food.
  • The frist reports showed that bacterial spores were not always inactivated by pressure, while vegetative bacteria were usually killed. Later it was discovered that using moderate pressures was more effective in eliminating spores than using higher pressures.
  • Experiments were also performed with anthrax, typhoid, and tuberculosis, which was a potential health risk for the researchers.

Key Terms

  • bridgmanization: Pascalization is also known as bridgmanization, named for physicist Percy Williams Bridgman.

Under very high hydrostatic pressure of up to 700 MPa (100,000 psi), water inactivates pathogens such as Listeria, E. coli and Salmonella.

High pressure processing (HPP), pascalization or bridgmanization, is a method of preserving and sterilizing food, in which a product is processed under very high pressure, leading to the inactivation of certain microorganisms and enzymes in the food. The technique was named after Blaise Pascal, whose work included detailing the effects of pressure on fluids. Pascalization is preferred over heat treatment in the food industry as it eliminates changes in the quality of foods due to thermal degradation, resulting in fresher taste, texture, appearance and nutrition. Processing conveniently takes place at ambient or refrigeration temperatures.

Experiments into the effects of pressure on microorganisms were first recorded in the late nineteenth century. The frist reports showed that bacterial spores were not always inactivated by pressure, while vegetative bacteria were usually killed. Around 1970, researchers renewed their efforts in studying bacterial spores after it was discovered that using moderate pressures was more effective than using higher pressures. These spores, which caused a lack of preservation in the earlier experiments, were inactivated faster by moderate pressure, but in a manner different from what occurred with vegetative microbes. When subjected to moderate pressures, bacterial spores germinate, and the resulting spores are easily killed using pressure, heat, or ionizing radiation.

Research into the effects of high pressures on microorganisms was largely focused on deep-sea organisms until the 1980s, when advancements in ceramic processing were made. This resulted in the production of machinery that allowed for processing foods at high pressures at a large scale, and generated some interest in the technique, especially in Japan. Although commercial products preserved by pascalization first emerged in 1990, the technology behind pascalization is still being perfected for widespread use.

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Japanese miso soup: Miso soup is sterilized with high pressure.

In pascalization, food products are sealed and placed into a steel compartment containing a liquid, often water, and pumps are used to create pressure. The pumps may apply pressure constantly or intermittently. During pascalization, more than 50,000 pounds per square inch (340 MPa) may be applied for around fifteen minutes, leading to the inactivation of yeast, mold, and bacteria. In the process, the food’s proteins are denatured, hydrogen bonds are fortified, and noncovalent bonds in the food are disrupted, while the product’s main structure remains intact. Because pascalization is not heat-based, covalent bonds are not affected, causing no change in the food’s taste.

Experiments were also performed with anthrax, typhoid, and tuberculosis, which was a potential health risk for the researchers. Indeed, before the process was improved, one employee of the Experimental Station became ill with typhoid fever.

Desiccation

Desiccation is the state of extreme dryness, or the process of extreme drying and can be used to control microbial growth.

Learning Objectives

Show how desiccation inhibits microbes

Key Takeaways

Key Points

  • Microorganisms cannot grow and divide when desiccated, but can survive for certain periods of time, depending on their features. After the addition of water, the bacteria will start growing again, so desiccation does not provide complete sterilization.
  • Pharmaceutical companies often use freeze-drying as a desiccation tool to increase the shelf life of products, such as vaccines and other injectables. Drying is also a method for food preservation.
  • Freeze-drying is performed using special equipment.

Key Terms

  • desiccation: the state of drying
  • freeze-drying: Freeze-drying, also known as lyophilisation, lyophilization, or cryodesiccation, is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport.

Desiccation is the state of extreme dryness, or the process of extreme drying. In biology and ecology, desiccation refers to the drying out of a living organism. Microorganisms cannot grow and divide when desiccated, but can survive for certain periods of time, depending on their features. After the addition of water, the bacteria will start growing again, so desiccation does not provide complete sterilization.

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SMART Freeze Dryer: New freeze dryer which is equipped with systems for immediate feedback on the properties of the dried product, eliminating the lengthy trial-and-error approach.

Some bacteria, such as Deinococcus radiodurans and Mycobacterium , are extremely resistant to damage from prolonged desiccation while others, such as Neisseria gonorrhoeae, can survive only short periods of desiccation.

Pharmaceutical companies often use freeze-drying as a desiccation tool to increase the shelf life of products, such as vaccines and other injectables. By removing the water from the material and sealing the material in a vial, the material can be easily stored, shipped, and later reconstituted to its original form. Preservation is possible because the greatly reduced water content inhibits the action of microorganisms and enzymes that would normally spoil or degrade the substance. Another example from the pharmaceutical industry is the use of freeze-drying to produce tablets or wafers.

Drying is also a method for food preservation that works by removing water from the food, which inhibits the growth of microorganisms. Open air drying using sun and wind has been practiced since ancient times to preserve food. A solar or electric food dehydrator can greatly speed the drying process and ensure more consistent results. Water is usually removed by evaporation (air drying, sun drying, smoking, or wind drying) but, in the case of freeze-drying, food is first frozen and then the water is removed by sublimation. Bacteria, yeasts, and molds need the water in the food to grow, and drying effectively prevents them from surviving in food.

Freeze-drying is performed using special equipment. Two components are common to all types of freeze-dryers: a vacuum pump to reduce the ambient gas pressure in a vessel containing the substance to be dried, and a condenser to remove the moisture by condensation on a surface cooled to −40º to −80ºC.

Osmotic Pressure

Osmotic pressure is the pressure which must be applied to a solution to prevent the inward flow of water across a semipermeable membrane.

Learning Objectives

Interpret osmotic pressure as a means of microbial control

Key Takeaways

Key Points

  • Osmotic pressure is of vital importance in biology as the cell’s membrane is selective toward many of the solutes found in living organisms.
  • When a cell is placed in a hypertonic solution, water actually flows out of the cell into the surrounding solution thereby causing the cells to shrink and lose its turgidity. Hypertonic solutions are used for antimicrobial control.
  • Salt and sugar are used to create hypertonic environment for microorganisms and are commonly used as food preservatives.

Key Terms

  • turgidity: Turgidity (turgor pressure) pushes the plasma membrane against the cell wall of plant, bacteria, and fungi cells as well as those protiat cells which have cell walls.

Osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane. It is also defined as the minimum pressure needed to nullify osmosis.The phenomenon of osmotic pressure arises from the tendency of a pure solvent to move through a semi-permeable membrane and into a solution containing a solute to which the membrane is impermeable. This process is of vital importance in biology as the cell’s membrane is selective toward many of the solutes found in living organisms.

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Salami: The original meaning of the word is: all kind of salted (meats).

Osmosis causes water to flow from an area of low solute concentration to an area of high solute concentration until the two areas have an equal ratio of solute to water. Normally, the solute diffuses toward equilibrium as well; however, all cells are surrounded by a lipid bilayer cell membrane which permits the flow of water in and out of the cell but restricts the flow of solute under many circumstances. As a result, when a cell is placed in a hypotonic solution, water rushes into the membrane, increasing its volume. Eventually, the cell’s membrane is enlarged such that it pushes against the cell’s rigid wall. In an isotonic solution, water flows into the cell at the same rate it flows out. When a cell is placed in a hypertonic solution, water actually flows out of the cell into the surrounding solution causing the cells to shrink and lose its turgidity. Two of the most common substances used to create hypertonic environment for microorganisms and prevent them from growing are salt and sugar. They are widely applied in food preservation.

Table salt (sodium chloride) is the primary ingredient used in meat curing. Removal of water and addition of salt to meat creates a solute-rich environment where osmotic pressure draws water out of microorganisms, thereby retarding their growth. Doing this requires a concentration of salt of nearly 20%.

Sugar is used to preserve fruits, either in syrup with fruit such as apples, pears, peaches, apricots, plums or in crystallized form where the preserved material is cooked in sugar to the point of crystallisation and the resultant product is then stored dry. The purpose of sugaring is to create an environment hostile to microbial life and prevent food spoilage. From time to time, sugaring has also been used for non-food preservation. For example, honey was used as part of the mummification process in some ancient Egyptian rites. However, the growth of molds and fungi is not suppressed as efficiently as the growth of bacteria.

Filtration

Fluids that would be damaged by heat, irradiation, or chemical sterilization can be sterilized by microfiltration using membrane filters.

Learning Objectives

Demonstrate microbial control using filtration

Key Takeaways

Key Points

  • A typical microfiltration membrane pore size range is 0.1-10 µm, with the most commonly used being 0.2 µm and 0.45 µm, which is sufficient to eliminate bacteria and fungi.
  • Quite often, when biological samples are processed, viruses must be removed or inactivated. Nanofilters with smaller pore sizes of 20-50 nm (nanofiltration) are used.
  • Filtration is commonly used for heat labile pharmaceuticals and protein solutions in processing medicines. It is also increasingly used in the treatment of drinking water.

Key Terms

  • ester: An ester is a chemical compound consisting of a carbonyl group adjacent to an ether linkage.
  • polyethersulfone: Thermoplastic polymers that have low protein retention. They contain the subunit aryl-SO2-aryl, the defining feature of which is the sulfone group.

Fluids that would be damaged by heat (such as fluids containing proteins like large molecule drug products, but also wine and beer), irradiation, or chemical sterilization can only be sterilized by microfiltration using membrane filters. This method is commonly used for heat labile pharmaceuticals and for protein solutions in processing medicines.

The typical microfiltration membrane pore size range is 0.1-10 µm, with the most commonly used being 0.2 µm; and 0.45 µm is sufficient to eliminate bacteria and fungi.

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Syringe Filter: A syringe filter with a pore size of 0.22 micrometers, small enough to capture and retain bacterial and fungal cells.

Microfiltration is increasingly used in drinking water treatment. It effectively removes major pathogens and contaminants such as Giardia lamblia cysts, Cryptosporidium oocysts, and large bacteria. For this application, the filter has to be rated for 0.2 µm or smaller pore size.

Quite often when biological samples are processed, viruses must be removed or inactivated. Nanofilters with smaller pore sizes of 20-50 nm (nanofiltration) are used. The smaller the pore size, the lower the flow rate. To achieve higher total throughput or avoid premature blockage, pre-filters might be used to protect small pore membrane filters. Some studies have shown that prions can be removed or reduced by filtration.

Membrane filters used in production processes are commonly made from materials such as mixed cellulose ester or polyethersulfone. The filtration equipment and the filters may be purchased as pre-sterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure proper functioning of the filter, the membrane filters are integrity tested post-use or sometimes pre-use. A non-destructive integrity test assures the filter is undamaged, and is also a regulatory requirement enforced by agencies like the Food and Drug Administration, the European Medicines Agency, and others.