Controlling Microorganisms

A Glimpse of History

• British physician Joseph Lister (1827 to 1912) revolutionized surgery by introducing methods to prevent wound infection.
• Impressed with Pasteur's work, Lister considered that 'minute organisms' might be responsible for infections.
• Lister applied phenol directly onto damaged tissues, which prevented infections.
• He improved methods further by sterilizing instruments and maintaining a clean operating environment.
• Oral antiseptic Listerine named for Lister in 1879 when it was introduced as a surgical antiseptic.

Approaches to Control

• Sterilization involves removing or destroying all microorganisms and viruses, resulting in a sterile item which is free of microbes, including endospores.
• Disinfection is the elimination of most or all pathogens, where some viable microbes may remain.
• Disinfectants are chemicals used on inanimate objects and are often called germicides.
• Bactericides kill bacteria, fungicides kill fungi, and virucides inactivate viruses.
• Antiseptics are chemicals used on living tissues.
• Decontamination reduces the number of pathogens to a safe level.
• Washing, use of heat, or chemicals can be used to decontaminate.
• Sanitization substantially reduces microbial population to meet accepted health standards in order to minimize spread of disease, but is not a specific level of control.
• Preservation is the process of delaying spoilage of perishable products which can be done by choosing storage conditions to slow growth or adding bacteriostatic (growth-inhibiting) preservatives.
• Pasteurization involves brief heating to reduce the number of spoilage organisms and destroy pathogens without changing characteristics of product.
• Daily life control methods include washing and scrubbing with soaps and detergents which achieves routine control.
• Soap aids in mechanical removal of organisms.
• Beneficial skin microbiota reside deeper on underlying layers of skin, hair follicles, and are not adversely affected by regular washing.
• Other control methods used in daily life include cooking foods, cleaning surfaces, and refrigeration.
• Controlling microbes in healthcare settings is very important due to the danger of healthcare-associated infections (HAIs), also known as nosocomial infections.
• Weakened patients are more susceptible to infection and may undergo invasive procedures like surgery.
• Pathogens are more likely found in a hospital setting, like feces, urine, respiratory droplets, and bodily secretions.
• Healthcare facilities must protect personnel.
• The COVID-19 pandemic demonstrated that healthcare workers are at risk of contracting infectious disease from patients.
• Standard Precautions are used in patient care to prevent infection of both patients and personnel.
• Transmission-Based Precautions are used if a patient might be infected with a highly transmissible or epidemiologically important pathogen.
• Special care must be taken to control microorganisms in operating rooms.
• Instruments must be sterilized to avoid introducing infection to deep tissues during surgery.
• Microbiology laboratories routinely work with microbial cultures and use rigorous methods of control.
• Microbial contamination must be eliminated to both experimental samples and the environment.
• Careful treatment both before work includes use of sterile materials, and after work includes sterilizing cultures and waste.
• Aseptic technique is used to prevent contamination of samples, workers, and the environment.
• The U.S. Centers for Disease Control and Prevention (CDC) has established biosafety levels (BSLs) for laboratories working with microorganisms.
• BSL-1 involves procedures for work with microbes not known to cause disease in healthy people.
• BSL-2 involves procedures for work with moderate-risk microbes that cause disease but have limited potential for transmission.
• BSL-3 involves procedures for work with pathogens that cause serious or potentially fatal disease through inhalation.
• BSL-4 involves procedures for work with easily transmitted deadly pathogens; there are only four BSL-4 labs in the USA.
• Perishables retain quality longer when contaminating microbes destroyed, removed, inhibited in food and food production facilities.
• Heat treatment is the most common and reliable mechanism, although it can alter flavor and appearance of products.
• Irradiation and high pressure can be used to treat certain foods without altering it.
• Chemical additives can prevent spoilage, but are regulated by FDA due to risk of toxicity.
• Food-processing facilities must keep surfaces and machinery clean and relatively free of microorganisms.
• Water treatment facilities ensure drinking water is free of pathogens.
• Chlorine is traditionally used to disinfect water, but can react with naturally occurring chemicals and form disinfection by-products (DBPs).
• Some DBPs can be linked to long-term health risks.
• Some organisms are resistant to chemical disinfectants.
• Cryptosporidium hominis is an intestinal pathogen that causes diarrhea.
• Regulations require facilities to minimize DBPs and C. hominis in treated water.
• Other industries such as pharmaceuticals, cosmetics, and deodorants must not carry microbial contamination.

Types of Microbes

• Bacterial endospores are the most resistant and are only destroyed by extreme heat or chemical treatment.
• Protozoan cysts and oocystsare resistant to disinfectants, excreted in feces, cause diarrheal disease if ingested, but are destroyed by boiling.
• Mycobacterium species have waxy cell walls and are resistant to many chemical treatments.
• Pseudomonas species are resistant to and can actually grow in some disinfectants.
• Non-enveloped viruses lack a lipid envelope and are more resistant to disinfectants.

Number of Microbes

• Only a fraction of a microbial population dies during a given time interval, so it takes more time to destroy a large population than a small one.
• Removing some organisms by washing reduces the time needed to sterilize or disinfect a product.
• Scrubbing helps remove biofilms because organisms in a biofilm are resistant to disinfectants.
• Decimal reduction time (D value) is the time required to kill 90% of a population under specific conditions.

Risk for Infection

• Medical instruments are categorized according to the risk for transmitting infectious agents.
• Critical items contact body tissues, must be sterile, and include needles and scalpels.
• Semi-critical instruments contact mucous membranes but do not penetrate body tissues, must be free of microorganisms and viruses, where some endospores may remain but are often blocked by mucous membranes, and include endoscopes and endotracheal tubes.
• Non-critical instruments and surfaces contact unbroken skin only, have a low risk of transmission, and include countertops, stethoscopes, and blood pressure cuffs.

Composition of an Item

• Some sterilization and disinfection methods inappropriate for certain items.
• Heat can damage plastics and other materials.
• Irradiation provides an alternative, but damages some types of plastic.
• Moist heat and liquid chemical disinfectants cannot be used to treat moisture-sensitive material.

Moist Heat

• Moist heat irreversibly denatures proteins.
• Boiling destroys most microorganisms and viruses but does not sterilize because endospores can survive.
• Pasteurization destroys heat-sensitive pathogens and spoilage organisms.
  • High-temperature-short-time (HTST) method: Milk - 72 degrees Celsius for 15 seconds; ice cream - 82 degrees Celsius for 20 seconds.
  • Ultra-high-temperature (UHT) method: Shelf-stable boxed juice and milk; known as “ultra-pasteurization."
  • Milk – 140 degrees Celsius for a few seconds, then rapidly cooled.

Sterilization Using Pressurized Steam

• Autoclaves are used to sterilize using pressurized steam.
• Increased pressure raises steam temperature and kills endospores.
• Sterilization typically occurs at 121 degrees Celsius and 15 pounds per square inch in 15 minutes.
• Longer times are needed for larger volumes
• To be effective, steam must enter item and displace air.
• Place long, thin containers on their sides and do not close containers tightly.
• Tape with a heat-sensitive indicator can confirm heating.
• Biological indicators can confirm lack of microbial growth after autoclaving.
• The commercial canning process uses an industrial-sized autoclave called a retort.
• Retorts are designed to destroy Clostridium botulinum endospores.
• The goal is to reduce 1012 endospores to only 1, which is a 12 D process.
• It is critical because endospores could germinate in canned foods, cells grow in low-acid anaerobic conditions and produce botulinum toxin.
• Canned food is considered commercially sterile.
• Endospores of some thermophiles may survive but it is usually not a concern, because they only grow at temperatures well above normal storage.

Dry Heat

• Incineration (destruction by burning) oxidizes components to ashes.
• In microbiology laboratories, the wire loops continually reused to transfer bacterial cultures are sterilized by flaming or heating in a benchtop incinerator.
• Incineration is also used to destroy medical wastes and contaminated animal carcasses.
• Hot air ovens kill microbes by destroying cell components and denaturing proteins.
• Requires higher temperature and longer times than moist heat because dry heat takes longer to penetrate and is less efficient at killing microbes.

Filtration

• Membrane filtration in a liquid medium retains bacteria while allowing the fluid to pass through.
• It is used extensively to remove organisms from heat-sensitive fluids.
• Membrane filters or microfilters have a small pore size (0.2 micrometer) to remove bacteria.
• Vacuum is used to move fluid through a filter.
• Depth filters use thick porous filtration material such as cellulose.
• Electrical charges trap cells.
• High-efficiency particulate air (HEPA) filters remove nearly all microbes over 0.3 micrometer from air.

Irradiation

• Electromagnetic radiation includes radio waves, microwaves, visible and ultraviolet light, X rays, and gamma rays.
• Wavelength is inversely proportional to frequency.
• High frequency has more energy than low frequency.
• Ionizing radiation (gamma rays, X rays) directly destroys DNA, damages cytoplasmic membranes, and also reacts with O₂ to produce reactive oxygen species.
• D10 = Radiation dose (Gray) to reduce surviving fraction to 10% (kill 90% of population).
• High energy gamma-rays are used to sterilize heat-sensitive materials, including packaging and some foods.
• Ultraviolet radiation destroys microbes directly.
• It damages DNA and can be used to destroy microbes in air, water, and on surfaces.
• Ultraviolet radiation has poor penetrating power; its effects are limited by thin films or coverings.
• It cannot kill microbes in solids or turbid liquids and is blocked by most glass and plastic.
• It must be carefully used since damaging to skin and eyes.
• Microwaves do not affect microorganisms, but the heat they generate can be lethal.
• Microwave ovens heat food unevenly, so cells can survive.

Chemicals Methods Used to Destroy Microorganisms and Viruses

• Germicidal chemicals can disinfect and, in some cases, sterilize.
• They react irreversibly with proteins, DNA, cytoplasmic membranes, or viral envelopes, but their exact mechanisms of action are often poorly understood.
• They are less reliable than heat, but are useful for treating large surfaces and heat-sensitive items.
• Some are sufficiently non-toxic to be used as antiseptics.
• In the United States, the FDA is responsible for ensuring that chemicals used to treat medical devices work and that drug products, including antiseptics, are safe and effective.
• Antiseptic washes are antimicrobial soaps.
• Antiseptic rubs are hand sanitizers.
• Topical antiseptic products are used by healthcare professionals to disinfect patients' skin in preparation for injections or surgery.
• With any chemical, the toxicity, or benefits must be weighed against the risk of use.
• Liquids cannot be not used on electrical equipment.

Categories of Germicidal Potency

• Sterilants destroy all microbes and are also called sporocides.
  • Used for heat-sensitive critical instruments.
• High-level disinfectants destroy viruses and vegetative cells.
  • They do not reliably kill endospores and are used for semi-critical instruments.
• Intermediate-level disinfectants destroy vegetative bacteria, mycobacteria, fungi, and most viruses. - They are used for to disinfect non-critical instruments.
• Low-level disinfectants destroy fungi and vegetative bacteria except mycobacteria, as well as enveloped viruses.
  • They do not kill endospores and non-enveloped viruses, and disinfect furniture, floors, walls.

Classes of Germicidal Chemicals

• Alcohols such as 60 to 80% aqueous solutions of ethanol or isopropanol can destroy vegetative bacteria and fungi, but are not reliable against endospores and non-enveloped viruses.
• Alcohols denature essential proteins and damage membranes.
  • Proteins are more soluble in water; pure alcohol less effective.
• Commonly used as antiseptic and disinfectant, as they are non-toxic, inexpensive, and leave no residue.
• Limitations include: evaporation quickly, limiting contact time, and can damage rubber, some plastics, and others.
• Tincture is an antimicrobial chemical dissolved in alcohol.
• Aldehydes include glutaraldehyde, formaldehyde, and ortho-phthalaldehyde (OPA).
• Aldehydes Inactivate proteins and nucleic acids and may include:
  • 2% alkaline glutaraldehyde - Common liquid sterilant, immersion of medical items for 10 to 12 hours, toxic; requires thorough rinsing after use.
  • Ortho-phthalaldehyde - Less toxic, but turns skin grey.
  • Formaldehyde - Used as gas or as formalin (37% solution), effective germicide that kills most microbes quickly used to kill bacteria and inactivate viruses for vaccines used to preserve specimens, and is a probable carcinogen.
• Biguanides such as chlorhexidine is most effective.
  • Chlorhexidine has extensive use as antiseptics, stays on skin and mucous membranes, relatively low toxicity, destroys vegetative bacteria, fungi, some enveloped viruses, and common in many products: skin cream and prescription mouthwashes.
• Ethylene oxide is a gaseous sterilant for heat- or moisture-sensitive items.
• Ethylene oxide destroys microbes, including endospores and viruses, by chemically modifying proteins and nucleic acids.
  • It penetrates fabrics, equipments, and implantable devices.
• Ethylene oxide is used for many disposable laboratory items and applied in special chamber resembling autoclave.
• It has limitations, as it is explosive, toxic, potentially carcinogenic.
• Halogens are oxidizing agents which react with proteins and cellular components.
  • Chlorine destroys all microorganisms, endospores and viruses.
    • It is caustic to skin and mucous membranes, 1:100 dilution of household bleach effective, and very low levels disinfect drinking water.
    • Cryptosporidium oocysts and Giardia cysts can survive chlorine.
    • Chlorine can react with organic compounds in water, disrupting germicidal activity and can produce potential carcinogens.
  • Chlorine dioxide (ClO₂) is used as disinfectant and sterilant.
  • Iodine kills vegetative cells and is unreliable on endospores.
• Metal compounds combine with sulfhydryl groups (-SH) of proteins.
  • High concentrations of these compounds are too toxic to be used medically.
  • Silver is used in creams and bandages.
  • Antibiotics replaced silver nitrate eye drops once given at birth to prevent Neisseria gonorrhoeae infections.
• Compounds of mercury, tin, and copper and others were once widely used as preservatives.
  • Extensive use led to environmental pollution and now strictly regulated.
• Ozone (O3) is an unstable form of oxygen.
  • Decomposes quickly, so is generated on-site and is a powerful oxidizing agent.
  • Used as alternative to chlorine and is a disinfectant for drinking and wastewater.
• Peroxygens are powerful oxidizers used as sterilants.
  • They are readily biodegradable, leave no residue, and are less toxic than ethylene oxide and glutaraldehyde.
  • Hydrogen peroxide effectiveness depends on surface. -Aerobic cells (such as humans) produce the enzyme catalase which breaks down H2O2 to O2 + H₂O,
  • More effective on inanimate objects and doesn't damage most materials, with no residue.
  • Peracetic acid is more potent than H2O2, sterilizes in less than 1 hour, and is effective in presence of organic compounds, with no residue, used on wide range of materials.
• Phenolic Compounds (Phenolics), such as Phenol, was one of earliest disinfectants.
  • It has unpleasant odor and irritates skin.
• Phenols destroy cytoplasmic membranes and denature proteins, and kill most vegetative bacteria and it is not reliable on all.

virus groups.

• Phenols leave antimicrobial residue.
• Hexachlorophene and triclosan were have been widely used in medical and personal care products.
• FDA recently limited some use of triclosan because of contact dermatitis as well as skin irritation, and an increased risk of allergic reactions, especially in children.
• Quaternary Ammonium Compounds (Quats) are cationic detergents.
• Quats exhibit low toxicity and provide disinfection of food preparation surfaces.
  • Charged hydrophilic and uncharged hydrophobic regions aid in removal of dirt, organic matter, organisms. -Positive charges of quats attracted to negative charges of cell surface.
  • They React with membrane and destroys vegetative bacteria and enveloped viruses. -Found in many personal care products.
• Pseudomonas resists, can grow in solutions.

Preservation of Perishable Products

• Chemical preservatives prevent or slow microbial growth and extend shelf life.
• Food preservatives must be non-toxic for safe ingestion.
• Weak organic acids (benzoic, sorbic, and propionic) affect cell membrane function and control molds and bacteria in foods.
• Nitrate and nitrite are used in processed meats.
  • They inhibit endospore germination and vegetative cell growth and higher concentrations give meats a pink color. - Can be carcinogenic when converted to nitrosamines by cooking or intestinal bacteria.
• Low-Temperature Storage
  • Refrigeration inhibits growth of pathogens and spoilage organisms by slowing or stopping enzyme reactions and psychrotrophs and psychrophilic organisms can still grow.
  • Freezing preserves by stopping microbial growth and some microbial cells are killed by ice crystal formation, but many survive and can grow once thawed.
• Reducing the Available Water
  • Accomplished by salting, adding sugar, or drying food.
  • Addition of salt or sugar increases environmental solutes, drawing water out of cells and dehydrating them.
  • Some bacteria grow in high salt environments; such as Staphylococcus aureus.
  • Drying is often supplemented by salting.
• Lyophilization (freeze drying) is used for foods like coffee, milk, meats, and vegetables.
  • Drying stops microbial growth but does not reliably kill.
  • Cases of salmonellosis have been seen from dried eggs.