Test 3 - Microbial Control

Types of Disinfectants: Surface-active agents (surfactants)

(detergents) decrease the surface tension of a liquid and include soaps and detergents. They emulsify oils and are good degerming agents. Acid-ionic sanitizers are important for cleaning dairy equipment. Deodorant soaps contain compounds such as triclocarban which inhibit gram-positive bacteria.

Types of Disinfectants: Halogens

(group 7A on the periodic table. They work well. Combine with AA tyrosine, causing protein its a part of to have a shape change so it doesn't work anymore) Iodine is effective against all kinds of bacteria, many endospores, fungi and some viruses. Its mechanism of action is due to its combination with the amino acid tyrosine in enzymes and cellular proteins. Tincture of iodine is an aqueous alcohol solution. Iodophors are a combination of iodine and an organic molecule. Iodophors do not stain and are less irritating than iodine. Common iodophors include Betadine, Isodine, Ioprep, Wescodyne and others. They are often used as surgical hand scrubs and pre-operative skin preparations. Chlorine is used as a gas or in combination with other chemicals. The gas is used in disinfecting municipal water supplies, swimming pools and sewage. Sodium hypochlorite, ordinary household bleach, is a good disinfectant. It is commonly used to disinfect benchtops, floors and other inanimate objects. A 10% solution readily kills the HIV virus. Chloramines are chemicals consisting of chlorine and ammonia. They are highly stable. The germicidal action of all chlorines is based on the formation of hypochlorous acid in water. Its neutral charge allows it to diffuse as rapidly as water in the cell.

How does moist heat kill (2 ways)?

1) denaturation/coagulation of cellular proteins/enzymes 2) altering the physical state of lipids (making membranes impermeable or leaky)

How does dry heat kill (2 ways)?

1) oxidation of cell components 2) altering the physical state of lipids

How does low temperature kill (4 ways)?

1) usually inhibits growth 2) by coagulation/denaturation of cellular proteins/enzymes 3) altering the physical state of lipids 4) decreased chemical reactions or metabolism

Chemicals can also be classified according to their method of action on bacterial cells:

1. Chemicals that alter membrane permeability (surfactants) 2. Chemicals that denature or coagulate cellular proteins and enzymes 3. Chemicals that affect nucleic acids

Types of Disinfectants (12)

1. Phenol and Phenol derivatives (phenolics) 2. Halogens 3. Alcohols 4. Heavy metals and their compounds 5. Surface-active agents (surfactants) 6. Quaternary Ammonium Compounds 7. Organic acids and derivatives 8. Aldehydes 9. Gaseous Chemosterilizers 10. Peroxides 11. Super D Hydrogen Peroxide 12. Ozone

Moist heat sterilization: Boiling water or free flowing steam.

100C for 15 minutes. Effective against intestinal bacteria and other vegetative bacterial cells (killed in 10 mins), but not spores (can resist boiling for up to 10 hours). HBV (hepatitis B virus) is killed within 30 minutes. Uses: dishes, basins, pitchers and various equipment such as dental hard pieces and carbon dental instruments.

Moist heat sterilization: Autoclaving (steam under pressure)

121C (249.5F) @ 15 pounds of pressure per square inch for 15-20 minutes. Probably the most widely used sterilization technique. It is not the pressure that kills but the fact that the pressure raises the temperature of water above the boiling point. This creates steam which rapidly penetrates cells and spores coagulating cellular proteins and enzymes. Uses: Sterilization of microbiological media, terminal sterilization of cultures, solutions, syringes, needles and other instruments and objects that can withstand the temperature and pressure.

Dry heat: Hot air (hot oven) sterilization:

170F-180F for 2 hours. The time required for dry sterilization is much longer because cellular components must oxidize. Uses: Glassware, mineral oil, Vaseline, waxes, powders and other materials which must be kept dry or which will not allow water to penetrate. Used in dental for cotton pellets and paper points.

Classic Pasteurization

60oC for 30 minutes

HTST (high temperature short time) Pasteurization

72oC for 15 seconds

Additional methods of physical control: Microwave irradiation

915 and 2,450 mega Hertz cause negatively charged ions to accelerate and collide with other molecules or cause dipoles to attempt to rotate and line up with the rapidly alternating electrical field. This generates sufficient heat to cook foods as well as kill bacteria and fungi. Studies have shown that water is necessary for effective sterilization and uses have been limited to terminal sterilization of cultures.

Define: Disinfectant

A chemical or physical agent that kills pathogens, usually on inanimate objects. Usually do not sterilize but may be sporicidal.

Define: Sterilization

A process that removes or destroys all life forms

Define: Disinfection

A process that usually kills pathogenic microbes, usually on inanimate (nonliving) objects.

Types of Disinfectants: Organic acids and derivatives

All of these organic acids inhibit enzymatic or metabolic activity. Sorbic acid (potassium sorbate) inhibits mold spoilage in foods and cheese. Benzoic acid (sodium benzoate) is an antifungal used in soft drinks and other acidic foods. Methylparaben and propylparaben, which are derivatives of benzoic acid, work at a neutral pH. They inhibit molds in liquid cosmetics and shampoos. Calcium propionate prevents mold growth in bread.

Factors Affecting Physical and Chemical Control of Microorganisms: Type of Organism

G+, G-, spore former, virus, fungus, protozoan

Types of Disinfectants: Peroxides

Hydrogen peroxide is a common household antiseptic. It is not a good antiseptic for open wounds but is useful in deep anaerobic wounds where the oxygen released by the action of the enzyme catalase in tissue is effective against anaerobes such as Clostridum spp. Benzoyl peroxide is also useful in treatment of deep wounds, although it is better known as the main ingredient in many acne preparations.

Which physical method of control cannot be relied on for sterilization?

Low temperature

Additional methods of physical control: Dessication

Microbes require water for growth, and adequately dried foods will not support their growth. Drying of fruits and meats preserved these items before refrigeration. ~control amt of water present. Remove the moisture from the food item. Idea is that bacterium can't live there if there is no water.

Pasteurization

Originally a method of physical disinfection using moist heat. Developed by Louis Pasteur to kill pathogens such as Mycobacterium tuberculosis, Streptococcus pyogenes and later Coxiella burnetti in milk and other dairy products without destroying the microbes responsible for the flavor and consistency of the product. Used to disinfect fruit juices, wines and other consumable products. Has been one of the major contributing causes to reduced incidence of tuberculosis, streptococcal sore throat and diphtheria.

Types of Disinfectants: Phenol and Phenol derivatives (phenolics)

Phenol (5% carbolic acid) is seldom used today with the exception of benchtop disinfection (Bad, harsh, gives off vapors). Phenolics are widely used. Phenolics injure cell membranes, inactivate enzymes or denature proteins. They are stable (hang around for a while), persistant (effect organisms for a while) and are not sensitive to organic matter. O-phenylphenol is the main ingredient of Lysol. Hexachlorophene is effective against Staphylococci and Streptococci that cause skin infections. Problems of neurological damage to infants from excessive exposure have occurred. Products which contain more than 1% of this chemical or must be obtained by prescription. Chlorhexidine is not a phenol but its structure and applications resemble hexachlorophene. It is frequently found as a 4% solution in isopropyl alcohol and known as Hibiclens.

Additional methods of physical control: Osmotic Pressure

Placing material in a hypertonic solution such as a strong salt or sugar solution causes cells to dehydrate and die. Salting meat was an early form of preservation before refrigeration. Thick sugar solutions are used in preserving fruits. ~control amt of water present.

Factors Affecting Physical and Chemical Control of Microorganisms: Temperature

Q10 - the effect of a 10oC rise in temperature on the killing rate. Electric dishwashers are more effective than hand washing because hands cannot tolerate the higher temperature. Many dishwashers have a sanitizing cycle for effective removal & destruction.

What is one of the most widely used techniques for killing or inhibiting microbes?

Temperature

Additional methods of physical control: Filtration

The passage of a liquid or gas through a screen-like material with pores small enough to retain bacteria and viruses is an effective means of sterilization. The process is used to sterilize heat-sensitive (labile) substances such as some culture media, sera, enzymes, vaccines and antibiotic solutions. Various types are available. Membranes composed of cellulose esters or plastic polymers are highly popular because of their uniform size and range from 0.45 to 0.01 μm. HEPA (high efficiency particulate air) remove almost all microbes larger than 0.3 μm in diameter. These are used in air supply systems servicing such hospital areas as operating rooms, nurseries, intensive care units and laboratories. ~Biggest use is air. Steralize air to reduce contamination and infection.

Additional methods of physical control: Ultrasonic Sound (high frequency vibrations)

Used to clean instruments and disrupt cells for enzymatic studies. The vibrations cause the formation of bubbles which bombard surfaces. This bombardment or cavitation removes materials from surfaces such as dental scalers, dentures and jewelry.

Define: Antiseptic

a chemical agent that inhibits the growth of microbes, usually on living tissues.

Define: Log Death Curve

a graphic representation of the death of a specific population number per interval of disinfection/sterilization time.

Types of Disinfectants: Ozone

a highly reactive form of oxygen that is generated by high-voltage electric discharges and is a potential replacement for chlorination in water treatment. ~Oxygen in the atmosphere is O2, ozone is O3. Three oxygen attached to each other. The sharp smell that follows an electrical storm. Ozone used for water supplies.

Factors Affecting Physical and Chemical Control of Microorganisms: Concentration

a low concentration of a chemical may actually stimulate growth (Arndt-Schultz Law)

Phenol Coefficient Test

a measure of a chemical compound's effectiveness as a disinfectant as compared with phenol. If a product has a PC of 12, it can be diluted 12x more than phenol and have the same effect. This test is used only on phenol-like chemicals. ~Use to be the classic test for efficiency. Only use this test now if chemical in question is similar to phenol. Phenol was the first chemical to be used to disinfect. Dr. Listor discovered that MD's were killing patient b/c not disinfecting.

Use Dilution Test

a method that determines how much a chemical can be diluted and still remain effective. Establishes appropriate dilutions of an agent for actual conditions. The test organisms are commonly Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella chlorasuis. Stainless steel cylinders are contaminated with specified numbers of the test bacterium. After drying, the cylinders are placed in a test disinfectant. At least ten samples are used. They are exposed to the disinfectant for 10 minutes, allowed to drain and are transferred to culture media tubes are and incubated for 48 hours. After incubation, the tubes are examined for growth. No growth indicates an effective concentration. ~Question is, how much of the chemical should I use and the answer is always the smallest amount. This test determines the smallest amount needed.

Define: Decontamination

a process in which both living pathogens & their toxic products are removed.

Define: Asepsis

a process of preventing putrefaction or contamination.

Define: Sanitization

a process that kills or removes 99.9% of growing bacteria from an object, usually eating utensils, dishes, etc.

Types of Disinfectants: Heavy metals and their compounds

all exhibit oligodynamic action - they combine with cellular proteins and enzymes, denaturing or coagulating them. Silver is bacteriocidal and is mainly used in two forms. Silver Nitrate is used in the eyes of newborns to prevent the development of opthalmia neonatorium (gonorrheal eye infections). Silver Nitrate is expensive because there is only one manufacturer. The AMA has approved the use of antibiotic drops such as tetracyclines or erythromycin in infants. Silverdine is a combination of silver and a sulfonamide used to treat burn victims. Mercury is not in disinfection or antisepsis because it is toxic to all life forms. It combines with sulfhydryl groups, disrupting sulfide bonds in proteins. Organic mercury compounds such as Mercurochrome and Methiolate are less iriritating and toxic than inorganic mercury and are used as antiseptics. Copper sulfate is used to destroy green algae in reservoirs and other water supplies. Zinc chloride is used in mouthwashes. Zinc oxide is used in paints as an antifungal agent. Calamine lotion contains zinc oxide and ferric oxide and serves as a soothing lotion for insect bites and poison ivy because of its antiseptic and astringent properties (removes water)

Types of Disinfectants: Aldehydes

among the most effective antimicrobials such as formaldehyde. Formaldehyde - In the form of an aqueous solution, this gas is called formalin and is used to preserve biological specimens. Used for embalming. Glutaraldehyde is a less irritating form. In a 2% solution (Cidex), it is bacteriocidal, virucidal and tuberculocidal in 10 minutes. It is sporocidal after about 3 to 10 hours of contact. Used for embalming. Cidex is used for scrubbing dental chairs and respiratory anesthesia equipment.

Factors Affecting Physical and Chemical Control of Microorganisms: pH

bacteria are more resistant at their optimum pH. Many disinfectant are at a pH that is not optimum for bacteria, therefore killing them more easily.

Types of Disinfectants: Alcohols

both ethanol and isopropyl alcohol are widely used, normally at 70%. Concentrations of 60% to 95% are effective. They are bacteriocidal and fungicidal but are not effective against endospores or nonenveloped viruses. Alcohol, used as tincture, enhances the effectiveness of other chemicals.

Define: Degermation (degerming)

careful tissue cleansing with an antiseptic. It may reduce the total bacterial population a thousand fold. Replaces the term skin disinfection.

Low temperature: Lyophilization (freeze drying)

cultures are brought down to -70oC and dehydrated forming a fine powder or pellet. Cultures can be preserved for long periods of time. They can be rehydrated by placing them in an enriched broth solution and incubating them for 24-48 hours before placing them on agar slants. Although 99.9% of the cells are killed by this process, if you start with 1 billion cells, there will be 100,000 survivors. Sera and some pharmaceuticals are also processed this way to prolong their shelf life.

Low temperature: Freezing

cultures kept at 0oC can be maintained for a minimum of 1 year. Often cultures are frozen in milk or slants and overlaid with sterile mineral oil. Freezing kills many bacteria by the formation of ice crystals that disrupt cells.

Low temperature: Refrigeration

cultures kept at 2-8oC can be maintained for several months. This slows down their metabolic rate and inhibits growth.

4 test to evaluate for efficiency of a chemical agent

initial testing, phenol coefficient test, use dilution test, filter paper disk method

UHT (ultra high treatment) Pasteurization

milk and other products may now be sterilized by this methods. Involves heating milk, etc to 140oC for 3 seconds and then reducing the temperature to 74oC. This equivalent treatment sterilized the product without coagulating milk components. The shelf life of the product is much longer and refrigeration may not be necessary until opening the container.

What are three physical methods of control?

moist heat, dry heat, low temperature (refrigeration, freezing, lypophilization)

Factors Affecting Physical and Chemical Control of Microorganisms: Medium

most chemicals act by interfering with organic molecules. Media with extraneous organic matter may hinder the effectiveness of the chemical

Factors Affecting Physical and Chemical Control of Microorganisms: Contact

most disinfectants and antiseptics act by contact with the microbial cell. Many use a wetting agent such as alcohol to get the chemical in close contact with a cell. Alcohol is used as a tincture in many preparations.

Types of Disinfectants: Quaternary Ammonium Compounds

most effective against gram-positive bacteria and less effective against gram negatives. These cationic detergents have good fungicidal, amoebicidal and viricidal (enveloped viruses) activity, but they are not sporocidal. They are colorless, odorless, tasteless, nontoxic and stable. However, they are inactivated by organic matter, soap, detergents and rough surfaces such as gauze. They may even support the growth of Pseudomonas aeruginosa. They act by disrupting the cell membrane and denaturing enzymes. Widely used examples are Benzalkonium chloride (Zephiran, Roccal) & Cetylpyrimidium chloride (Cepacol).

Filter Paper Disk Method

often used in teaching laboratories to evaluate the efficiency of a chemical agent. A disk is soaked in a specified concentration of disinfectant. The disk is placed on a Petri dish containing the test bacterium. After incubation, a zone of inhibition will appear around the disk if the disinfectant is effective. The larger the zone of inhibition, the greater the effectiveness of the agent.

Factors Affecting Physical and Chemical Control of Microorganisms: Time

one of the most important and most frequently overlooked factors in control. Any microbial population consists of individual cells with varying degrees of resistance. The exposure time must be sufficient to kill even the most resistant members of the population, The phase of the growth cycle also affects control; lag phase cells are most susceptible and decline phase cells are the most resistant.

Define: Cidal Agent

one that causes fairly prompt death (irreversible damage). E.g. Bactericide, fungicide, virucide, germicide, microbiocide.

Define: Static Agent

one that causes inhibition of growth (reversible damage). E.g. Bacteriostatic agent, fungistatic agent

Types of Disinfectants: Super D Hydrogen Peroxide

stabilized form; does not decompose and may be used to disinfect soft contact lenses, utensils, heat sensitive equipment and food processing equipment.

Types of Disinfectants: Gaseous Chemosterilizers

such as Ethylene Oxide inactivate proteins by combining with organic functional groups. (used to treat things that can't stand autoclave heat). It is a sterilant that kills all microbes and endospores and has great penetrating power. It is used to sterilize disposable medical supplies and equipment used in hospitals when heat treatment is not practical. Propylene oxide and beta-propiolactone are also important gaseous sterilants. These gases are suspected carcinogens.

Additional methods of physical control: Ionizing Radiation

such as gamma rays or high-energy electron beams travels through a cell so quickly that it ionizes water resulting in reactive hydroxyl and peroxide radicals that damage DNA. The two most commonly used radioisotopes are cobalt (60Co) and Cesium (137Cs). Uses: sterilization of pharmaceuticals, plasticware, disposable medical supplies (syringes, catheters, suturing materials) and irradiation of foods and radiopasteurization.

Additional methods of physical control: Nonionizing radiation

such as ultraviolet (UV) light has a longer wavelength and less energy. The most efficient bacteriocidal wavelength is 265 nm (because DNA absorbs this best). UV irradiation causes the formation of thymine dimers which distort the DNA molecule leading to mutations and death. It is used to sterilize sera and vaccines, in the meat packing industry to control microbial spoilage and may be found in hospitals and industries as germicidal lamps. The major disadvantage of UV irradiation is its poor penetrating power. It may also cause blindness and skin cancer. Photoreactivation is a phenomenon in which UV irradiated bacterial cells will repair damaged DNA when exposed to a strong light source. It involves a light-activated enzymatic repair system in which the thymine dimers are removed and replaced. This may also lead to mutations via an SOS repair mechanism.

Initial testing

testing disinfectants or sterilizers using pure cultures to define their range of usefulness

Define: Temperature Coefficient (Q10)

the effect of a 10o rise in temperature on the killing rate

Define: Antispesis

the inhibition of growth of microorganisms on living tissues.

Define: Microbial load (bioburden)

the number of microbes in an object to be treated

Define: Thermal Death Time

the shortest period of time required to kill a suspension of bacteria at a specific temperature under specific conditions

Define: Thermal Death Point

the temperature at which a given suspension of bacteria are killed in 10 minutes

Define: Decimal Reduction Time (DRT)

the time required for 90% of microbes to be killed

Dry heat: Direct flaming

used on inoculation loops and transfer needles

Dry heat: incineration

used to sterilize contaminated paper products, dressings, cadavers, bags, wipes, anything that is disposable and no longer of value.