Topic 6

Sterilization

The removal of destruction of all living microorganisms

Sterilant

A sterilizing agent

Disinfection

• Destruction of vegetative pathogens

• Not the same as complete sterility

• Make use of chemicals, UV, radiation, boiling water, steam

• Most commonly applied to the use of a chemical to treat an inert surface or substance

Antipsepsis

When the treatment is directed at living tissue

Degerming

Results in mechanical removal rather than killing most of the microbes in a limited area.

Ex: Before the animal receive an injection, the skin is swabbed with alcohol

Sanitization

Intended to lower microbial counts to safe public health levels and minimize the chances of disease transmission from one user to another.

Ex: High-temperature washing

Suffix —cide

• Meaning “kill”

• Biocide/Germicide - kills microorganisms

• Fungicide

• Virucide

Suffix —stat or —stasis

Treatments that only inhibit the growth and multiplication of bacteria.

Ex: Bacteriostatic agent

Sepsis

From the Greek for decay or putrid; Indicates bacterial contamination

Aseptic

An object or area is free of pathogens

Asepsis

The absence of significant contamination

Sterilization

Destruction or removal of all forms of microbial life, including endospores but with the possible exception of prions.

Usually done by steam under pressure or a sterilizing gas, such as ethylene oxide.

Commercial Sterilization

Sufficient heat treatment to kill endospores of Clostridium botulinum in canned food.

More-resistant endospores of thermophilic bacteria may survive, but they will not germinate and grow under normal storage conditions.

Disinfection

Destruction of vegetative pathogens on inanimate objects.

May make use of physical or chemical methods.

Antisepsis

Destruction of vegetative pathogens on living tissue.

Treatment is almost always by chemical antimicrobials

Degerming

Removal of microbes from a limited area, such as the skin around an injection site.

Mostly a mechanical removal by soap and water or an alcohol soaked swab.

Sanitization

Treatment is intended to lower microbial counts on eating and drinking utensils to safe public health levels.

May be done with high temperature washing or by dipping into a chemical disinfectant.

Number of microbes

More microbes = longer time to eliminate

Environmental Influence

• Most disinfectants work better in warm solutions

• Presence of inorganic matter (blood, vomitus, feces) inhibits the action of chemical antimicrobials

• Nature of suspending medium (fats and protein) can be protective for microbes.

Time exposure

Extended exposure can affect more resistant microbes or endospores

Alteration of Membrane Permeability

Damage to the lipids or proteins of the plasma membrane by antimicrobial agents causes cellular contents to leak into the surrounding medium and interferes with the growth of the cell.

Damage to Protein and Nucleic Acids

• The nucleic acids DNA and RNA are the carriers of the cell’s genetic information

• Thus, damage to these by heat radiation or chemical is frequently lethal to the cell

• The cell can no longer replicate, nor can it carry out normal metabolic functions such as the synthesis of enzymes

Heat

• May inactivate certain vitamins or antibiotics in a solution

• One of the most common methods of food preservation

• Usually used to sterilize laboratory media and glassware and hospital instruments

• Kills microorganisms by denaturing their enzymes

• Heat resistance varies among different microbes

Thermal Death Time (TDT)

The minimal length of the time for all bacteria in a particular liquid culture to be killed at a given temperature

Decimal Reduction Time (DRT)

The time, in minutes, in which 90% of a population of bacteria at a given temperature will be killed

Boiling or Flowing Steam

(Protein Denaturation) Kills vegetative bacterial and fungal pathogens and many viruses within 10min; less effective on endospore.

Autoclaving

(Protein Denaturation) Very effective method of sterilization; at about 15psi of pressure (121C), all vegetative cells and their endospores are killed in about 15mins

Pasteurization

(Protein denaturation) Heat treatment for milk (72C for about 15sec) that kills all pathogens and most nonpathogens.

Direct Flaming

• Burning contaminants to ashes

• Very effective method of sterilization.

• Used for inoculating loops.

Incineration

• Burning to ashes

• Very effective method of sterilization.

• Used for disposal of contaminated dressings, animal carcasses and paper.

Hot-air sterilization

• Oxidation

• Very effective method of sterilization but requires temperature of 170C for about 2hr.

• Used for empty glassware

Autoclaving

• Preferred method of sterilization in healthcare environments, unless the materials to be sterilized can be damaged by heat or moisture.

• Higher pressure = Higher temperature

• Used to sterilize culture medium media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items that can withstand high temperatures and pressures.

Pasteurization

• By Louis Pasteur

• Mild heating, which was sufficient to kill the organisms that caused the particular spoilage problem without seriously damaging the taste of the product

• Later applied to milk

• Eliminates pathogenic microbes, lowers microbial number

Ultra High Temperature (UHT) Treatment

Can be stored for several months without refrigeration. 140C for 4 seconds.

Filtration

• The passage of a liquid or gas through a screenlike material with pores small enough to retain microorganisms.

• Used to sterilize heat-sensitive materials, such as some culture media, enzymes, vaccines, and antibiotic solution.

Refrigeration

Decreased chemical reactions and possible changes in proteins.

Has a bacteriostatic effect.

Deep-freezing

Decreased chemical reaction and possible changes in proteins.

An effective method for preserving microbial cultures, food, and drugs.

Lyophilization

Decreased chemical reaction and possible changes in proteins.

Most effective method for long-term preservation of microbial cultures, food, and drugs.

High Pressure

• Alters the molecular structures of proteins and carbohydrates, resulting in the rapid inactivation of vegetative bacterial cells.

• Endospores are relatively resistant to high pressure.

• An advantage is that these treatments preserve flavors, colors, and nutrient values of the products.

Desiccation

• In the absence of water, microorganisms cannot grow or reproduce but can remain valuable for years. Then, when water is made available to the them, they can resume their growth and division.

• The principle that underlies lyophilization, or freeze-drying, a laboratory process for preserving microbes.

• Ex: Gonorrhea bacterium can withstand desiccation for only about an hour, but the tuberculosis bacterium can remain viable for months.

• Viruses are generally resistant to this but they are not as resistant as bacterial endospores, some of which have survived for centuries.

Osmotic Pressure

• Use of high concentration of salts and sugars to preserve food is based on this effect.

• High concentrations of these substances create a hypertonic environment causes water to leave the microbial cell

• Ex: Concentrated salt solutions are used to cure meats, and thick sugar solutions are used to preserve fruits.

Ionizing Radiation

• Have shorter wavelength (less than 1nm)

• The principal effect of this is ionization of water - forming highly reactive hydroxyl radicals (toxic from of oxygen) - kills organisms by reacting with organic cellular components especially DNA.

• Ex: Gamma rays, X-rays, High energy electron beams

Nonionizing Radiations

• Have longer wavelength (greater than 1nm).

• Damages the DNA of exposed cells by causing bond to form between adjacent pyrimidine bases, usually thymines, in DNA chains.

• The thymine dimers inhibit correct replication of the DNA during cell reproduction.

• Most effective wavelength for killing microorganisms are about 260nm.

• Ex: UV Light

Microwaves

• Do not have much direct effect on microorganisms, and bacteria can readily be isolated from the interior of recently operated microwave ovens.

• Moisture-containing foods are heated by microwave action, and the heat will kill most vegetative pathogens.

Principles of Effective Disinfection

• Reading the label

• Concentration of disinfectant affects its action

• Consider the material being disinfected

• Will the disinfectant easily make contact with microbes

• A disinfectant might need to be left on a surface for several hours

Dilution Tests

• Current standard by American Official Analytical Chemist

• Metal or glass cylinders (8mm x 10mm) are dipped into standard cultures of the test bacteria grown in liquid media, removed and dried at 37C for a short time.

• The dried cultures are transferred to a medium that permits the growth of the surviving bacteria.

The Disk-Diffusion Method

• Used in teaching laboratories to evaluate the efficacy of a chemical reagent.

• A disk or filter paper is soaked with a chemical and placed in an agar plate previously inoculated with test organism.

• A clear zone representing inhibition of growth can be seen around the disk.

Phenols

• Irritated the skin and gas disagreeable odor.

• Often used in throat lozenges for its local anesthetic effect but has little antimicrobial effect at low concentrations.

• Concentration above 1% (in some throat sprays) = significant antibacterial effect.

Phenolics

• Derivatives of phenols that has been chemically altered to reduce its irritating qualities or increase its antibacterial activity in combination with a soap or detergent.

• Effective on the cell wall of mycobacteria which are rich in lipids.

• Remain inactive in the presence of organic compounds, stable and persist for long periods after application.

• Suitable agents for disinfecting pus, saliva and feces.

MOA

Injuring lipid-containing plasma membranes, which results in leakage of cellular contents.

Bisphenols

Derivative of phenol

Hexachlorphene

Ingredient of a prescription lotion used for surgical and hospital microbial control.

Gram-positive staphylococci and streptococci is susceptible.

Triclosan

Ingredient in antibacterial soaps, toothpastes, and mouthwashes; its uses is now widespread that resistant bacteria have been reported; it inhibits an enzyme needed for the biosynthesis of fatty acids which mainly affects the integrity of the plasma membrane.

Biguanides

• Have a broad spectrum of activity.

• Affects the bacterial cell membrane.

• Especially effective against gram-positive bacteria and also gram-negative bacteria except of most pseudomonas.

• Not sporicidal but have some activity against enveloped viruses.

Chlorhexidine

Biguanides frequently used for microbial control on skin and mucous membranes; often used for surgical hand scrubs.

Essential Oils

• Mixture of hydrocarbons extracted from plants.

• Used in traditional medicine and preserving foods.

• Antimicrobial action is primarily due to phenolics and terpenes.

• Generally have greater activity against gram-positive bacteria than against gram-negative bacteria.

Iodine

• One of the oldest and most effective antiseptics.

• Active against all kinds of bacteria.

• Impairs protein synthesis and alters cell membranes.

• Available as tincture, iodophor and povidone-iodine.

• Used mainly for skin disinfection and wound treatment.

Chlorine

• As gas or in combination with other chemicals.

• Hypochlorous acid (HOCl) that forms when chlorine is added to water.

• Liquid form of compressed chlorine gas for disinfecting swimming pools and sewage.

Calcium Hypochlorite

Used to disinfect dairy equipment and restaurant eating utensils.

Sodium Hypochlorite

Household disinfectant (bleach)

Alcohols

• Effectively kill bacteria and fungi but not endospores and nonenveloped viruses.

• Denatures protein, disrupt membranes and dissolve many lipids including lipid component of enveloped viruses.

• Unsatisfactory antiseptics when applied to wounds.

Ethanol

• 70% - recommended optimum concentration.

• When it is pure, it is less effective than aqueous solutions because denaturation requires water.

Isopropanol

Often sold as rubbing alcohol; slightly superior to ethanol as an antiseptic and disinfectant. Less volatile, less expensive, more easily obtained than ethanol.

Heavy Metals and their Compounds

Can be biocidal or antiseptic.

Oligodynamic Action

The ability of very small amounts of heavy metals to exert antimicrobial activity.

Silver-sulfadiazine

Topical cream for use on burns.

Inorganic Mercury Compounds (Mercuric Chloride)

Bacteriostatic, broad-spectrum but now is limited because of toxicity and ineffectiveness in organic matter.

Copper Sulfate

Chiefly used to destroy green algae.

Silver-impregnated dressings

Useful against antibiotic-resistant bacteria.

Surface Active Agents

Can decrease surface tension among molecules of a liquid.

Ex: soap and detergents - mechanical removing of microbes through scrubbing; breaks oily film (emulsification)

Quaternary Ammonium Compounds (Quats)

• Most widely used; cationic detergents

• Cleansing ability is related to positively charged portion

• Name is derived from four-valence ammonium (NH4+)

• Also fungicidal, amebicidal, virucidal against enveloped viruses.

• Do not kill endospores or mycobacterium.

• Ex: benzalkonium chloride, cetylpyridium chloride

Sulfur Dioxide

Wine making

Sodium Benzoate

Prevents molds from growing in cheese and soft drinks

Sodium Nitrate and Sodium Nitrite

Added to meat products like ham, bacon, hotdogs, and sausage.

• Preserves the read color of meat by reactive with blood components in the meat.

• Prevent germination and growth of botulism endospores that might be present.

Nisin

Antibiotic added to cheese to inhibit the growth of certain endospore-forming spoilage bacteria.

Natamycin (pimaricin)

Antifungal antibiotic mostly used in cheese.

Aldehyde

Among the most effective antimicrobials

Formaldehyde (formalin (37%) aqueous solution)

Used to preserve biological specimens and inactive bacteria and viruses in vaccines.

Glutaraldehyde

• Less irritating and more effective than formaldehyde.

• Used to disinfect hospital instruments

• 2% solution is bactericidal, tuberculocidal and virucidal in 10min and sporicidal in 3-10 hrs.

• One of the hew liquid chemical disinfectants that can be considered a sterilizing agent.

Gaseous Chemosterilants

Frequently used as substitutes for physical sterilization processes. Requires a closed chamber similar to a steam autoclave.

Ethylene Oxide

Kills all microbes and endospores but requires a lengthy exposure period of several hours. It is toxic and explosive in its pure for so its usually mixed with nonflammable gas such as CO2.

Chlorine Dioxide

• Short-lived gas

• Has been used to fumigate enclosed building areas contaminated with endospores of anthrax.

• Has broad spectrum of activity against bacteria and viruses.

Plasma Sterilization

• Used for sterilizing long, hollow tubes.

• The instruments are placed in a container in which a combination of a vacuum, electromagnetic field, and chemicals such as hydrogen peroxide form the plasma.

Plasma

A state of matter in which a gas is excited by an electromagnetic field.

Hydrogen Peroxide

• Not a good antiseptic for open wounds.

• Quickly broken down to water and gaseous oxygen by the action of the enzyme catalase, which is present in human or animal cells.

• Does effectively disinfect inanimate objects.

• Even sporicidal at high concentrations.

• Heated, gaseous hydrogen peroxide can be used as a sterilant of atmosphere and surfaces.

Paracetic Acid (Peroxyacetic Acid) PAA

• One of the most effective liquid chemical sporicides available and can be used as a sterilant.

• Generally effective on endospores and viruses within 30 minutes and kills vegetative bacteria and fungi in less than 5 minutes.

• Disinfection of food-processing and medical equipment, especially endoscopes, because it leaves no toxic residues.