CPB227 - Microbiology - Module 2 - Sterilization and disinfection

Disinfection

Act of reducing the number of pathogenic organisms on objects or in material so that they pose no threat of disease Typically applied to inanimate objects

ATPase Bioluminescence

All prokaryotic and eukaryotic cells use the molecule adenosinitriphosphate (ATP) for energy This system can detect how much ATP is present on a surface Good test of detecting organic material It is used to detect how clean a surface is.

Antimicrobial agents - Proteins

Alteration of protein structure is call denaturation (hydrogen and disulfide bonds are disrupted, and the functional shape of the protein molecule is destroyed) Any agent that denatures proteins prevents them from carrying out their normal functions

Beneficial biofilms

Aquariums use biological filters laden with biofilms to maintain water quality Fish produce nitrogen waste (ammonia containing compounds) Ammonia in high quantities are toxic to fish

Sterilization - Moist heat

Autoclave Penetrating properties Widely used physical sterilization method Autoclave - water heated under pressure, its boiling point is elevated, so temps above 100 C can be reached Complete sterilization can be achieve after 15 minutes in an autoclave at a pressure of 15 psi and 121 C

Organisms killed

Bacteria and fungi can exist in two forms 1. Actively growing vegetative form 2. Static spore form Spores are highly resistant Mycobacteria re more resistant than most other vegetative forms

Quaternaries

Bactericide - gram positive bacteria and gram - negative organisms. Not effective against bacterial spores Have been shown to be fungicidal Many applications as disinfectants; sanitizing agents Germicidal and detergent action Low toxicity high solubility in water Stability in solution Noncorrosiveness Damaging effects and denaturation of proteins, interference of glycolysis and membrane damage Most likely site of damage - cytoplasmic membrane

Biological indicators

CDC recommends weekly autoclaving of a culture containing heat resistant endospores, such as those of Geogacillus stearothermophilus, to check autoclave performance Endospore strips are commercially available to make this task easy

Ultraviolet (UV) light (Non-ionizing)

Consists of light waves between 40 and 390 nanometers (nm) Especially effective for inactivating viruses Kills fewer bacteria than one would expect Penetrates air-reducing number of air-borne microorganisms and killing them on surfaces in operating rooms and rooms that will contain caged animals Limited use and does not penetrate glass, cloth, paper or most other materials, and it does not go around corner or under lab benches

Autoclave indicator tape

Consists of yellow or white indicator strips and pressure -sensitive adhesive Stripes will turn brown or black Complete color change occurs in 15 to 20 minutes at 121 C Not fully reliable - do not indicate how long appropriate conditions were maintained

Aldehydes (Formaldehyde and flutaraldehyde)

Highly microbial and sporcidal Formaldehyde - solid substance and room temperature Vet Med used on aqueous solution as formalin Preserve tissue specimens Fumes are noxious and irritating to tissues and eyes Glutaraldehyde 2% solution - wide spectrum of antimicrobial activity Effective against: vegetative bacteria, fungi, bacterial and fungal spores, and viruses

Organisms inhibited

Ideally organisms should be killed

Aseptic techniques

Involves practices and procedures performed under controlled conditions with the goal of minimizing contamination and decreasing the likelihood of transmitting an infection to a patient

Proper sanitation

It is important to use proper sanitation procedures prior to autoclaving or applying the disinfectant Every surface must be cleaned to remove any organic material Typically involves the use of soap and water. Once surface has completely dried, the object or surface may be either autoclaved or disinfected, as appropriate

Antimicrobial agents - membranes

Membranes contain proteins, so can be altered by all preceding reactions Membranes also contain lipid Surfactants include alcohols, detergents, and Quaternary ammonium compounds, such as benzalkonium chloride, which dissolve lipids

Antimicrobial agents - Cell components

Nucleic acid and energy producing systems Alkylanine agents can replace hydrogen or amino or alcohol group in nucleic acid Certain dyes interfere with cell wall formation Some substances (lactic acid and propionic acid - ie end products of fermentation) inhibit fermentation and thus prevent energy production in certain bacteria, molds, and some other organisms

Factors that can influence the effectiveness (potency) of a chemical antimicrobial agent

Number of microorganisms present, time, temperature, pH and concentration Time-adequate time should always be allowed for an agent to kill the maximum number of organisms Temperature - increasing temperature doubles the rate of chemical reactions and thereby increases the potency of the chemical agent pH = acidic or alkaline pH can increase or decrease the agents' potency. A pH that increases the degree of ionization of a chemical agent often increases its ability to penetrate a cell. Such a pH can also alter the contents of the cell itself Concentration - increasing concentration may increase the effects of most antimicrobial chemical agents, with the exception of 100% ethanol High concentrations may be bactericidal, whereas lower concentrations may be bacteriostatic (growth inhibiting) Follow label instructions !!

Halogens (Iodine)

Oldest and most effective topical antiseptics Used as a germicidal agent tincture of iodine Iodophors (Iodine and carrier molecules) release iodine over time Germicidal characteristics of iodine Effective against bacterial sports Mainly used as surgical scrubs and among the best antiseptics Iodine- oxidizing agent; which may account for its antimicrobial activity Can irreversibly oxidize and thus inactivate essential metabolic compounds (proteins and sulfhydryl groups)

Factors considered when selecting an antiseptic or disinfectant

Organisms killed Organisms inhibited Rate of action Side effects Additional considerations

Sterilization - Dry heat

Oven Penetrates substances more slowly than moist (ie steam) heat Usually used to sterilize metal objects and glassware Only suitable means of sterilizing oils and powders 171 C for 1 hour, 160 C for 2 hours or longer, or 121 C for 10-16 hours or longer depending on the volume of the load Open flame - used to sterilize inoculating loops and the mouths of culture tubes

Sterilization method - Filtration

Passage of a material through a filter, or straining device Used mostly for liquids and requires filters with exceedingly small ports

Ways VT can reduce the chances of transmitting infection to a patient

Proper hand washing (single most effective method) Aseptic techniques Proper sanitation Disinfection

Rate of action

Rate of action differs greatly among the various chemical agents Time required for a particular agent to produce its maximum effect is the disinfection time Influenced by: Nature of material being disinfected Degree of soil and microbial contamination Concentraton of disinfectant

Additional considerations on antiseptics and disinfectants

Reasonably priced Maintain potency for long periods of time Soluble in water and stable in aqueous solution

Removal of organic matter

Removal typically involves the use of soap and water Even the best autoclaves and disinfectants will fail to eliminate or reduce, respectively, the amount of microbes on a surface or in an object if there is too much organic material present

Assessing success of disinfection in a clinical setting

Replicate Organism Detection and Counting (RODAC) plates ATPase Bioluminescence

Chlorine and chlorine compounds

Represent one of the most widely used disinfectants Available in many different compounds Hypochlorides (chlorinate lime, bleach) Chloramines - stability 5% sodium hypochlorite (bleach) disinfectant of choice in most small animal clinics to kill parvo virus Inactivated by the presence of organic matter Work by forming hypochlorous acid. Oxygen released is a strong oxidizing agent, results, microorganisms are destroyed

Side effect of an antiseptic or disinfectant

Toxicity Possession of an irritating vapor Undesirable staining properties Destructive effects on instruments and fabrics All antiseptics or disinfectants should be relatively non-toxic

Removal of biofilm

Ultrasonic cleaners - break apart and dispense biofilms off objects such as surgical instruments Manual scrubbing can help reduce the amount of biofilm

Sterilization method - sonication

Ultrasonic vibration Mechanical method of microbial control used to remove microbes form the surface of instruments and teeth Utilizes high frequency sound waves to generate "shock waves" that are conducted through living structures or liquids Force exerted by these shock waves leads to the rupture of microbial membranes, and their removal from surfaces Used to clean surgical instruments, dental instruments, teeth, pacemakers, hearing aids, test tubes, and small electronics

Phenols & phenolic compounds

Very effective disinfectants Spectrum of activity Phenol 5% rapidly kills the vegetative cells of bacteria and fungi, spores re much more resistant Mode of action - not clear Phenolics can have a variety of effects such as disruption of cells, precipitation of cell protein, inactivation of enzymes and leakage of amino acids from the cells Lethal effect is associated with physical damage to the membrane structures in the cell surface

Ionizing radiation

X-rays - wavelengths of 0.1 to 40 nm and gamma rays - even shorter wavelengths Damages DNA and produces peroxides, which act as powerful oxidizing agents in cells Can also kill or cause mutations in human cells if it reaches them Used to sterilize plastic laboratory and medical equipment as well as pharmaceutical products

Alcohols

70% - bacteriocidal for vegetative cells but are practically inert against bacterial sports Active against enveloped and non-enveloped viruses Not active against fungi Effective in reducing the microbial flora of skin and the disinfection of clinical thermometers Protein denaturates - account for their antimicrobial activity Solvents for lipids and may damage lipid complexes in the cell membrane

Sterilization method - radiation

Four general types : Ultraviolet (UV) light (non-ionizing) Ionizing radiation (X-rays) Microwave radiation Strong visible Light

Ethylene Oxide

Gas - powerful sterilizing agent Used to sterilize heat - or moisture - sensitive material Power to penetrate Believed to be alkylation reactions with enzymes and other proteins. Alkylation consists of the replacement of an active hydrogen atom in an organic compound

Biofilm

Grouping of bacteria with a distinct architectural structure Made up of two main parts Core of bacteria Outer layer of extracellular polysaccarides Bacteria produce these extracellular polysaccarieds; they are very sticky and help in the attachment of the biofilm to surfaces like the inside of water pipes Relatively resistant to antibiotics

Label directions

Higher concentrations - may be bactericidal (kills the bacteria) Lower concentrations - may be bacteriostatic (growth inhibiting)

Proper hand washing

Single most effective method to reduce the chances of transmitting infection to a patient Good techniques - all surfaces of the hands, fingers, and nails are adequately scrubbed Contact time - prior to veterinary patient - minimum of 20 seconds Pre-surgical at least 10 minutes

Replicate Organism Detection and Counting (RODAC) plates

Solid agar microbial media plates used for the detection and enumeration of aerobic (oxygen loving) bacteria present on a surface (eg. surgery table)