Dr. R.

Tan

Primary target of microbial control Microorganisms capable of causing infection or spoilage that are constantly present in the external environment and on the human body Relative Resistance of Microbial Forms
Highest resistance
bacterial endospore (Bacillus & Clostridium)

Moderate resistance
protozoan cyst some fungal spores some naked virus vegetative bacteria that have higher resistance ( M. tuberculosis, S.aureus, Pseudomonas)

Least resistance
most bacterial vegetative cells ordinary fungal spores & hypae enveloped virus Yeasts Trophozoites

Definition of Frequently Used Terms

Sterilization - any process, physical or chemical, that will destroy or remove all viable microorganisms (bacteria, fungi, spores, and virus) from an object or from a particular environment - total inactivation of all forms of microbial life in terms of the organism s ability to reproduce Sanitization - is any cleansing technique that mechanically removes microorganisms or reduce the level of contaminants or microbial population to a safe level as determined by public health standards

Sepsis - growth of microorganisms or the presence of microbial toxins in the blood and other tissues Asepsis - any practice that prevents the entry of infectious agents into sterile tissues thus preventing infection Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery and also in the food industry Antiseptics - are products/chemical agents used for the asepsis of living tissues applied directly to exposed body surfaces (skin, wounds, mucosa) and surgical incision to destroy or inhibit vegetative pathogens - applied topically to living tissues

Disinfection - refers to the use of physical process or chemical agent (disinfectants) that promotes killing, inhibition, or removal of microorganisms (vegetative or growing form) but not the resistant spores of bacteria

Disinfectant - is an agent used to reduce the viability of a microbial population below a threshold level that causes infection - are used on surfaces of inanimate objects (floors, tables)

suffix cide/cidal - meaninig to kill - added when a killing action is implied - a suffix indicating that the antimicrobial agent will kill or destroy a certain group of microorganism Germicide: An agent that kills certain microorganisms  Bactericide: An agent that kills bacteria. Most do not kill endospores.  Viricide: An agent that inactivates viruses.  Fungicide: An agent that kills fungi.  Sporicide: An agent that kills bacterial endospores and of fungal spores.

Suffix static/stasis - meaning to stand still / stop - the agent will prevent the growth or multiplication of the type of organism but are not killed outright Bacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them Fungistatic agent prevents the growth of fungi

Dynamics of Sterilization & Disinfection

Microbes are not killed instantly when exposed to a lethal agent The kinetics of death of a microbial population is exponential: the number of survivors decreases with time (exponential killing)

the larger the initial number of cells to be killed, the more intense or prolonged is the treatment required for sterilization

Evaluation of Disinfectants
Phenol Coefficient Test Phenol p reference standard agent official method used to test disinfectant potency designed to determine the ratio of the highest dilution of the germicide that will kill the test organism within a specified time to the greatest dilution of phenol showing the same result indicates disinfecting ability compared to that of phenol

METHODS OF MICROBIAL CONTROL

Physical Agents Chemical Agents Chemotherapeutic Agents

Physical Agents
I. Heat II. Freezing III. Radiation IV. Filtration V. SonicVibration

I. HEAT - most reliable and universally applied method of sterilization - whenever possible, should be the method of choice - 2 kinds of heat : 1. Dry 2. Moist Thermal Death Time - refers to minimum time required to kill all microbes at a specified temperature in a specified environment - sterilization time is directly related to the number of organisms in a suspension - time required inversely related to temperature of exposure Thermal Death Point : lowest temperature required to kill all microbes when time is held constant

DRY HEAT
Sterilization that requires higher temperature and longer period of heating denotes air with a low moisture content that has been heated by flame or electric heating coil Temp. ranges from 160C several thousand C MOA : denaturation of proteins oxidative damage toxicity effects of elevated levels of electrolytes Used for sterilization of glasswares and materials such as oils, jellies, and powders that are impervious to steam

Forms of Dry Heat:

1. Direct flaming
- bacteriological loop, needle, glass rods through the flame of a Bunsen burner
Outer non luminous flame oxidizing region (incomplete combustion) Hottest part of the flame inner blue cone (reducing region of intense combustion) Unburnt gas and air region

Red heat: Inoculating loops & needles, points of forceps and spatulas in Bunsen burner flame till red hot Flaming: Scalpel blades, needles, mouths of culture tubes, bottles, glass slides, cover slips, and glass rods by passing through Bunsen flame without red hot

2. Incineration
- Soiled dressing and hospital waste materials are physically destroyed by burning them to ashes - the ultimate sterilization

3. Hot air oven

- most widely used type of dry heat - oven heat set 1800C for 2 hours - temp. enough to kill all type of organism, including the sporeformers - For glasswares, metal instruments, sealed materials, swab sticks are sterilized - It is electrically heated and fitted with thermostat that maintains chamber air at chosen temperature and fan than distributes hot air in chamber

MOIST HEAT
more effective than dry heat, and kills bacteria faster temp ranges from 60-135C Mesophilic nonsporeformers: 60C for 30mins 60C for 60mins: S. aureus / S. faecalis 80C for 5-10mins: Vegetative form of all bacteria, yeast & fungi 120C for 4mins: Sporeformers (C. botulinum) MOA: denaturation and coagulation of proteins production of single-stranded breaks of DNA loss of functional integrity of membrane and leakage of small molecules

Methods of moist heat: 1. Pasteurization - impt. application: * make food and beverages safe for consumption * pasteurization of milk and preparation of bacterial vaccines - 60-65C for 30 minutes followed by rapid cooling to 130C or lower - Heat labile liquids may be disinfected but NOT STERLIZED by heating below 1000C - primary target: non spore forming pathogens(Salmonella sp., Campylobacter jejuni, Listeria monocytogenes, Brucella sp., Coxiella sp. and Mycobacterium sp.)

2. Boiling - can be relied only for disinfection and not for sterilization - 30 minutes boiling 1000C adequate to kill vegetative cell p Bacteria, Fungi, Protozoa - inactive against viruses - sporeformer 5 hours - ineffective against many bacterial and fungal spores - useful for reducing viable levels if no better method is available 3. Live Steam - is steam under pressure, obtained by heating water in a boiler (Arnold sterilizer) - An apparatus that employs steam under pressure at 100C - same sterilizing action with boiling - for sterilization of specialized bacteriological culture media

4. Tyndallization (Fractional / Intermittent sterilization )

- free flowing steam 80-1000C 30minutes for 3 consecutive days involves exposing the material to elevated temp (killing the vegetative cell) incubate at 37C (to allow spores to germinate to form new vegetative cell) expose to elevated temp again (to kill the newly germinated vegetative cells) - for sterilization of certain liquid or semisolid materials that are easily destroyed by heat and for materials that are not suited to the autoclave - it is used to sterilize heat-sensitive culture media containing materials such as carbohydrates, egg or serum - kills both sporeformer & non-sporeformers

5. Steam Sterilization (Autoclave) - the whole of the material to be sterilized remain in contact with saturated steam at the required temperature & time - 1210C for 20 minutes (15 lbs steam pressure per sq. inch) - most efficient & dependable moist heat method ery effective against vegetative cells & bacterial endospores - commonly used for sterilizing : 1. surgical instruments and dressings 2. Bacteriological media not destroyed by heat 3. biohazard wastes - Preferred method of sterilization, unless material is damaged by heat, moisture, or high pressure

II. FREEZING
not a reliable method of sterilization the formation of ice crystals outside the cell causes the withdrawal of water from the cell interior, resulting in an increased intracellular electrolyte concentration and denaturation of proteins Primarily used in the preservation of bacterial cultures Lyophilization ( Freeze-drying ) - for preserving biological materials Refrigeration (0 to 7oC ) Bacteriostatic effect: Reduces metabolic rate of most microbes so they cannot reproduce or produce toxins

III. RADIATION
Sunlight - bactericidal activity - impt. role in spontaneous sterilization under natural conditions

defined as energy emitted from atomic activities and dispensed at high velocity through matter or space Inactivation of microorganisms occurs either: - direct ionization of a vital cellular molecule - indirectly through the reaction of the free radicals produced in the cellular fluid

Ionizing radiation Radiation that have sufficient energy to remove electrons out of microbial molecules X-ray, alpha, beta & gamma rays uses short wavelength much higher energy content than UV rays has greater power penetration can penetrate a solid barrier, bombard a cell, enter it, and dislodge electrons from molecules breakage of DNA creates massive mutations greater capacity to produce lethal effects used for sterilization of sutures, plastics, syringes, catheters, prosthesis & plastic tubings Disadvantages: - Penetrates human tissues - May cause genetic mutations in humans

Non-ionizing radiation
Energy absorbed by the molecule cannot remove an electron completely, the excitation produced often leads to photochemical changes Ex. Ultraviolet light - Effectiveness of UV light as a lethal and mutagenic agent is closely related to its wavelength (240-280nm); optimum at about 260nm which corresponds with the maximum absorption of DNA - Exposure to UV light causes thymine molecules to link together forming thymine dimers Energy of UV radiation is low, penetrating ability is very poor primarily used to control airborne infections, where it is used for the disinfection of enclosed areas such as nurseries, hospital wards & OR

IV.

FILTRATION

process of separating microorganism from contaminated solution an effective method to remove microbes from air and liquids used to prepare liquids that cannot withstand heat, including serum and other blood products, vaccines, drugs, IV fluids, enzymes and culture media useful for trapping microorganism only
types of membrane filters: 1.Seitz asbestose cellulose 2.Sintered glass glass filaments 3.Chamberland unglazed porcelain 4.Berkefeld diatomaceous earth

High Efficiency Particulate Air Filters (HEPA) - composed of a mat of randomly arranged fibers composed of fiberglass between 0.5 - 2.0 m in diameter - used to provide a flow of sterile air to hospital /sterile rooms - critical in the prevention of the spread of airborne bacterial and viral organisms - medical-use HEPA filtration systems also incorporate highenergy ultra-violet light units to kill off the live bacteria and viruses trapped by the filter media. Membrane Filters - Used in industry and research 0.22 and 0.45um Pores: Used to filter most bacteria Don t retain spirochetes, mycoplasmas and virus 0.01 um Pores: Retain all viruses and some large proteins

V. SONIC VIBRATIONS Sound vibration at high frequency, in an upper audible and ultrasonic range (20-1000 kHz), provide a useful technique for disruption & disintegration of the cell The passage of sound thru a liquid produces alternating pressure changes, which, if the sound intensity is sufficiently great, causes cavities to form in the liquid the cavities grow in size until they collapse violently leading to cell disintegration No practical value in sterilization & disinfection uses 1. research laboratories 2. treating sewage H2O

Chemical Agents
I. Agents that damage cell membrane II. Agents that denatures protein III. Agents that modify the functional group of protein & nucleic acid

Factors affecting disinfectant potency

1. Number of microorganisms 2. Nature of microorganism 3. Temperature 4. pH 5. Time 6. Mode of action of the agents 7. Concentration of the agent 8. Presence of exogenous materials - alter disinfectant activity by: surface absorption of the disinfectant by protein colloids formation of a chemically inert or less active compound binding of the disinfectant by active groups of foreign protein

Distinctive Properties of Antiseptics and Disinfectants They should be: Able to kill or slow growth of microbes Nontoxic to humans and animals Soluble in water Storable Effective quickly and at low concentration

Agents that Damage the Cell Membrane

destroys structural integrity of cell membrane (protein & lipids) interferes with normal membrane function the net effect is the release of small metabolites from the cell and interfere with the active transport and energy metabolism Agents: Surface active agents Phenolic compound Alcohol

I. Surface active agents (Surfactants)

- Substances that alter the energy relationship at interfaces producing a reduction of surface tension

- disrupt the integrity of cell membrane 1. Anionic agents - produce electrically (-) colloidal ions in the solution 2. Cationic agents - produce electrically (+) ions in the solution 3. Nonionic agents - produce electrically neutral colloidal particles in the solution; Not effective 4. Ampholytic or amphoteric agents - capable of acting either as anionic or cationic detergents

Anionic Agents
includes soap and the largest portion of modern synthetic detergents

agents are most active in an acid pH Causes gross disruption of the lipoprotein framework of the cell wall help remove microbes by rubbing anionic agent + acid a very effective acid-anion surfactants: sanitizers , commercial detergents display very rapid bactericidal action Effective against gram (+) organisms relatively ineffective among gram (-) species because of their lipopolysaccharide outer membrane

Cationic Agents a) Quarternary ammonium compound (QUATS) most important antibacterial surface-active agent Not effective in the presence of organic compounds Suitable for cleaning water systems and smooth surfaces used as disinfectants, surfactants, and fabric softeners Bactericidal against gram positive organisms Have littler effect against fungi and bacterial spores Ex. Benzalkonium chloride (Zephiran) application many include: - disinfecting instruments - used primarily in hand or face washes - preserving drugs in low concentration form

b) Biguanides two cationic groups separated by a hydrophobic bridging structure Wide range of antimicrobial activity: - bactericidal against gram (+) & gram (-) bacteria, yeasts, protozoans and enveloped viruses - not effective against spores, protozoan cysts MOA: disrupt cytoplasmic membrane to cause cell leakage and may enter cells to cause coagulation of the cell cytoplasm Ex. Chlorhexidine - skin cleansers for surgical scrub, preoperative skin preparation, cleansing skin wounds - oral hygiene products as an anti-plaque agent

II. Phenolic compound

at low concentration, these compounds are rapidly bactericidal causing leakage of cell contents and irreversible inactivation of membrane-bound oxidases and dehydrogenases Parent compound : Carbolic acid ( phenol ) was first used by Lister as a disinfectant excellent for disinfecting feces, blood, pus, sputum & other proteinaceous material Rarely used today because it is a skin irritant, expensive and has strong odor primarily use for testing new bactericidal agent replaced by Phenol derivatives

Phenol derivatives active in the presence of organic matter its activity persists for a long period of time ideal for surfaces and concrete floors have no detergent activity greater germicidal activity lower toxicity than phenol very effective against bacteria but not so good against viruses or spore producing bacteria They are usually quite cheap Some phenols contain chlorine which adds properties of quick action. Ex. Cresols (Lysol, Creolin) Xylenol
Biphenol Triclosan

Cresols - Derived from coal tar - are used to dissolve other chemicals, as disinfectants and deodorizers, and to make specific chemicals that kill insect pests - sold under the trade names: * Creolin - septic tank disinfectant * Lysol
LYSOL Hospital disinfectant-deodorant is highly effective against TB, MRSA, and HIV Tuberculocidal, virucidal, fungicidal, bactericidal Minimizes concern over the spread of germs in public facilities

Xylenols - known as Dimethylphenols - Highly toxic, combustible crystals; slightly soluble in water, soluble in most organic solvents - important class of phenolics with great industrial importance - are used as : - pesticides - manufacture of antioxidants - disinfectant - solvent - fungicide

Biphenols (Diphenyl compound) - the halogenated diphenyl compounds exhibit unique antibacterial properties - Effective against gram (+) staphylococci and streptococci - not effective against gram-negative organisms -The most important, is the chlorinated derivative, Hexachlorophene
very useful as a topical anti-infective, anti-bacterial agent often used in soaps bacteriostatic skin cleanser used as a preservative in cosmetic products
pHisoHex, was widely used as a very effective antibacterial skin cleanser in the treatment of acne

Triclosan - polychlorinated phenoxy phenols - potent wide spectrum antibacterial and antifungal agent - found in: - soaps (0.10-1.00%) - deodorants - toothpastes - shaving creams - mouth washes - cleaning supplies

III Alcohol Alcohols provide an insight into the interaction of organic solvents with lipid membranes; dehydrate cells, disrupt membranes and cause coagulation of protein A 70% aqueous solution is more effective at killing microbes than absolute alcohols 70% conc. penetrate the bacterial cell wall and denature the proteins and enzymes inside of the cell 95% conc. coagulates the protein on the outside of the cell wall and prevents any alcohol from entering the cell effective against vegetative bacteria, fungi and some viruses bacterial endospores and many viruses are unaffected

Ethyl alcohol -Used as skin disinfectant because of their bactericidal action and ability to remove lipids from the skin surfaces - unable to kill spores; not relied for sterilization of instruments - Uses: - to sterilize skin before cutaneous injections - to disinfect thermometers Isopropyl alcohol - most effective type of alcohol - bactericidal activity is slightly greater than ethanol - less volatile for this reason, it had been recommended as replacement for the sterilization of thermometers - Toxic effect is greater and long lasting - Necrosis may result from absorption of vapors through the lungs during alcohol sponge bath

Agents that denature Proteins

In its native state, each protein possesses a characteristic conformation that is required for its proper function Agents that alter the conformation of the proteins by denaturation cause an unfolding of the polypeptide chain so that the chain becomes randomly and irregularly looped or coiled

Agents that causes denaturation of proteins: 1. Acid & alkalies - exert their antibacterial activity through their free H+ and OH- ions, through undissociated molecules or by altering the pH of organism s environment - Most acids and alkalis are either too caustic to tissues or are relatively inefficient bactericides Benzoic acid Lactic acid used in the food and cosmetic industry to prevent growth of microorganisms Acetic acid Propionic acid 2. Alcohol 3. Acetone

Agents that Modify the Functional Group of Proteins and Nucleic Acid
The catalytic site of an enzyme contains specific functional groups that bind the substrate and initiate the catalytic events Inhibition of enzyme activity results in one or more of these functional groups to be altered or destroyed Important functional groups of the cell wall, membrane and nucleic acids are also susceptible to inactivation Agents: Heavy metals Oxidizing agents Dyes Aldehydes

I. Heavy Metals Include copper, selenium, mercury, silver, and zinc salts of most metals are generally too toxic to use on living tissues Oligodynamic action: Very tiny amounts are effective Some complex organic salts in alcohol solution are highly bacteriostatic and make useful wound disinfectants soluble salts of heavy metals poison enzyme activity by forming mercaptides with the sulfhydryl groups of cysteine residue They disrupt cellular metabolism thus killing the microbes

 Silver Silver nitrate

highly bactericidal for gonococci and routinely used as prophylaxis against opthalmia neonatorum topical cream for burn patients

Silver sulfadiazine

Mercury Mercuric chloride very toxic disinfectant, not used today Organic mercury compounds like Metaphen, Merthiolate and Mercurochrome less toxic but unreliable as skin
disinfectant

Copper Copper sulfate

is used to kill algae in pools and fish tanks

Selenium Kills fungi and their spores Used for fungal infections like Tinea vesicolor infection, sebborheic dermatitis and dandruff Selinium sulfide - used in dandruff shampoos (Selsun blue) Zinc Zinc chloride is used in mouthwashes Zinc oxide is used as antifungal agent in paints widely used to treat a variety of other skin conditions, in products such as baby powder and barrier creams to treat diaper rashes (Desitin) calamine cream, anti-dandruff shampoos, and antiseptic ointments

II. Oxidizing agents Halogens Chlorine- and iodine-based compounds are the most significant microbicidal halogens used in the clinic/hospitals and have been traditionally used for both antiseptic and disinfectant purposes Hydrogen peroxide These are the new broad spectrum disinfectants that are highly active against most microorganisms

Iodine Based Compounds iodine is rapidly bactericidal, fungicidal, tuberculcidal, virucidal, and sporicidal Tinctures a solution of alcohol or of alcohol and water, containing animal, vegetable, or chemical drugs Iodophors are complexes of iodine and a solubilizing agent or carrier, which acts as a reservoir of the active free iodine - very effective against viruses and bacteria - less active against certain fungi and spores than are tinctures - the most widely used: Povidone-iodine (Betadine)

 Povidone-iodine (Betadine) - broad spectrum topical iodophor microbicide - available in "Swab Aid" pads, Swab Sticks and as a Surgical Scrub - Indications: - antiseptic treatment of the skin and mucous memb. - surgical site preparation - abrasions and lacerations

Chlorine based compounds Chlorine is widely used as water disinfectant 3 types of chlorine compounds:
Hypochlorites organic chloramines inorganic chloramines

Disinfectant action is due to the liberation of free chlorine hypochlorites and elemental chlorine + H2O = hypochlorous acid a strong oxidizing agent, and an effective disinfectant very active against viruses and bacteria

 Hypochlorite solution widely used for sanitizing dairy products and food processing equipment employed as sanitizers in most households, hospitals, and public buildings Used as a bleaching agent marketed as: Chlorox, Zonrox, Purex

Hydrogen peroxide broad-spectrum efficacy against viruses, bacteria, yeasts, and bacterial & fungal spores clear, colorless liquid that is commercially available in a variety of concentrations ranging from 3 to 90%
 3% solution, it is harmless but very weak antiseptic whose primary clinical use is in the cleansing of wound  10 to 30% solution and longer contact time are required for sporicidal activity

antibacterial action: oxidizing ability by producing hydroxyl free radicals which attack essential cell components Transient action; poor penetration; acts only on the surface

III. Dyes some of the coal-tar dyes not only stain bacteria but are inhibitory at very high dilutions within the usual pH range, the basic dyes are the most effective their current medical use is limited primarily to the treatment of dermatologic lesions

 Triphenyl methane dyes

Crystal violet, Malachite green, Brilliant green Highly selective for gram (+) organisms Used in the lab in the formulation of selective culture media

Acridine dyes Often referred as Flavines because of their yellow color

bactericidal and bacteriastatic effect upon a number of org. Compounds of clinical use : proflavine, acriflavine used in wound antisepsis

IV. Aldehydes 3 agents: formaldehyde glutaraldehyde ethelene oxide The lethal effect results from their alkylating action on proteins resulting in irreversible, enzyme modification and inhibition of enzyme activity making them nonfunctional Antimicrobial activity occurs as a result of cross-linking of: proteins and nucleic acids in fungi, protozoa and bacteria capsid and nucleic acid complexes in viruses They are active against bacteria and their spores, viruses, fungi and protozoa

Formaldehyde Excellent disinfectant ; least agents acting on proteins commercially available in: Containing 37% formaldehyde (Formalin) - used for preserving fresh tissues - major component of embalming fluids - Irritates mucous membranes, strong odor - when used in high concentration, it destroys all organisms, including spore Containing 91-99% Paraformaldehyde ( a polymer) Containing 0.2-0.4% - Used to inactivate virus / bacteria in the preparation of vaccines As GAS - used to decontaminate rooms, buildings, fabrics, and instruments

Glutaraldehyde - used as cold sterilant for surgical instruments - 10x more effective than formaldehyde as a bactericidal and sporocidal agent and less toxic - Less irritating - One of the few chemical disinfectants that is a sterilizing agent 2% solution of glutaraldehyde (Cidex) is: - Bactericidal, tuberculocidal, and viricidal in 10min. - Sporicidal in 3 to 10 hours - Commonly used to disinfect hospital instruments esp. respiratory therapy equipment

Ethylene oxide - Employed in gaseous sterilization - Denature proteins, by replacing functional groups with alkyl group - Chemicals that sterilize in a chamber - Highly penetrating - Active against all types of bacteria, including spores & TB bacilli -Kills all microbes and endospores, but requires exposure of 4 to 18 hours - used especially on materials that would be damaged by heat (polyethylene tubings, electronic & medical instruments, biologicals and drugs) - used in sterilization of heart-lung machines