Dr. R. Tan

Microbial

Control

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 FormsHighest resistance

Moderate resistance

Least resistance

bacterial endospore (Bacillus & Clostridium)

protozoan cystsome fungal sporessome naked virusvegetative bacteria that have higher resistance ( M. tuberculosis, S.aureus, Pseudomonas)

most bacterial vegetative cellsordinary fungal spores & hypaeenveloped virusYeastsTrophozoites

Sterilization - process of destruction or removal of all viable microorganisms 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

Disinfection - refers to the use of physical process or chemical agent

(disinfectants) that promotes killing, inhibition, or removal of pathogenic microorganisms (usually on inanimate objects) but not bacterial endospore

Sanitization - is any cleansing technique that mechanically removes microorg.

to reduce the level of contaminants / microbial population to a safe level as determined by public health standards

Definition of Frequently Used Terms

Sepsis – growth of microorganisms or the presence of microbial toxins in the blood and other tissues

Asepsis - refers to any practice that prevents the entry of infectious agents into sterile tissues and thus prevents infection

Antisepsis – aseptic technique practiced in health care, range from sterile methods that uses chemical agents (antiseptics) which are applied directly to exposed body surfaces, wounds, and surgical incision to destroy or inhibit vegetative pathogens

Disinfectant - is an agent used to reduce the viability of a microbial population below a threshold level that causes infection

- Disinfectants are used on inanimate objects

Antiseptic - chemical agents that opposes sepsis or putrefaction either by killing microorganisms or by preventing

their growth - Term commonly used for agents that are applied

topically to living tissues

This distinguishes them from antiseptics that serve the same purpose but are used on living tissues without causing toxicity.

suffix “cide” – 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 viricide – destroys virus fungicide – destroys fungi

bactericide – destroys bacteriagermicide – (or disinfectant) – agent that kills

microorg. capable of producing an infection

Suffix “static/stasis” – meaning to stand still - a suffix indicating that the agent will prevent the growth or multiplication of the type of organism but are not killed outright

bacteriostatic - prevents the growth of bacteriafungistatic – prevents the growth of fungi

Dynamics of Sterilization & Disinfectionmicroorganisms 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)because of the exponential form of survivor-time

curve, the larger the initial number of cells to be killed, the more intense or prolonged is the

treatment required for sterilizationThe rate of disinfection varies with the

concentration of the disinfectant

Factors affecting disinfectant potency1. Number of microorganisms

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

2. Nature of microorganism- the efficacy of a chemical agent depends on these properties: specie , growth phase of culture, and presence of special structure (such as spores and capsules), and the number of the organisms in the test system

3. Temperature - the killing of bacteria by chemical agents increases with an increase in temperature - for each 10°C increase in temperature, there is doubling of the death rate

4. pH - Hydrogen ion concentration influences bactericidal action by affecting both the organism and the chemical agent - Some disinfectants are more effective at alkaline pH (glutaraldehyde) while others are more effective at acid pH (e.g. phenols)

5. Time- when bacteria are exposed to a specific concentration of a bacterial agent, even in excess, not all organisms die at the same time; rather, there is a gradual decrease in the number of living cells

6. Mode of action of the agents damage cell membrane function denatures protein induce extensive nucleic acid damage

7. Concentration of the agent - the concentration required to produce a given effect varies with the disinfectant, the organism and the method of testing - high conc. lethal to bacteria - low conc. may stimulate, retard, or even kill the organism - An effective concentration must be used

8. Presence of exogenous materials - presence of organic matter (serum, blood, pus) alter

disinfectant activity - 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

Evaluation of DisinfectantsAssociation of Official Analytical Chemists (AOAC)

methodsApproved protocol for evaluation purposes

Phenol Coefficient TestPhenol 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

Methods of Microbial Control

Physical Agents Chemical Agents

Chemotherapeutic Agents

• Heat• Freezing• Radiation• Filtration• Ultrasonic and SonicVibration

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 - time required inversely related to temperature of

exposure - sterilization time is directly related to the number of

organisms in a suspension

Thermal Death Point - lowest temperature required to kill all microbes when

time is held constant

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 160°C – several thousand °CMOA :

denaturation of proteinsoxidative damage toxicity effects of elevated levels of electrolytes

Its use is limited primarily to sterilization of glasswares and such materials as oils, jellies, and powders that are impervious to steam

DRY HEAT

Forms:1. Direct flaming

- bacteriological loop, needle, glass rods through the flame of a Bunsen burner

2. Incineration / cremation - burn to ashes - useful for decontaminating infected lab. Animals

3. Hot air oven - most widely used type of dry heat- oven heat set 1800C for 2-4 hours - temp. enough to kill all type of organism,

including the sporeformer

MOIST HEAT kills bacteria faster than dry heat preferred because of its more rapid killing temp ranges from 60-135°C MOA:

denaturation and coagulation of proteins production of single-stranded breaks of DNA loss of functional integrity of membrane and

leakage of small moleculesmicroorganism Time required for

sterilizationMost mesophilic nonsporeformers 60°C for 30minS. Aureus & S. faecalis 60°C for 60minVegetative form of all bacteria, fungi & yeast

80°C for 5-10min

Heat resistant sporeformers 120°C for 4min100°C for 5.5min is required

Forms:1. Boiling

- can be relied only for disinfection and not for sterilization - 30 minutes boiling 1000C adequate to kill vegetative cell Bacteria, Fungi, Protozoa - sporeformer 5½ hours

2. Free – Flowing steam - live steam Arnold sterilizer - same sterilizing action with boiling

3. Steam Under Pressure - steam sterilization is carried out in an autoclave using pressured steam - the principal requirement : the whole of the material

to be sterilized remain in contact with saturated steam at the required temperature for the necessary period of time

- 1210C 15 – 20 minutes (15lbs steam pressure per sq. inch)

- most dependable moist heat method of sterilization for hospital clinical lab. & research

facilities - very effective against vegetative cells and most

bacterial endospores - commonly used for sterilizing :

1. Hospital instruments ( surgical & medical )

2. Bacteriological media not destroyed by heat

3. Hospital supplies

4. Tyndallization - Fractional /Intermittent sterilization - free flowing steam 80-1000C 30 minutes for 3 consecutive days involves exposing the material to elevated temp

(killing the vegetative cell)

incubate at 37°C (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

- it is used to sterilize heat-sensitive culture media containing such materials ac carbohydrates, egg or serum

- kills both sporeformer & non-sporeformer bacteria

5. Pasteurization - applied to fresh beverages such as milk, milk products, beer and wine - heat is applied to these liquids to kill potential agents of infection and spoilage at the same time retaining the liquid`s flavor and food value - 63-66°C for 30 minutes followed by rapid cooling - the temperature does not sterilize the milk, but it does kill all disease producing bacteria commonly transmitted by milk - primary target: non spore forming pathogens (Salmonella sp., Campylobacter jejuni, Listeria monocytogenes, Brucella sp., Coxiella sp. and Mycobacterium sp.)

FREEZINGIs not a reliable method of sterilizationPrimarily used in the preservation of bacterial culturesIn freezing, 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

The cell membrane is damaged, and a leakage of intracellular organic compounds ensues

Lyophilization ( Freeze-drying ) A process used for preserving biological material, by

removing the water from the sample, which involves first freezing the sample and then drying it, under a vacuum, at very low temperatures

RADIATION- defined as energy emitted from atomic activities and dispensed at high velocity through matter or space-Sunlight possesses appreciable bactericidal activity and plays an important role in the spontaneous sterilization that occurs under natural conditions-2 typesIonizing radiation: Radiation that have sufficient energy to remove an electron completely from an atom and produce an electrical charge (ionization)Nonionizing radiation: Energy absorbed by the molecule cannot remove an electron completely, the excitation produced often leads to photochemical changes

Ionizing radiation - Ex: electromagnetic rays: 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 cutgut , nylon sutures, plastic,

syringe, catheter, prosthesis & plastic tubings

Non-ionizing radiation- 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 absorption maximum of DNA- Lethal effect on bacteria is attributed to absorption and resultant damage of DNA formation of abnormal bonds

(damage) killing of the organism - 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

FILTRATION-an effective method to remove microbes from air and liquids- process of separating microorganism from contaminated solution - filtration sterilization is used to prepare liquids that cannot withstand heat (heat-labile), including serum and other blood products, vaccines, drugs, IV fluids, enzymes and culture media- useful for trapping microorganism only - types filters

1. Seitz – asbestose – cellulose 2. Sintered glass – glass filaments 3. Chamberland – unglazed porcelain 4. Berkefeld – diatomaceous earth 5. Membrane filter – cellulose ester

-High efficiency particulate air (HEPA) filters are used to provide a flow of sterile air to hospital /sterile rooms

ULTRASONIC AND SONIC VIBRATIONS- Sound vibration at high frequency, in an upper audible and

ultrasonic range (20-1000 kc), 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

destroys structural integrity of cell membrane (protein & lipids)

interferes w/ 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

• Cationic – most effective• Anionic• Non-ionic – not effective• Amphoteric

Phenolic compound Alcohol

I. Surface active agents- Substances that alter the energy relationship at

interfaces producing a reduction of surface or interfacial tension

- Compounds that possess both water-attracting (hydrophilic) and water-repelling (hydrophobic) groups

- They disrupt the integrity of cell membrane This results in the loss of  molecules from the

cytoplasm and affects the proton motive force which provides energy for solute transport

- The interface between the lipid-containing membrane of a bacterial cell and the surrounding aqueous medium provides a susceptible target site for agents of this type

Cationic Agents Quarternary ammonium compound- QAC are any of a group of ammonium salts in which organic

radicals have been substituted for all four hydrogens of the original ammonium cation

- This is the most important antibacterial surface-active agent- Bactericidal for a wide range of organisms, gram (+) species are

more susceptible- Ex. Benzalkonium chloride ( Zephiran )

used primarily in hand or face washes must not be applied to areas which have not been fully

rinsed as it is inactivated by organic compounds Benzalkonium application many include disinfecting

instruments and preserving drugs in low concentration form

Anionic Agents - Among the anionic detergents are soaps and fatty acids that

dissociate to yield a negatively charged ion- agents are most active in an acid pH- Causes gross disruption of the lipoprotein framework of the

cell wall- By combining an anionic agent with acid, a very effective acid-

anion surfactant sanitizer - display very rapid bactericidal action (within 30sec)

- Effective against gram (+) organisms but are relatively ineffective among gram (-) species because of their lipopolysaccharide outer membrane

The activity of all four Surface active agents may be enhanced by incorporating other disinfectants such as the diguanide

Diguanides - have antimicrobial activity against vegetative bacteria, yeasts,

protozoans and enveloped viruses but not against spores, protozoan cysts and mycobacteria

- They function by disrupting cytoplasmic membrane to cause cell leakage and may enter cells to cause coagulation of the cell cytoplasm

- It may be used together with surface active disinfectants at a concentration of 1 – 4%

- Ex. chlorhexidine which is more effective at pH 7 – 8 used as a safe antiseptic to apply to prevent body infection

and in oral rinses for treating sore gums and mouth ulcers and preventing plaque on teeth

Chlorhexidine digluconate (GynePro feminine wash)

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 )

- excellent for disinfecting feces, blood, pus, sputum & other proteinaceous material

- primarily use for testing new bactericidal agent - It had been replaced as a practical disinfectant by less caustic and less toxic phenol derivative

- Cresols- Xylenols

- Diphenyl compound - Phenolics are effective against bacteria, fungi and virus

Phenol derivatives:• Cresols - the simplest of the alkyl phenols- Ortho-, meta-, paracresols – are applicably more active than phenol- usually employed as a mixtureCresols obtained industrially by the distillation of coal tar -- usual source of these products - Cresols are used to dissolve other chemicals, as disinfectants and deodorizers, and to make specific chemicals that kill Insect pests- sold under the trade names: Lysol and Creolin • Xylenols- Dimethylphenols- important class of phenolics with great industrial importance - are used as pesticides and in the manufacture of antioxidants

Kills 99.9% of germs in 30 seconds

• Hospital disinfectant-deodorant is highly effective against TB, MRSA, and HIV-1• Tuberculocidal, virucidal, fungicidal, bactericidal• Minimizes concern over the spread of germs in public facilities• Prevents odors and growth of mold and mildew

• A general purpose Disinfectant• Kills most bacteria on surface• Destroys odors, cleans and disinfects against germs and

against staph• Good septic tank disinfectant

• Diphenyl compound - the halogenated diphenyl compounds exhibit unique antibacterial properties- effective against gram + bacteria ( staph & strep ); offers no protection against gram-negative infections-The most impt, is the chlorinated derivative, Hexachlorophene

• very useful as a topical anti-infective, anti-bacterial agent, often used in soaps, toothpaste and antiperspirant

• also used in agriculture as a soil fungicide, plant bactericide • 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

III Alcohol -Alcohols provide an insight into the interaction of organic solvents with lipid membranes- they disorganize lipid structure by penetrating into the hydrocarbon region-Alcohols are normally used at concentrations between 50%-90% and have optimal activity at 70% - 75% as the presence of water helps to denature membranes, dissolve cell membrane lipids causing cell lysis - In addition to their effect on the cell membrane, alcohols also denature proteins- effective against vegetative bacteria, fungi and viruses - have little sporicidal activity - usually used to disinfect work surfaces and, as antiseptics on skin- Antiseptic activity may be enhanced by incorporating other antimicrobial compounds: chlorhexidine (0.5%)

iodine (1% to 2%)

Ethanol / Ethyl alcohol- Used as skin disinfectant because of their bactericidal action and ability to remove lipids from the skin surfaces- their action as disinfectant is restricted by their inability at normal temperatures to kill spores not relied for sterilization of instruments- Uses: - to sterilize skin before cutaneous injections - to disinfect thermometers- Most effective at 50-70%- effective against gram (+) , gram (-) , AF bacteria

Isopropyl alcohol - Most effective at 50-70%- 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

- 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: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 environmentBenzoic acidLactic acid used as food preservative Acetic acid Propionic acid

2. Alcohol 3. Acetone

- 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 is altered or destroyed- Important functional groups of the cell wall, membrane and nucleic acids are also susceptible to inactivation-Agents:

1. Heavy metals2. Oxidizing agents3. Dyes4. Alkylating agents

Heavy Metals – soluble salts of mercury, silver , arsenic and other heavy metals poison enzyme activity by forming mercaptides with the sulfhydryl groups of cysteine residue• Mercurials

• Mercuric chloride – very toxic disinfectant, not used today• Metaphen, Merthiolate, Mercuchrome – less toxic, unreliable as skin disinfectant

• Silver compounds • widely used as antiseptic ; either as soluble silver salts or as colloidal preparations• inorganic silver salts - efficient bactericidal agents but has irritant and caustic effects• Silver nitrate – highly bactericdal for gonococci and routinely used as prophylaxis against

opthalmia neonatorum in neonates• Silver sulfadiazine – topical cream for for burn patients

Oxidizing agents • most useful antimicrobial agents: halogens (iodine, chlorine)

hydrogen peroxide

• Halogens - chlorine and iodine are among the most useful disinfectants - Almost exclusively bactericidal and effective against sporulating bacteria, virus, fungi and protozoa (1) iodine

- exist principally in the form of I2 at pH value below 6 where maximal bactericidal action is manifested

- iodine tinctures came into wide use as microbicides - Iodine quickly kills a broad range of microorganisms- destroys many microorganisms and viruses within 3-5 minutes- used in cleansing skin or disinfecting small wounds but

presented with some problems - strong smell and can stain skin and clothing. - react to metals and are rather unstable

- skin irritant

Betadine Solution- various mixtures and solutions of

iodine, called Iodaphores, have been formulated that greatly lessen such disadvantages and dangers

- enhance stability and microbe killing properties.

-Contains 10% povidone-iodine- broad spectrum topical iodophor microbicide- available in "Swab Aid" pads, Swab Sticks and as a Surgical Scrub - It is a fast-acting, broad-spectrum antiseptic that kills bacteria (including antibiotic resistant organisms), as well as fungi/yeasts, viruses and protozoa- It is indicated for degerming skin, wounds and mucous membranes- widely use in hospitals, sanitation and water purification, veterinary industries, food service and also the brewing industries

-

(2) chlorine- 3 types of Chlorine compound: hypochlorite,

inorganic & inorganic chloramines

- The active moiety of hypochlorites and chlorine is hypochlorous acid which is stable at a low pH, which is a

strong oxidizing agent and an effective disinfectant

- Water disinfectant - hypochlorites - most useful of the chlorine compound

- widely used for sanitizing dairy products and food processing equipment

- employed as sanitizers in most households, hospitals, and public buildngs

- marketed as: Chlorox, Zonrox, Purex

• Hydrogen peroxide - antibacterial action is secondary to its oxidizing

ability as well as formation of a more toxic free hydroxyl radical from the peroxide in an iron-dependent reaction

- It is a weak acid- In a 3% solution, it is harmless but very weak antiseptic whose primary clinical use is in the cleansing of wounds- It has strong oxidizing properties and is therefore a powerful bleaching agent that is mostly used for bleaching paper

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

• aniline dye derivatives especially Crystal violet, Malachite green, Brilliant green • Highly selective for gram (+) organisms• Used in the laboratory in the formulation of selective culture media

• Acidine dyes • Often referred as Flavines because of their yellow color • exert a bactericidal and bacteriastatic effect upon a number of organisms• Compounds of clinical use : proflavine, acriflavine• used in wound antisepsis

Alkalyting agent - The lethal effect results from their alkylating action on proteins- Glutaraldehydes and formaldehydes are the most commonly used aldehydes-They are active against bacteria and their spores, viruses, fungi and protozoa-Antimicrobial activity occurs as a result of cross-linking proteins and nucleic acids in fungi, protozoa and bacteria and capsid nucleic acid complexes in viruses -These effects are mediated predominantly via amine, sulfurhydryl

and carboxyl groups on microbial surface proteins

Formaldehyde - One of the least agents acting on proteins- commercially available in:

• aqueous solution containing 37% formaldehyde (Formalin) • paraformaldehyde a poymer (contain 91-99% formaldehde)

- Formalin - used for preserving fresh tissues- major component of embalming fluids- when used in high concentration, it destroys all organisms, including spore

- Used to inactivate virus in the preparation of vaccines- As a 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

Ethylene oxide - Employed in gaseous sterilization, especially on materials that would be damaged by heat (polyethylene tubings, electric & medical instruments,b iologicals and drugs)- Active against all types of bacteria, including spores & TB bacilli