Lecture 6 Microbial Control

8/6/2019 Lecture 6 Microbial Control

1/63

Dr. R. Tan


2/63

Primary target of microbial control

Microorganisms capable of causing infection or spoilagethat are constantly present in the external environmentand on the human body

Relative Resistance of Microbial FormsHighest resistance Moderate resistance Least resistance

bacterial endospore(Bacillus & Clostridium)

protozoan cystsome fungal sporessome naked virus

vegetative bacteria thathave higher resistance

( M. tuberculosis,S.aureus, Pseudomonas)

most bacterial vegetative cellsordinary fungal spores & hypaeenveloped virus

YeastsTrophozoites


3/63

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 particularenvironment

- total inactivation of all forms of microbial life in terms ofthe organisms ability to reproduce

Sanitization- is any cleansing technique that mechanically removes

microorganisms or reduce the level of contaminants ormicrobial population to a safe level as determined by public

health standards

Definition of Frequently Used Terms


4/63

Sepsis - growth of microorganisms or the presence of

microbial toxins in the blood and other tissues

Asepsis - any practice that prevents the entry of infectiousagents into sterile tissues thus preventing infection

Aseptic techniques are used to prevent contamination ofsurgical instruments, medical personnel, and thepatient during surgery and also in the food industry

Antiseptics-

are products/chemical agents used for theasepsis of living tissues applied directly to exposed bodsurfaces (skin, wounds, mucosa) and surgical incision to destroor inhibit vegetative pathogens

- applied topically to living tissues


5/63

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

(disinfectants) that promotes killing, inhibition, orremoval of microorganisms (vegetative or growingform) 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)


6/63

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 notkill endospores.

Viricide: An agent that inactivates viruses.

Fungicide:An agent that kills fungi.

Sporicide: An agent that kills bacterial endospores andof fungal spores.


7/63

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 ofbacteria, but does not necessarily kill them

Fungistatic agent prevents the growth of fungi


8/63

Dynamics of Sterilization & Disinfection

Microbes are not killed instantly when exposed to a lethalagent

The kinetics of death of a microbial population isexponential: the number of survivors decreases with time

(exponential killing)

the larger the initial number of cells to be killed, the moreintense or prolonged is the treatment required forsterilization


9/63

Evaluation of Disinfectants

Phenol Coefficient Test

Phenolp reference standard agent

official method used to test disinfectant potency

designed to determine the ratio of the highest dilution ofthe germicide that will kill the test organism within aspecified time to the greatest dilution of phenol showing

the same result indicates disinfecting ability compared to that of phenol


10/63

METHODS OFMICROBIALCONTROL

Physical Agents

Chemical AgentsChemotherapeutic Agents


11/63


12/63

I. Heat

II. FreezingIII. RadiationIV. FiltrationV. SonicVibration

Physical AgentsPhysical Agents


13/63

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 aspecified temperature in a specified environment- sterilization time is directly related to the number oforganisms 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


14/63

DRY HEAT

Sterilization that requires higher temperature and longerperiod of heating

denotes air with a low moisture content that has beenheated 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 asoils, jellies, and powders that are impervious to steam


15/63

Forms of Dry Heat:

1. Direct flaming- bacteriological loop, needle, glass rods through theflame of a Bunsen burner

Red heat:Inoculating loops & needles, points of forceps and spatulas in Bunsen

burner flame till red hotFlaming: Scalpel blades, needles, mouths of culture tubes, bottles, glass

slides, cover slips, and glass rods by passing through Bunsen

flame without red hot

Outer non luminous flame

oxidizing region (incomplete combustion)

Hottest part of the flame

inner blue cone (reducing region regionof intense combustion)

Unburnt gas and air


16/63

2. Incineration- Soiled dressing and hospital waste materials arephysically 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, sealedmaterials, swab sticks are sterilized

- It is electrically heated and fitted withthermostat that maintains chamber air at chosen

temperature and fan than distributes hot air in chamber


17/63

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 ofsmall molecules


18/63

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 1000

C- primary target: non spore formingpathogens(Salmonella sp., Campylobacter jejuni, Listeriamonocytogenes, Brucella sp., Coxiella sp. andMycobacterium sp.)


19/63

2.Boiling-

can be relied only for disinfection and not for sterilization- 30 minutes boiling 1000C adequate to kill vegetative cellp 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


20/63

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 areeasily 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


21/63

5.Steam Sterilization (Autoclave)

- the whole of the material to be sterilizedremain in contact with saturated steamat the required temperature & time

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

- most efficient & dependable moist heat method -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 isdamaged by heat, moisture, or high pressure


22/63

II. FREEZING

not a reliable method of sterilization

the formation of ice crystals outside the cell causes thewithdrawal of water from the cell interior, resulting in anincreased 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 mostmicrobes so they cannot reproduce or produce toxins


23/63

III. RADIATION

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 producedin the cellular fluid

Sunlight- bactericidal activity- impt. role inspontaneoussterilization undernatural conditions


24/63

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, anddislodge 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


25/63

Non-ionizing radiation

Energy absorbed by the molecule cannot remove an electroncompletely, the excitation produced often leads to

photochemical changes

Ex. Ultraviolet light

-

Effectiveness of UV light as a lethal and mutagenic agent isclosely related to its wavelength (240-280nm); optimum atabout260nm which corresponds with the maximumabsorption 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 usedfor the disinfection of enclosed areas such as nurseries,hospital wards & OR


26/63

IV. FILTRATION

process of separating microorganism from contaminatedsolution

an effective method to remove microbes from air andliquids

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 cellulose2.Sintered glass glass filaments3.Chamberland unglazed porcelain4.Berkefeld diatomaceous earth


27/63


28/63

V. SONICVIBRATIONS

Sound vibration at high frequency, in an upper audible andultrasonic range (20-1000 kHz), provide a useful techniquefor disruption & disintegration of the cell

The passage of sound thru a liquid produces alternating

pressure changes, which, if the sound intensity is sufficientlygreat, causes cavities to form in the liquid

the cavities grow in size until they collapse violently leadingto cell disintegration

No practical value in sterilization & disinfection

uses 1. research laboratories

2. treating sewage H2O


29/63

I.I. AgentsAgents that damage cell membranethat damage cell membrane

II. Agents that denatures protein

III. Agents that modify the functional

group of protein & nucleic acid

Chemical AgentsChemical Agents


30/63

Factors affecting disinfectant potency

1. Number of microorganisms2. 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 foreignprotein


31/63

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


32/63

destroys structural integrity of cellmembrane (protein & lipids)

interferes with normal membrane function

the net effect is the release of smallmetabolites from the cell and interfere withthe active transport and energy metabolism

Agents:

Surface active agents Phenolic compound Alcohol

Agents that Damage theC

ell Membrane


33/63

I. Surface active agents (Surfactants)

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

- disrupt the integrity of cell membrane

1. Anionic agents-

produce electrically (-) colloidal ions inthe solution

2. Cationic agents - produce electrically (+) ions in the solution

3. Nonionic agents - produce electrically neutral colloidalparticles in the solution; Not effective

4. Ampholytic or amphoteric agents - capable of acting eitheras anionic or cationic detergents


34/63

Anionic Agents

includes soap and the largest portion of modern syntheticdetergents

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 oftheir lipopolysaccharide outer membrane


35/63

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 sporesEx.Benzalkonium chloride (Zephiran)

application many include:

- disinfecting instruments- used primarily in hand or face

washes

- preserving drugs in lowconcentration form


36/63

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 leakageand may enter cells to cause coagulation of the cellcytoplasm

Ex. Chlorhexidine- skin cleansers for surgical scrub,

preoperative skin preparation,cleansing skin wounds

- oral hygiene products as an anti-plaque


37/63

II. Phenolic compound

at low concentration, these compounds are rapidlybactericidal causing leakage of cell contents andirreversible inactivation of membrane-bound oxidasesand 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


38/63

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 ofquick action.

Ex. Cresols (Lysol, Creolin) XylenolBiphenol Triclosan


39/63

Cresols

- Derived from coal tar- are used to dissolve other chemicals, as disinfectants anddeodorizers, and to make specific chemicals that kill insectpests- sold under the trade names:

* Creolin - septic tank disinfectant* Lysol

LYSOL

Hospital disinfectant-deodorant is highly effectiveagainst TB, MRSA, and HIV

Tuberculocidal, virucidal, fungicidal, bactericidal

Minimizes concern over the spread of germs in public

facilities


40/63

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


41/63

Biphenols (Diphenyl compound)

- the halogenated diphenyl compounds exhibit uniqueantibacterial 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 avery effective antibacterial skincleanser in the treatment of acne


42/63

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


43/63

III Alcohol

Alcohols provide an insight into the interaction of organicsolvents 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 enzymesinside of the cell

95% conc. coagulates the protein on the outside of thecell wall and prevents any alcohol from entering the cell

effective against vegetative bacteria, fungi and someviruses

bacterial endospores and many viruses are unaffected


44/63

Ethyl alcohol-Used as skin disinfectant because of their

bactericidal action and ability to removelipids 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


45/63

In its native state, each protein possessesa characteristic conformation that is

required for its proper function Agents that alter the conformation of the

proteins by denaturation cause anunfolding of the polypeptide chain sothat the chain becomes randomly andirregularly looped or coiled

Agents that denature

Proteins


46/63

Agents that causes denaturation of proteins:

1.Acid & alkalies- exert their antibacterial activity through their free

H+ and OH- ions, through undissociated moleculesor by altering the pH of organisms environment

-Most acids and alkalis are either too caustic to tissuesor are relatively inefficient bactericides

Benzoic acid

Lactic acid

Acetic acid Propionic acid

2.Alcohol

3.Acetone

used in the food and cosmetic industry to

prevent growth of microorganisms


47/63

The catalytic site of an enzyme contains specificfunctional groups that bind the substrate andinitiate the catalytic events

Inhibition of enzyme activity results in one or

more of these functional groups to be altered ordestroyed

Important functional groups of the cell wall,membrane and nucleic acids are also susceptible toinactivation

Agents:

Heavy metals Oxidizing agents

Dyes Aldehydes

Agents that Modify the FunctionalGroup of Proteins andNucleic Acid


48/63

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 highlybacteriostatic and make useful wound disinfectants

soluble salts of heavy metals poison enzyme activity by

forming mercaptides with the sulfhydryl groups ofcysteine residue

They disrupt cellular metabolism thus killing the microbes


49/63

Silver

Silver nitrate highly bactericidal for gonococci and routinelyused as prophylaxis against opthalmia neonatorum

Silver sulfadiazine topical cream for burn patients

MercuryMercuric chloride very toxic disinfectant, not used today

Organic mercury compounds like Metaphen, Merthiolateand Mercurochrome less toxic but unreliable as skin

disinfectant

Copper

Copper sulfate is used to kill algae in pools and fish tanks


50/63

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 powderand barrier creams to treat diaper rashes (Desitin)calamine cream, anti-dandruff shampoos, andantiseptic ointments


51/63

II. Oxidizing agents

Halogens

Chlorine- and iodine-based compounds are the mostsignificant microbicidal halogens used in theclinic/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


52/63

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 solubilizingagent or carrier, which acts as a reservoir of the activefree iodine

- very effective against viruses and bacteria

- less active against certain fungi and spores than aretinctures

- the most widely used: Povidone-iodine (Betadine)


53/63

Povidone-iodine (Betadine)

- broad spectrum topical iodophormicrobicide

- available in "Swab Aid" pads,Swab Sticks and as a Surgical Scrub

- Indications:

- antiseptic treatment of the skin and mucousmemb.

- surgical site preparation

- abrasions and lacerations


54/63

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 effectivedisinfectant

very active against viruses and bacteria


55/63

Hypochlorite solution

widely used for sanitizing dairy productsand food processing equipment

employed as sanitizers in most households,hospitals, and public buildings

Used as a bleaching agent

marketed as: Chlorox, Zonrox, Purex


56/63

Hydrogen peroxide

broad-spectrum efficacy against viruses, bacteria, yeasts,and bacterial & fungal spores

clear, colorless liquid that is commercially available in avariety of concentrations ranging from 3 to 90%

3% solution, it is harmless but very weak antiseptic whoseprimary clinical use is in the cleansing of wound

10 to 30% solution and longer contact time are requiredfor 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


57/63

III. Dyes

some of the coal-tar dyes not only stain bacteria but areinhibitory at very high dilutions

within the usual pH range, the basic dyes are the mosteffective

their current medical use is limited primarily to thetreatment of dermatologic lesions


58/63

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


59/63

IV. Aldehydes

3 agents: formaldehydeglutaraldehyde

ethelene oxide

The lethal effect results from their alkylating action on

proteins resulting in irreversible, enzyme modification andinhibition 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


60/63

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 allorganisms, 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


61/63

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 instrumentsesp. respiratory therapy equipment


62/63

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 exposureof 4 to 18 hours

- used especially on materials that would be damaged byheat (polyethylene tubings, electronic & medicalinstruments, biologicals and drugs)

- used in sterilization of heart-lung machines


63/63

Lecture 6 Microbial Control

Documents

Transcript of Lecture 6 Microbial Control