Physical Methods of Microbial Control

8/3/2019 Physical Methods of Microbial Control

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MICROBIAL CONTROL

Ms. Nerralyn N. Muni R.M.T.


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Biosafety Levels

LevelLevel RiskRisk Types of Types of

AgentsAgents

Example ofExample of

AgentsAgents

Precautions requiredPrecautions required

II MinimalMinimal

biohazardbiohazard

Those notThose not

known toknown to

cause diseasecause disease

in healthyin healthyadultsadults

Bacillus subtilis,Bacillus subtilis,

MycobacteriumMycobacterium

gordonaegordonae, soil, soil

microbesmicrobes

No special equipmentNo special equipment

required. Work can berequired. Work can be

done on opendone on open

benchtops.benchtops.

IIII ModerateModerate

biohazardbiohazard

CommonCommon

pathogenspathogens

found infound inhumanhuman

blood, bodyblood, body

fluids, andfluids, and

tissuestissues

Escherichia coli,Escherichia coli,

SalmonellaSalmonella, HIV,, HIV,

hepatitis B virus,hepatitis B virus,influenzainfluenza

Procedures with theProcedures with the

potential for aerosolspotential for aerosols

or splashes must beor splashes must beconducted in aconducted in a

biologic safety cabinet.biologic safety cabinet.

Access toAccess to

laboratory must belaboratory must be


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Biosafety Levels Continued


AgentsAgents


AgentsAgents


Limited when work isLimited when work is

being conducted. Anbeing conducted. An

autoclave must beautoclave must beavailableavailable

IIIIII HighHigh

biohazardbiohazard

Agents thatAgents that

may causemay cause

serious orserious or

lethal diseaselethal diseasefromfrom

exposure viaexposure via

inhalation.inhalation.

EffectiveEffective

Bacillus anthracisBacillus anthracis

Francisella,Francisella,

Brucella,Brucella,

MycobacteriumMycobacteriumtuberculosis,tuberculosis,

Rickettsia rickettsiiRickettsia rickettsii,,

Manipulation ofManipulation of

infectious materialsinfectious materials

must be conducted inmust be conducted in

class II biologic safetyclass II biologic safetycabinet by personnelcabinet by personnel

wearing personalwearing personal

protective clothing.protective clothing.

Access toAccess to


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AgentsAgents


AgentsAgents


TreatmentTreatment

availableavailable

Coxiella burnetii,Coxiella burnetii,

mold stages ofmold stages of

systematic fungisystematic fungi

laboratory must belaboratory must be

controlled.controlled.

Specialized ventilationSpecialized ventilationminimizing release ofminimizing release of

infectious aerosolsinfectious aerosols

requiredrequired

IVIV ExtremeExtreme

biohazardbiohazard

Agents thatAgents that

pose a highpose a highrisk of liferisk of life--

threateningthreatening

disease,disease,

which maywhich may

Ebola virus,Ebola virus,

Lassa virus, and

Lassa virus, andothers that causeothers that cause

hemorrhagichemorrhagic

feversfevers

Requires use of classRequires use of class

III biologic safetyIII biologic safetycabinet or fullcabinet or full--body,body,

airair--supplied positivesupplied positive

pressure suit andpressure suit and

independent unit withindependent unit with


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Level Risk Types of

Agents

Example of

Agents

Precautions

required

be transmitted

by aerosolsand for which

there is no

vaccine or

therapy

specialized

ventilation andwaste management

systems


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Biologic Safety Cabinets

ClassClass Description Description Use Use

II Unsterilized room air passes into theUnsterilized room air passes into the

cabinet. Air passes through a highcabinet. Air passes through a high--

efficiency particulate air (HEPA) filterefficiency particulate air (HEPA) filter

before being exhaustedbefore being exhausted

Provides minimalProvides minimal

personnel protection.personnel protection.

Doesnt protect workDoesnt protect work

surface.surface.

IIII Laminar flow cabinets with variableLaminar flow cabinets with variable

sash opening. Air passes through onesash opening. Air passes through one

HEPA filter before reaching the workHEPA filter before reaching the work

surface and another before beingsurface and another before beingexhausted.exhausted.

Type most commonlyType most commonly

used in hospitalused in hospital

microbiologymicrobiology

laboratories. Provideslaboratories. Providesprotection for both theprotection for both the

laboratorian and thelaboratorian and the

work. Must maintain anwork. Must maintain an

open area for 3 feetopen area for 3 feet


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Biologic Safety Cabinets Continued

ClassClass Description Description Use Use

From cabinet duringFrom cabinet during

operation. Must beoperation. Must be

certified initially,certified initially,whenever moved morewhenever moved more

than 18 inches, andthan 18 inches, and

annually thereafter.annually thereafter.

IIIIII Self Self--contained system, completelycontained system, completely

enclosed, with negative pressure. Air isenclosed, with negative pressure. Air isfilter sterilized coming in and goingfilter sterilized coming in and going

out. Gloves are attached to the frontout. Gloves are attached to the front

for manipulations inside the hood.for manipulations inside the hood.

Provides maximumProvides maximum

protection. Usedprotection. Usedextensively in researchextensively in research

facilities.facilities.


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Physical Methods of Microbial

Control


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Dry Heat Sterilization

kills spores, vegetative forms

ideal for sterilizing glasswares, metalwares,

and anh

ydrous oils Oxidizing to ashes

Burns cell constituents

Denaturing proteins


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Direct flaming

Incineration

Oven

160-170 deg. Celsius- 2-3 hours

200 deg. Celsius 1 hours


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Moist Heat Sterilization

more effective than dry heat method

coagulation of the cell protein


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Autoclaving

most effective method of moist heat sterilization

for vegetative cells, spores

121 deg. Celsius- 30 mins. 15 psi


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Boiling

for vegetative cells and viruses

100 deg. Celsius-10 mins


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Pasteurization

Destroy spoilage organism in wine, vinegar,

milk, juices

62 deg. Celsius- 30 mins 72 deg. Celsius- 15 secs (HTST)

82 deg. Celsius- 20 secs-ice cream

104-150 deg. Celsius- 2-5 secs (UHT)


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Fractional Sterilization Method

for vegetative forms of microorganisms

Tyndallization

Inspissation


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DayDayTemperTemper

atureatureTime Time PurposePurpose

11 8585CC 60 minutes60 minutesDrying of the medium and killing theDrying of the medium and killing the

organisms in their vegetative formorganisms in their vegetative form

Time inTime in

betweenbetween

overnightovernight

incubationincubationGrowth of vegetative forms from sporesGrowth of vegetative forms from spores

2275 to75 to

8080CC20 minutes20 minutes killing the organisms in their vegetative formkilling the organisms in their vegetative form

Time inTime in

betweenbetween

overnightovernight

incubationincubation

Growth of vegetative forms from any sporesGrowth of vegetative forms from any spores

remainingremaining

3375 to75 to

8585CC20 minutes20 minutes killing the organisms in their vegetative formkilling the organisms in their vegetative form


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Filtration

nitrocellulose or cellulose acetate


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Cold

The effectiveness of low temperatures

depends on the particular microorganisms and

the intensity of the application


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Dessication

Removal of H2O


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Osmotic pressure

high concentrations of salts and sugars

undergo plasmolysis


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Radiation

Ultraviolet radiation

reduction of air borne contamination

has poor penetration capability

Ionization Radiation

high energy emitted from radioactive isotopes

such as cobalt 60 (gamma rays) or by cathode or

beta rays


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Chemical Methods of Microbial

Control


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Principles of Effective Disinfection

Careful attention should be paid to the

properties and concentration of the

disinfectant to be used

The presence of organic matter, degree of

contact with microorganisms, and

temperature should also be considered


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Phenol and Phenolics

injuring plasma membranes

inactivating enzymes

denaturing proteins

Triclosan- phenolic compound toothpaste,

lotions, deodorant soaps


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Biguanides (Chlorhexidine)

damages plasma membranes of vegetative

cells

Soaps, lotions, cath

eters, surgical mesh


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Halogens

Iodine combines with the amino acid tyrosine

Tincture

Iodophore

Chlorine is added to water forming

hypochlorous acid

Water treatment


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Alcohols

denaturing proteins

dissolving lipids

Antiseptic, disinfectant


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Heavy Metals and Their Compounds

Silver, mercury, copper, and zinc

Opthalmia neonatorum


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Quaternary Ammonium Compounds

(Benzalkonium chloride, Cetylpyridinium chloride)

disrupting plasma membranes most effective against gram-positive bacteria

Disinfectant, non-food substances


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Organic Acids and Derivatives

Sorbic acid, benzoic acid, and propionic acid

inhibit fungal metabolism


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Aldehydes

inactivating proteins

Gluteraldehyde medical instruments

Formalydehyde vaccine preparation


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Gaseous Chemosterilizers

protein denaturation

Ethylene oxide gas

Easily penetratesh

ard-to-reach

places and fabricsand does not damage moisture-sensitive material

Toxic, explosive and potentially carcinogenic

For medical devices


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Oxidizing Agents

Ozone and peroxide are used as antimicrobial

agents.

oxidizing molecules inside cells


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Antimicrobial Action


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Principles

1. The agent must be in an active form.

This is ensured through the *pharmacodynamic

design of the drug, which takes into account the

route through which the patient will receive the

agent (e.g., orally, intramuscularly, intravenously).

* The study of the action or effects of drugs on living

organisms.


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2. The antibiotic must also be able to achievesufficient levels or concentrations at the siteof infection so that it has a chance to exert an

antibacterial effect (i.e., be in anatomicapproximation with the infecting bacteria).

The ability to achieve adequate levels depends onthe *pharmacokinetic properties of the agent.

* The process by which a drug is absorbed,distributed, metabolized, and eliminated by thebody.


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3. The remaining steps in antimicrobial action

relate to direct interactions between the

antibacterial agent and the bacterial cell


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Mode of Action of Antibacterial

Agents


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Inhibitors of Cell Wall Synthesis


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Beta-Lactam Antimicrobial Agents

Beta-lactam antibiotics are those that contain

the four-membered, nitro-gen-containing,

beta-lactam ring at the core of their structure.

The beta-lactam ring is key to the mode of

action of these drugs that target and inhibit

cell wall synthesis by binding the enzymes

involved in synthesis.


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AntibioticsAntibioticAntibiotic Mode of Mode of

ActionAction

ActivityActivity Comments Comments

BetaBeta--LactamsLactams

PenicillinsPenicillins

Penicillin,Penicillin, ampicillinampicillin,,mezlocillinmezlocillin,,

piperacillinpiperacillin,, AugmentinAugmentin

CephalosporinsCephalosporins

CefazolinCefazolin,, cofotetancofotetan,,

ceftriaxoneceftriaxone,, cefotaximecefotaxime,,

ceftazidimeceftazidime

AztreonamAztreonam

ImipenemImipenem

InhibitionInhibition

of cell wallof cell wall

synthesissynthesis

EffectiveEffective

against gramagainst gram--

positives andpositives and

gramgram--

negativesnegatives

UsuallyUsually

bactericidal.bactericidal.

Low toxicity.Low toxicity.


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Glycopeptides

Glycopeptides bind to precursors of cell wall

synthesis, the binding interferes with the

ability of the PBP enzymes, such as

transpeptidases and transglycosylases, to

incorporate the precursors into the growing

cell wall


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Antibiotics Continued

AntibioticAntibiotic Mode of Action Mode of Action Activity Activity Comments Comments

GlycopeptidesGlycopeptides

VancomycinVancomycin

Inhibition of cellInhibition of cell

wall synthesiswall synthesis

EffectiveEffective

against gramagainst gram--

positivespositives

resistant toresistant to

other betaother beta--

lactams.lactams.

UsuallyUsually

ineffectiveineffective

against gramagainst gram--negatives.negatives.

Used to treatUsed to treat

methicillinmethicillin

resistantresistant

staphstaph


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Inhibitors of Cell Membrane

Function


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Inhibitors ofProtein Synthesis


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Aminoglycosides

Aminoglycosides binds to protein receptors on

the organisms 30S ribosomal subunit

gentamicin.

tobramycin, amikacin, netilmicin,

streptomycin, and kanamycin


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Macrolide-Lincosamide-Streptogramin

(MLS) Group

macrolides, such as erythromycin,

azithromycin, and clar-ithromycin, and

clindamycin (a lincosamide).

binds to receptors on the bacterial 5OS

ribosomal subunit


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Oxazolidones

currently represented by linezolid

Linezolid is a synthetic agent that inhibits

protein synthesis through a unique

mecha-nism.


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Chloramphenicol

binds to the 5OS ribosomal subunit


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Tetracyclines

binds to the 30S ribosomal subunit


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Inhibitors ofDNA and RNA

Synthesis


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Fluoroquinolones

simply referred to as quinolones, are

derivatives of nalidixic acid, an older

antibacterial agent.

bind to and interfere withDNA gyrase

enzymes


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Metronidazole

direct interactions between the activated drug

and DNA that results in breakage ofDNA

strands


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Rifampin

binds to the enzyme DNA-dependent RNA

polymerase and inhibits synthesis of the RNA


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Inhibitors of Other Metabolic

Processes


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Sulfonamides

bind to dihydropteroate synthase and disrupt

the folic acid pathway


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Trimethoprim

dihydrofolate reductase is inhibited


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Nitrofurantoin

have several targets involved in bacterial

protein and enzyme & synthesis and the drug

also may directly damage DNA


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Testing Methods


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Disk Diffusion/Kirby Bauer Technique

Using the disk diffusion susceptibility test,

antimicrobial resistance is detected by

challenging bac-terial isolates with antibiotic

disks that are placed on the surface of an agar

plate that has been seeded with a lawn of

bacteria.


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BrothDilution Tests

Using the broth dilution test, the microorganismis grown in liquid media containing differentconcentration of antibiotics.

The minimum inhibitory concentration (MIC) isthe lowest concentration of chemotherapeuticagent capable of preventing microbial growth.

The minimum bactericidal concentration (MBC) is

the lowest concentration of chemotherapeuticagent that kills bacteria


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Broth Microdilution Methods

Commercial panels are designed to receive

the stan-dard inoculum and are incubated

using conditions and durations recommended

for conventional broth microdilution.

They are growth-based systems that require

overnight incubation, and NCCLS interpretive

criteria apply for interpretation of mostresults.


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Agar Dilution Derivations

Uses an instrument to apply antimicrobial

agent to the surface of an already prepared

agar plate in a concentric spiral fashion.

Start-ing in the center of the plate the

instrument deposits the highest concentration

of antibiotic and from that point drug

application proceeds to the periphery of theplate


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Serum bactericidal test

The serum bactericidal test (SBT) is analogous

to the MIC-MBC test except that the medium

used is patients serum that contains the

thera-peutic antimicrobial agent(s) the patient

has been receiving

Physical Methods of Microbial Control

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