BIOL 2460 chapter 13

Sterilization

removal/killing of ALL microbes from a fomite(inanimate object)

-Heat

-Pressure

-Filtration

-Chemical (sterilants)

Disinfection/Antisepsis

inactivation of microbes

Sanitization/Degerming

decreasing microbial load

BSL-1

Very little risk; Microorganisms not known to cause disease in healthy adults.

-sink for handwashing & door to close off lab

- Nonpathogenic E. coli and B. subtilis

BSL-2

Pose moderate risk; restrictive access

•BSL-1 plus PPE, self-closing doors, eyewash station, autoclave or sterilization method

•S. aureus, Salmonella spp.

•Viruses like hepatitis, mumps, and measles

•(Ex. Micro labs)

indigenous microbes

BSL-3

potential to cause lethal infections by inhalation

• BSL-2 plus respirator, biosafety cabinets, hands-free wash sink, two sets of doors, directional airflow

• Indigenous or "exotic" pathogens

• M. tuberculosis, B. anthracis

• West Nile Virus, HIV

BSL-4

Most dangerous; often fatal

• BSL-3 plus full biohazard suit, change clothing on entry, shower on exit, decontaminate all material on exit, lab must have its own air supply

• "Exotic" pathogens; Ebola and Marburg viruses

Critical

must be sterile; items used inside the body (i.e. sterile tissue or bloodstream)

• Ex. Surgical instruments, catheters, IV fluids

Semicritical

do not require high-level sterilization; items might contact non-sterile tissue (e.g. gut) but not penetrate tissue

• Ex. GI endoscope, respiratory therapy equipment

Noncritical

do not require sterilization; contact but do not penetrate intact skin

• Ex. Stethoscopes, bed linens, blood pressure cuffs

Disinfectant

Inactivation/kill of microbes on fomites

Example: Include vinegar and bleach

Antiseptic

acts on microbes but not organism/tissue

• Example: hydrogen peroxide, rubbing alcohol

Sanitization

reduce microbial load on fomite

• Usually with heat or chemicals

Degerming

reduce microbial load on living tissue

• Usually mechanical - washing hands, wiping with paper towel, etc.

-cides

to kill

-static

stop growth

Decimal reduction time (DRT)

how much time it takes to kill 90% (1 log reduction) of population

• 1 x 106 -> 1 x 105

Thermal Death Point

lowest temp that will kill in 10 min

Thermal Death Time

shortest length of time required to kill all test microbes at a specified temperature

Dry Heat

aka incineration; direct application of high heat (>250C)

• Bunsen burner

• Bacteria incinerator

Moist Heat

application of high-temperature liquid/vapor. Beneficial b/c penetrates cells better than dry

• Example: autoclave

Autoclave

raise the temperature of water above boiling point (~121C) by raising pressure to 15 psi

• Kills viruses & endospores

-gravity: uses stream to push out air

-prevacuum: vacuums out air first

Refrigeration & Freezing

Ultra-low temperatures (-80C) can be used for preservation

Slows metabolism but will grow when temps are raised.

Pascalization

high pressure used in food industry to kill microbes

Desiccation

drying or dehydration, doesn't kill all microbes, so they may regrow in favorable conditions

Lyophilization

freeze-drying; rapid freezing then placed under vacuum

Ionizing radiation

enters into cells and disrupts molecular structures such as DNA

• X-rays & gamma rays

• Can be used to sterilize non-autoclavableitems

• Can be an alternative to pasteurization in canned foods

Non-ionizing radiation

does not penetrate glass, plastics, etc. but can damage cells w/ direct exposure

• UV irradiation - forms thymine dimers in DNA causing lethal mutations

Sonication

High frequency sound waves to disrupt cell structure

• Causes bubbles to form inside cells and induce lysis

Membrane filtration

removes microbes from liquid samples

• Useful when media cannot be autoclaved(ex. Urea broth, antibiotic, and vitamin solutions)

• Filters usually have a pore size of 0.2 μm (or smaller for viruses)

Carbolic acid

first used by Joseph Lister forsurgical wounds

Lysol

original formulations (now is quaternary compound)

Triclosan

commonly used in hand soaps;banned by FDA in 2017

Mercury

treated syphilis but banned b/c of neural toxicity effects

Silver

used today to treat burn wounds, pediatric ophthalmianeo natorum, and in antibiotics

Copper sulfate

used as algicide to treat pools

Zinc

mouthwashes, calamine lotion, baby powder

Iodine

oxidizes cellular components;commonly used as a iodophor(complex with organic molecule)

Chlorine

• Hypochlorous acid - Cl + H2O; used to treat water

• Sodium hypochlorite - bleach

• Chloramine - Cl + NH3; very stable"swimming pool smell

Fluorine

Most recognizable with dental products

• Deposits in tooth enamel and provides disruption in microbial fermentation and processes

Alcohols

Used as disinfectants and antiseptics

• MOA: Disrupts membranes and denatures cytoplasmic proteins -> lysis

• Used as 70% to allow better cell penetration

• Only viricidal for enveloped viruses

Soaps

fatty acid salts, Not -cidal or -static but means of mechanical removal

Detergents

synthetic polar & non-polar molecules

• Anionic - neg. anion on chain

• Cationic - pos. anion on chain

Quaternary ammonium salts

-Cationic detergents

-Similar to phospholipids & can insert into lipid bilayer

-Common day Lysol

Bisbiguanides

•Cationic molecules that have antiseptic properties

•disrupt membrane & congeal cytoplasmic contents

•Not active against naked viruses, M. tuberculosis, and spores.

Chlorhexidine

common surgical scrub and longer lasting than iodophors

Alexidine

faster acting surgical scrub"up and coming"

Alkylating Agents

replace hydrogen atom with alkyl group

Formaldehyde

fixes specimens by cross-linkingproteins

Glutaraldehyde

acts faster than formaldehyde; common disinfectant of surgical equipment

Ethylene oxide

gaseous sterilizer that has high penetrating ability

β-propionolactone

clear liquid or vapor with strong odor; wide variety of sterilization; medical, tissue, milk, etc

Peroxygens

Oxidizing agents used as disinfectants or antiseptics; produceradical oxygen to disrupt macromolecules

Hydrogen peroxide

common & cheap disinfectant

Peracetic acid

more effective than H2O2 (hydrogen peroxide); immune to inactivation bycatalases and peroxidases

Benzoyl Peroxide

present in acne medications; very effective against Propionibacterium acnes

Carbamide peroxide

agent in toothpaste that combats biofilms

Ozone gas

used to clean air and water supply

Supercritical Fluids

Pressure and temp are increased in molecules to have properties between liquid and gas

Ex. Supercritical CO

Preservatives

Most inhibit microbial growth in food products

• In foods, important to be non-toxic (and flavorless)

Sorbic acid

inhibits various cellular enzymes (e.g. in CAC, catalases, and oxidases)

• Increases efficacy as pH decreases

• Added into a variety of foods; dairy, bread, fruit & veg

Benzoic acid

decreases intracellular pH, interferes with oxidative phosphorylation and AA uptake

• Found naturally in fruits, berries, spices, and fermented foods

Propionic acid

inhibit enzymes and decrease intracellular pH

• More effective at higher pH than sorbic or benzoic

• Naturally produced by some cheeses

• Added to other cheeses, and baked goods

• Added to raw dough to prevent contamination by B.mesentericus

Sulfur Dioxide

MOA unclear (inhibit protein formation or reduce intracellularpH?)

• Prevents browning of foods

• Used in winemaking since ancient times

• Dissolves in water readily (sulfites)

Nitrites

Nitric Oxide reacts with iron-sulfur groups (disrupts ETC)

• added to processed meats (maintains color; stops C. botulinum endospore germination)

• Nitrosamines (carcinogen) produced when nitrite-preserved meats are heated

Nisin

disrupts G+ cell wall production

• Produced by Lactococcus lactis

• Used to preserve cheeses, meats, and beverages

Natamycin

antifungal macrolide antibiotic

• Disrupts fungal cell membrane

• Prevents bacterial protein synthesis

• Used in cottage, sliced, and shredded cheese

High effectiveness

kill vegetative cells, fungi, viruses, and endospores

Intermediate effectiveness

less effective against endospores and viruse

Low effectiveness

kill only vegetative cells and enveloped viruses, not endospores

Phenol Coefficient

compares a chemical's antimicrobic properties to those of phenol

Phenol coefficient of:

• 1.0 = chemical has the same effectiveness as phenol

• <1.0 = chemical is less effective than phenol (formalin)

• >1.0 = chemical is more effective than phenol (chloramine)

Disk diffusion

a test with antibiotic impregnated paper discs on agar plates, measure resistance

Use-dilution test

determines agent's effectiveness on an inanimate surface

In-use test

determine whether disinfectant is contaminated

Heat sterilization

•Oldest and most common

•Alters membranes and/or denatures proteins

Pasteruization

Semi-sterilizes food, but does not ruin food quality

High Temperature Short Time (HTST)

72 C for 15 sec then bottled and refrigerated

Ultra High Temperature (UHT)

138 c for 2 or more seconds, then ssealed in airtight containers for up to 90 days without refrigeration.

Milkborne organisms killed by pasteurization.

Campylobacter jejuni, Coxiella Burnetii, Listeria monocytogenes, Escherichia coli O157:H7, Mycobacterium tuberculosis, M. paratuberculosis, Salmonella spp., Yersinia enterocolitica

thymine dimers

two neighboring thymines attached to one another by covalent bonds

High-efficiency particulate air (HEPA) filters

remove microbes >0.3 µm in diameter. used in households, biological safety cabinets(BSCs), as well as hospitals and surgical suites