BIOL 2460 Chapter 13

Main Goal

reduce microbial load and reduce infection of contamination

clean

it is all relevant

sterilization

removal/killing of ALL microbes

Disinfection/Antisepsis

inactivation of microbes

Sanitization/Degerming

decreasing microbial load

Who established the 4 levels of BSL

CDC, NIH, WHO

BSL 1

•Very little risk

•sink for handwashing & door to close off lab

•Agents that do not cause infection in healthy adults

•Nonpathogenic E. coli and B. subtilis

•(Ex. BIOL freshman labs)

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 measels

•(Ex. Micro labs)

BSL-3

•Potential to cause lethal infections by inhalation

•BSL-2 plus respirator, bio safety cabinets, hands-free wash sink, two sets of doors, directional air flow

•Indigenous or "exotic" pathogens

•M. tuberculosis, B. anthracis

•West Nile Virus, HIV

•(No example at UTA)

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 own air supply

•"Exotic" pathogens; Ebola and Marburg viruses

•(only 13 in USA)

critical cleaning

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

Ex: surgical instruments, catheters, IV fluids

semicritical cleaning

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

noncritical cleaning

do not require sterilization' items contact but do not penetrate intact skin (stethoscopes, bed lines, blood pressure cuffs)

Sterilization

Complete killing or removal of all microbes from fomite

Methods: Heat, pressure, filtration, chemical

aseptic technique

used to prevent sterile environment from becoming contaminated

disinfectant

inactivation/kill of microbes on fomites

(include vinegar and bleach)

some microbes may not be inactivated-- disinfection does not equal sterile

antiseptic

acts on microbes but not organism/tissue

(hydrogen peroxide, rubbing alcohol)

sanitization and degerming

Decrease of microbial load (amount of microbes)

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 towels, etc)

may be used in combination with disinfectant to maximize microbial reduction

common protocols for control of microbial growth

measuring success of control

-methods can partial or full kill various microbes

- cides: kill

- static: stop growth

Ex:

Bactericidal vs Bacteriostatic

Viricidal; vs Viristatic

Fungicidal vs Fungistatic

-degree of control can be observed with microbial death curve

-many factors affect success of control

-length of exposure; concentration of agent; population level

microbial death curve

measure of percentage of kill

decimal reduction time DRT

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

physical means of control

1. Temperature

2. Radiation

3. Filtration

4. Desiccation

5. Pressure

6. Sonication

Heat Sterilization

•Oldest and most common

•Alters membranes and/or denatures proteins

thermal death point

the lowest temperature required to kill all microbes in a sample in 10 minutes

thermal death time

the length of time required to kill all bacteria in a liquid culture at a given 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

Ex. autoclave

autocalve

raise temperature of water above boiling point 121C by raising pressure to 15 psi

kills viruses & endospores

Autoclave types

gravity: use steam to push out air

prevacuum: vacuums out air first

require periodic QC to check functionality: autoclave tape, spore tests, diack tubes, recorders

pasteurization

semi-sterilizes food but does not ruin food quality

many methods rely on "flash" heating foods to kill most mircobes

Refrigeration and Freezing

•Usually not sterilization method but static

•Slows metabolism but will grow when temps are raised

•(Reason why USDA recommends thawing at temps lower than optimal)

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

pascalization

high pressure used in food industry to kill microbes

hyperbaric chambers can be used to treat infections - counters hypoxia by saturating infection site with oxygen

used in combination with temperature in autoclaves

desiccation

drying or dehydration

method used for millennia to preserve foods (raisins, prunes, jerky, etc)

all cells including microbes require water for metabolism and survival

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

lyophilization

freeze-drying; rapid freezing then placed under vacuum

salts or sugars can used to lower water activity of foods/materials without physical drying (osmosis)

radiation

high energy radiation -used to kill or inhibit microbes

ionizing radiation

enters into cells and disrupts molecular structures such as DNA

X-rays & gamma rays

can be used to sterilize non-autoclavable items

can be 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

used in laboratory and clinical settings

filtration

use of barrier to physically separate microbes

useful when media cannot be autocalved (urea broth, antibiotic and vitamin solutions)

filters usually have pore size of .2 um or smaller for viruses

membrane filtration

remove microbes from liquid samples

Air is commonly filtered through

High-efficiency particulate air (HEPA) filters

used in households, biological safety cabinets, hospitals and surgical suites

physical methods of control

physical methods of control part 2

Chemical means of control

-Phenolics

-Heavy Metals

-Halogens

-Alcohols

-Surfactants

-Bisbiguanides

-alkylating agents

-peroxygens

-supercritical fluid

-food, cosmetic, and pharma preservatives

phenols

carbon molecule with benzene ring and -OH group

MOA: denature proteins and membranes

Ex: carbolic acid - first used by joseph lister for surgical wounds

lysol - original formulations (now is quaternary compound)

triclosan - commonly used in hand soaps, banned by FDA in 2017

heavy metals

MOA: binds to and inhibits proteins

not exclusive to mircobes

mercury

treated syphillis but banned b/c of neural toxicity effects

silver

used today to treat burn wounds, pediatric ophthalmia, neonatorum, and in antibiotics

sopper 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 recongizable with dental products

deposits in tooth enamel and provides disruption in microbial fermentation and processes

alcohols

used as disinfectants and solvents.

- MOA: disrupts membranes and denatures cytoplasmic proteins - lysis

- used as 70% to allow better cell pernetration

- only viricidal for enveloped

- can be used in combo with iodine

surfactants

chemicals that lower surface tension of after

in most soaps and detergents; aid in gegerming

soaps

fatty acids 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" (Bisbiquanides)

alkylating agents

strong disinfecting agents

- replace hydrogen atom with alkyl group

M

OA: inactivates enzymes and nucleic acids

formaldehyde

fixes specimens by cross-linking proteinsw

glutaraldehyde

acts faster than formaldehyde; common disinfectant of surgical equipment

ethlyene oxide

gaseous sterilizer that has high penetrating ability

B-propionolactone

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

peroxygens

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

hydrogen peroixde

common and cheap disinfectant

peracetic acid

more effective than immune to activiation by and peroxidases

Benzoyl peroxide

present in acne meds; very effective against Propionibacterium acnes

Carbamide peroxide

agent in toothpaste that combats bioflims

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 CO2

•Allows for easier cell penetration and formation of carbonic acid and increase acidity

•Non-reactive, non-toxic, & non-flammable

•Good for many vulnerable materials such as foods and some tissues

preservatives

most inhibit microbial growth in food products

in foods, important to be non-toxic and flavorless

- sorbic acid

- benzoic acid

- propionic acid

- sulfur dioxide

- nitrites

- nisin

- natamycin

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 intracellular Ph)

- prevents browning of foods

- used in winemaking since ancient times

- dissolves in water readily (sulffites)

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

Disinfectant/Preservative Effectiveness Testing

-Mandated by many agencies

-What level of kill can the agent have?

- how long does it last?

- what microbes does it work on?

3 levels of effectiveness

high - kill vegetative cells, fungi, viruses, and endospores

intermediate - less effective against endospores and viruses

low - kill only vegetative cells and enveloped viruses, not endospores

methods of effectiveness

-phenol coefficient

-disk diffusion

-use-dilution test

-in-use test

phenol coefficient

how strong is the agent relative to phenol

- staphyloccous aureus and salmonella enterica typhi used as test organisms

- exposed to agent diluted in water for 7.5 min

- phenol coefficent of:

1.0 = chemical has same effectiveness as phenol

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

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

disk diffusion method

measures degree of inhibition using sterile filter paper disks with chemicals

- places on an inoculated agar plate

- lawn of bacteria exhibits zones of inhibition

- larger zone diameter correlates to increased inhibition

use-dilution test

•determines agent's effectiveness on an inanimate surface

•Stainless steel cylinder dipped in target culture then dried

•Cylinder then dipped into various concentrations of disinfectant

•Cylinder then transferred to fresh media

•Turbidity = bacterial survival

in-use test

•determine whether disinfectant is contaminated

•Usually used in clinical settings