Microbiology Module III

Antiseptic

A disinfectant that is nontoxic enough to be used on skin

Aseptic Technique

Use of specific methods to exclude contaminating microorganisms from an environment

Bactericidal

kills bacteria

Bscteriostatic

prevents the growth of, but does not kill, bacteria

Decontamination

treatment used to reduce the number of disease-causing microbes to a level that is considered safe to handle

Disinfectant

A chemical used to destroy many microorganisms and viruses

Fungicide

kills fungi

Germicide

kills microorganisms and inactivates viruses

Pasteurization

a brief heat treatment used to reduce the number of spoilage organisms and to kill disease causing microbes

Preservation

the process if inhibiting the growth of microorganisms in products to delay spoilage

Sanitize

to reduce the number of microorganisms to a level that meets public health standards; implies cleanliness as well

Sterilant

a chemical used to destroy all microorganisms and viruses; an absolute term

Sterilization

the process of destroying all microorganisms and viruses, through physical or chemical means

Viricide

inactivates viruses

% Alcohol

62% bare minimum-60% in glycerin base

High-level disinfection

HLD= inactivation of most vegetative bacteria, fungi and a 6-log reduction of mycobacteria (go down to 1 or 99.999%)

Antiseptics

destruction or inhibition of microbes on living tissue; prevents sepsis (Prescence of microbes, toxins, or components in blood of host); less toxic than disinfectants

Selecting an antimicrobial Procedure

Type of microorganism, number of microorganisms, environmental conditions (ph, presence of organic matter, temperature), risk of infection, concentration of agent, duration of exposure

Viruses Fact

don’t replicate on inanimate objects→ only from host→ envelope viruses get peptidoglycan layer from host

Are herpes virus or rhinovirus more likely to be spread (in context of being on a surface)

Herpes (envelope); not survive on inanimate object as long b/c of phospholipid bilayer; temp. ph, soap, alcohol can break down bilayer which makes it easier to get rid of than a naked virus. Rhinovirus is a naked virus, protein surrounded, naked virus

Types of Microbes-Level of difficulty in killing; Descending order of resistance to germicidal chemicals

Bacterial spores→ mycobacteria→ nonlipid or small viruses→ fungi→ vegetative bacteria→ medium size or lipid viruses

Types of Microbes- Level of difficulty in killing (hardest-easiest)

Bacterial Spores→Mycobacteria→Naked Viruses (nonlipid) or Small Viruses→ Fungi→ Vegetative Bacteria→ Lipid or medium sized viruses

Bacterial Spores

Bacillus subtilis, Clostridium sporogenes

Mycobacteria

Mycobacterium tuberculosis var. bovis, Nontuberculous mycobacteria

Nonlipid (naked) or Small Viruses

Trichophyton spp., Cryptococcus spp., Candida spp.

Vegetative Bacteria

Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella choleraesuis, Enterococci

Lipid or Medium-size viruses (have envelope, peptidoglycan layer)

Herpes simplex virus, CMV, Respiratory synctial virus, HBV, HCV, HIV, Hantavirus, Ebola Virus

Kill ___ Log of Microbes

1

Microbes not immediately killed upon exposure to agents

true

Decimal reduction time (D-value)

time needed to jill 90% of microbes

Z value

temperature increase needed to reduce D to 1/10 value

F value

time (min) at some temp. needed to kill cells or spores

Number of Microbes: x-axis

time (min): domain, independent variable, abcissa

Number of Microbes: y-axis

Log10 (number of survivors): dependant variable, range, ordinate

Prion Sterilization and Disinfection

want 3 log reduction of prions in one hour; physical methods not optimal; chemical methods that work best are Cl and NaOH but corrosive

Prion Sterilization: Physical Methods (not effective)

temp, pH, grinding, pulverizing, heating, burning, handwashing

Prion-misfolded Protein

acquired ability to convert its normal, healthy counterparts into its own abnormal shape, leading to a chain of reaction of misfolding; starts out as a healthy protein (PrP^c)→ once 1 misfold happens if touch healthy proteins surrounding, will cause healthy proteins to fold = chain rxn.

Recommendations for Prion Sterilization and Disinfection

Keep instruments moist; decontaminate; use four possible options (2x diff. autoclaving or 2x diff. immersion)

Autoclave 132 C 18 minutes in ____

prevaccum

Autoclave 132 C 1 hour on

gravity cycle

Immerse in 1 N NaOH 1 hour, then sterilize 121 C gravity or `34 C _____

prevaccuum 1 hour

Immerse 1 N NaOH 1 hour, 121 C 30 minute gravity; clean again and do 121 C 15 minutes

True

Prion Sterilization and Disinfection Physical Methods

Heat (dry heat, moisture heat - autoclaving), pressure, filtration, radiation (uV, ionizing), removal of oxygen, freezing, dehydration, mechanical removal (washing)

Adiabatic: why standard autoclave fail to destroy prions

Newton’s law of cooling

Pv=nRT

Boyle’s Law (P1V1=P2V2) works on autoclave - if you squeeze a gas (inc. pressure) it takes up less space (decrease volume)

Autoclave Standard

121 degrees C at 15 ib/sq in for 15 minutes ; prions inactivated 132-134 deg C

Prion

a misfolded version of a normal protein found in the host; no nucleic acids (DNA or RNA)

Geobacillus stearothermophilus

endospores are used for quality control-purple to yellow, one in water, one in autoclave. In autoclave vial remains purple=no pH change and no germination = successfully killed

Geobacillus stearothermophilus: Purple

spres do not germinate = successfully killed

Geobacillus stearothermophilus: Yellow

spores germinate, change pH, dye changes color

Gravity displacement autoclave

steam admitted at the top or sides of chamber; steam forces air out bottom of chamber through the drain vent; less penetration, so longer sterilization time

High-speed pre-vaccuum sterilizer

fitted with a vacuum pump (or ejector); ensures air removal from sterilizing chamber before steam is admitted; increased penetration, short sterilization time

Pasteurization

significantly decreases numbers of heat sensitive microbes, including pathogens (Mycobacterium tuberculosis and Coxiella burnetii)

Pasteurization: LTH

Low temperature holding 62.8 deg C for 30 minutes

Pasteurization: HTST

high temperature, short time 72 deg C15 sec or 82 deg C 20 sec

Pasteurization: UHT

Ultra high temperature 141 deg C 2 sec

Dry Heat

160-170 deg C for 2-3 hours; oxidize cell contents (structural damage to cell); standard: 200 deg C for 1.5 hours; Moist heat is more effective in killing bacteria than dry heat

Gases: Betapropiolactone (BPL)

does not penetrate materials well; decomposes rapidly

gases: vaporized hydrogen peroxide

used at wide temp. range

gases: chlorine dioxide (ClO2)

high lvl disinfection (HLD); 1 mg/L at 60% relative humidity; effective between pH 4-10, destroys biofilms, spores, vegetative bacteria; affects proteins, membranes, nucleic acids

Filtration

Physically removes microbes; heat sensitive liquids (membrane filters - 0.45 um; 0.22 um)

Filtration: Air, HEPA

(high efficiency particulate air filter); 0.3um; removes particles with 99.97% efficiency; out of 1000 particles, if HEPA filter removes particle down to .3 micrometers with 99.97% accuracy; you remove 99.97 and left with 3 particles

Filtration: Laminar flow biosafety hoods

force air through HEPA filters; project vertical flow sterile air across opening; room air enters grid at front of hood; filtered air flows through grid (blown up and out through HEPA filters)

Radiation: UV (200-300nm)- non-ionizing

damages DNA; causes T-T dimers; little penetration; air, surface

Radiation: Gamme, X-rays -ionizing

produces reacting molecules in microbes; heat sensitive material; foods; measured in absorbed radiation dose (rad, 100 rad =1 Gray)

Non-ionzing radiation

his type of radiation has enough energy to excite electrons, causing molecules to vibrate or spin faster (which we feel as heat), but not enough energy to rip electrons away from atoms.

Ionizing Radiation

carries enough energy to eject electrons from atoms or molecules, creating ions (charged particles). This process directly breaks covalent bonds, including the backbone of DNA.

How much radiation do you get: Dental x-ray

0.5-3.0 mrem

How much radiation do you get: Smoke detector

< 1 mrem

How much radiation do you get: Cross country flight

5 mrem

How much radiation do you get: Xrays and gamma rays

0.1 gray=1 rad= 1 rem= 10 milliSieverts

Chemical methods

Disinfection (Some sterilize) large surface, heat sensitive; antisepsis, preservatives (weaker), affect membranes, enzymes

Category of germicide (kills germs):Sterilant

Bacteria:

endospores- yes

mycobacteria- yes

others-yes

Fungi: All - yes

Viruses:

nonlipid- yes

lipid- yes

Category of germicide (kills germs): High Level

Bacteria:

endospores- some

mycobacteria-yes

others-yes

fungi: all- yes

Viruses:

nonlipid- yes

lipid-yes

Category of germicide (kills germs):Intermediate level

bacteria:

endospores-no

mycobacteria-yes

others-yes

fungi: all- yes

viruses:

nonlipid-varies

lipid- yes

Category of germicide (kills germs): Low level

Bacteria:

endospores- no

mycobacteria-no

others-varies

Fungi: all- varies

viruses:

nonlipid- no

lipid-varies

Antimicrobial activity

refers to the ability of a substance to kill or inhibit the growth of microorganisms

Cytoplasmic Membrane

Biguanides, Phenolics, Quats

Proteins

Alcohols. Aldehydes, Halogens, Metals, Ozone, Peroxygens, Phenolics

DNA

Ethylene oxide, Aldehydes

Sufactants

affect membranes; cationic (quats), anionic

Surfactants: Cationic (quats)

interact with PO4³- groups; affect membranes; attracted to cell surface; detergents-amphipathic (contain hydrophobic and hydrophilic regions); react with phospholipids in membranes; unlike soaps, not derived from fats, clean inanimate objects, ineffective vs. endospores, mycobacteria, naked viruses

Surfactants: Anionic

some antimicrobial properties

Phenolics

denature proteins, affect membranes; remain active, effective; 5% solutions kills bacteria; skin, neurological damage (lysol can lyse cells - breaking down of a cell’s membrane causing it to bust and die ); phenol coefficient test used to determine efficacy of toxic substance; based on highest dilution of disinfectant that kills all bacteria after 10 minutes

Alchohols

lipid solvents (dissolve membrane lipids, coagulate proteins); bactericidal, fungicidal, not sporicidal, viricidal (70% soln) (not effective vs. naked viruses); disinfect surfaces; antiseptics (commons hand gels have 62.5% ethanol in glycerin base, better brands have 68-70%); evaporate

Coagulation of proteins

of protein denaturation—a critical mechanism that makes alcohols effective disinfectants, but also explains why they fail against highly resistant agents like prions and spores.

Halogens: Chlorine

oxidizes cell constituents (cell membranes, DNA, proteins -breaks these down); disinfectant; produces HCLO→HCL +O ; water, dairy food; cheap easy effective

Halogens: Iodine

linked as iodophore (slowly releases iodine over time)

Heavy Metals

interfere with S groups in proteins, Toxic, less effective; 1% soln AgNO3-opthalmic gonorrhoea; silver sulfadiazine (burns); CuSO4-algicide

Peroxide

3% solution hydrogen peroxide; some bacteria produce catalase (enzyme that breaks down peroxide); rendering inactive

Biguanides

chlorhexidine; antiseptics

Aldehydes

inactive proteins and DNA, glutarldehyde-sterilize; formaldehyde-preservation; ortho-phthaldehyde (HLD)

Did watson and crick discover DNA ?

NO

DNA:Deoxyribonucleic Acid

discovered in 1869 by Freidrich Meischer; acidic, with much phosphorous; found inside the cell nucleus

Nucleobases: DNA

Adenine, Guanine, Thymine, Cytosine

Nucleobases: RNA

Adenine, Guanine, Uracil, Guanine

Purines - Double Ring (6 Carbon)

Adenine and Guanine

Pyrimidine-Single Ring (6 Carbon)

Cytosine, Thymine, and Uracil

Is uracil a base or nucleoside or nucleotide

Base

Sanger Dideoxy Sequence

technique that uses 3’ and 5’