Microbiology Chapter 12: Sterilization and Disinfection

Drying

Preserves certain foods

Pressure: ___ ___ per square ____ (psi)

Pressure: 15 lbs per square inch (psi).

Mechanisms of Action of Chemical Agents

Protein damage, Lipid damage, Other cell components such as nucleic acids and energy production systems.

Uses of Low Temperatures

Refrigeration, Freezing, Drying, Freeze Drying (Lyophilization).

What two organisms are used for the Phenol Coefficient test?

Salmonella typhi G+ and Staphylococcus aureus G- (The PC is reported separately for each test organism)

Refrigeration

Slows down growth (4-5°C)

Specific Examples of Chemical Agents

Soaps and Detergents, Surfactants, Cationic detergents, Acids and Alkalis, Heavy Metals, Halogens, Phenol, Oxidizing agents, Alkylating Agents, Heat, Lyophilization, Radiation, Filtration, Autoclaving, Pasteurization, Radiation.

What are the most difficult microorganisms to destroy?

Spores.

Time required for sterilization

The fewer organisms present, the shorter the time needed to achieve sterility.

Sterilization

The killing or removal of all microorganisms in a material or on an object.

Disinfection

The reduction of the number of pathogenic microorganisms to the point where they pose no danger of disease.

Effects on Chemical Antimicrobial Potency

Time (longer), Temperature (hotter), pH (H ions), Concentration (heavily).

Mercury (Merthiolate, Mercurochrome)

To disinfect skin wounds. Often made as a tincture, dissolved in alcohol.

Radiation

UV light, X rays, gamma rays

When pressure ____, temperature _____.

When pressure Increases, temperature Increases.

Autoclaving, Steam _____.

When water is heated under pressure, its boiling point is elevated, so temperatures above 100°C can be reached. Steam penetrates. Excellent for sterilizing lab media. Not as good for sharp instruments, powders. Don't pack too tight.

Tincture Solutions

are characterized by the presence of alcohol mixed in with the main chemical.

Prions

are highly resistant and must be sterilized longer and at higher temperature.

Zone of inhibition

area on a plate where Micros won't/can't grow due to the presence of an antimicrobial agent.

Surfactants

binds to dirt and water, is able to be whisked away; lifts dirt out of crevices. Kills some viruses, but not spores.

Gluteraldehyde

can sterilize without heat.

Membrane (lipid) damage

caused by surfactants→soaps, detergents and alcohols

Copper Sulfate

controls algae growth (water quality)

Protein damage

denaturation (hydrogen and disulfide bonds are disrupted, and the functional shape of the protein is destroyed). Denaturation includes: Hydrolysis, Oxidation, The attachment of atoms or chemical groups

Labile

easily broken down or displaced.

Chlorine

in water (Hypochlorite, Bleach) kills bacteria and many viruses; is inactivated by organic materials (pools: more people, more chlorine)

Heat Killing Principle

killing with heat is caused by temperature and time.

Selenium Sulfide

kills fungi (dandruff, skin infections)

A Phenol Coefficient greater than >1

means that the disinfectant is more effective.

A Phenol Coefficient equal to =1

means the disinfectant has the same effectiveness as phenol.

A Phenol Coefficient less than <1

means the disinfectant is less effective than phenol.

Soaps and Detergents

mechanical removal of microbes, oils, and dirt; they do NOT sterilize. Easy to use, cheap, readily available.

Alcohols: Ethyl and Isopropyl (70-90%)

most effective when mixed with water (10%), Water helps better denature the proteins and dissolve lipids in the membrane. -Evaporation reduces effectiveness, doesn't stay in contact -Doesn't kill spores or all bacteria -Good for vegetative cells on skin -flammable.

Pasteurization kills

pathogens such as Salmonella and Mycobacterium

Hibiclens

surgical scrub good for a wide spectrum of microbes.

In an autoclave, is it the increased pressure or the increased temperature that kills the organisms?

the increased temperature.

The Phenol Coefficient

the ratio of the dilution of the agent being tested to the dilution of phenol that will kill all organisms in 10 minutes but not 5 minutes.

Silver Nitrate

to prevent gonorrhea

Open Flame

used for inoculating loops; incineration.

Iodine/Iodophor

usually in tincture used as an antiseptic for the skin.

Characteristics of an Ideal Disinfectant (10)

• Fast-acting • Stable around organics— stable predicts how its going to work, always the same • Non-toxic (Chlorine is very harsh and toxic) • Wide spectrum (good for all organisms) • Non-damaging to materials (floor, skin, shoes) • Easy to prepare (mixable with water) • Doesn't breakdown/change over time • Readily Available • Inexpensive • Has no offensive odor

HEPA (High efficiency particulate air filter) used in ventilation systems.

○ Only filtered air will go into Operating Rooms (+ pressure= air is filtered before entering) ○ Laminar flow hoods in labs ( - pressure= air being filtered on the way out)

Formaldehyde

"fixes" proteins. Inactivates without changing their antigenic properties. Used to make formalin, for vaccines and tissue preservation (biopsies, specimens for dissection).

U.V. light

-40-390 nm; damages proteins and nucleic acids. -Poor penetration -Good for killing airborne microbes -Used in biological/laminar flow hoods.

Filtration

-Can be used to sterilize substances that are destroyed by heat (drugs, serum, vitamins, sucrose) -Physical removal of microbials by small pores (smaller than viruses) -To separate viruses from bacteria (manufacture of vaccines) -To collect microorganisms from air and water samples (water quality testing)

Lyophilization

-Freeze drying; Rapid freezing under high vacumn to remove all water. -Good for long term preservation of bacteria.

Heat

-One of the most popular ways to destroy microbes (flame or boiling) -Moist or Dry heat may be used -Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells but not endospores. -Steam sterilization (autoclaving) is necessary to destroy the bacterial endospores.

Ionizing Radiation

-X-rays and gamma rays <40nm -Named because it can dislodge electrons from atoms, creating ions. -Damages DNA and produces peroxides (powerful oxidizing agents in cells) -An excellent sterilizing agent because of its ability to penetrate deep into objects. -Used for plastic lab and medical equipment; also certain foods. -Ionizing Radiation will destroy bacterial endospores and both prokaryotic and eukaryotic vegetative cells (sterilizes).

___ part phenol: ___ parts water -- kills population in ___ minutes.

1 part phenol: 9 parts water, kills population in 10 minutes

Bactericidal vs Bacteriostatic

1. BacteriCidal: is a substance that kills bacteria. Bactericides are disinfectants, antiseptics, or antibiotics. 2. BaterioStatic: a biological or chemical agent that stops bacteria from reproducing, while not necessarily killing them otherwise.

Principles of Controlling Microbial Growth

1. Proportional Death Rates, 2. Time required for sterilization, 3. Role of organic matter, 4. Microbial susceptibilities.

What temperature do prions need to be heated to be sterilized, and for how long.

134°C for 18 min.

Milk Pasteurization Holding Method

62.9°C for 30 minutes (not boiling)

Milk Pasteurization Ultra high temperature Method

74°C—>140°C—>74°C in 5 seconds

Dry heat oven temp.

@ 171°C for 1 hour. (350oF)

Antiseptic

A chemical agent that can safely be used externally on living tissue to destroy microorganisms or to inhibit their growth.

Sanitizer

A chemical agent typically used on food-handling equipment and eating utensils to reduce bacterial numbers so as to meet public health standards. Sanitization may simply refer to thorough washing with only soap or detergent.

Disinfectant

A chemical agent used on inanimate objects to destroy microorganisms. Most disinfectants do not kill spores.

Proportional death rates

A definite proportion of the organisms die in a given time interval

Pasteurization.

A process invented by Louis Pasteur to destroy microbes that caused wine to sour, does not achieve sterility.

Toxic agents

Alkylating agents can cause cancer and should not be used in situations when they might affect human cells.

Germicide

An agent capable of killing microbes rapidly; some such agents effectively kill certain microorganisms but only inhibit the growth of others.

Viricide

An agent that inactivates viruses.

Bacteriostatic Agent

An agent that inhibits the growth of bacteria.

Bactericide

An agent that kills bacteria. Most such agents do not kill spores.

Sporocide

An agent that kills bacterial endospores or fungal spores.

Fungicide

An agent that kills fungi

Cationic detergents

Are the most effective. Many are Quaternary Ammonium Compounds (QUATS) which act as DISINFECTING AGENTS (more chemistry= more powerful), They are often combined with other agents to increase their effectiveness.

Heat resistant spore cultures.

Are used as a quality check to make sure the autoclave is working properly.

Moist Heat

Boiling (unpressurized)

Hydrogen peroxide

Breaks down into oxygen and water. The oxygen kills strict anaerobes present in many wounds.

Freezing

Can stop chemical reactions, inhibiting growth

Halogens

Chlorine, Iodine

Phenols

Denatures proteins and disrupts membranes.

Alkylating Agents

Denatures proteins and nucleic acids.

Oxidizing Agents

Disrupts sulfide bonds in proteins and disrupts membranes.

Phenol Coefficient, Filter Paper Method, Dilution Assay

Evaluates the effectiveness of a new chemical to this old one, that we know its effectiveness at a certain level. Means of comparing all chemicals on one scale and standardizing.

Ethylene Oxide

Gas; needs to be in a fumigation chamber, Has very good penetrating power, sterilizes heat labiles, penetrates plastics, requires aeration, gas is topic explosive and carcinogenic.

Lysol, Hexachlorophene

Halogenated phenol, inhibits staphylococci sp.

Milk pasteurization Flash Method

Heated to 71.6°C for at least 15 seconds.

Heavy Metals

Inhibit Growth, Silver Nitrate, Mercury (Merthiolate), Selenium, Copper sulfate.

Iodophor

Iodine combined with surfactant for slow release; penetrates well, evaporates slower. ex) Betadine & Isodine.

Boiling (unpressurized)

Kills vegetative cells, inactivates some viruses, doesn't kill spores.

__-__ __(time)__ @ __°__

Less time, lower temp. 15-20 minutes at 121°C.

Microbial susceptibilities

Microorganisms different in their susceptibility to antimicrobial agents.

Reactions that affect other cell components

Nucleic acids and energy-production systems, CAN BE DANGEROUS TO HUMAN.

Acids

Organic acids lower the pH of materials, inhibiting bacteria. Several are used as food preservatives (benzoic acid, soft drinks.)

Role of organic matter

Organic material diminishes efficacy (effectiveness) of chemical agents.

Dry Heat

Oxidizes proteins (disrupts disulfide bonds), Good for glassware, metals, oils and powders.