Chapter 3.- Sterilization, Disinfection, and Antispesis

What are surfactants?

"Surface active" chemicals

What is antisepsis?

*Reduction* in the number of microorganisms and viruses, particularly potential pathogens, on *living* tissue Chemical used on skin or other tissue (antiseptic) Some antiseptics and disinfectants have same components, but disinfectants are more concentrated or can be left on surface for longer period.

Phenol

- Intermediate- to low-level disinfectants - Denature proteins and disrupt cell membranes - Effective in presence of organic matter - Remain active for prolonged time - Commonly used in health care settings, labs, and homes - Have disagreeable odor and possible side effects

How much radiation is given?

.02-.15 kilograce while to sterilize is about 70-100 kilograce

What are the 2 actions of Antimicrobial Agents ?

1) Alteration of cell walls and membranes. 2) Damage to proteins and nucleic acids

What are the factors affecting the efficacy of Antimicrobial Methods?

1) Nature of the sites to be treated 2) Degree of susceptibility of microorganisms involved 3) Pertaining environmental conditions 4) Organic material present 5) Number and resilience of the contaminating organisms 6) Type and concentration of disinfectant

What is the microbial activity of alcohol?

Bactericidal against vegetative bacteria, mycobacteria, some fungi, and lipid-containing viruses • Alcohols have no activity against bacterial spores, and poor activity against some fungi and non-lipid-containing viruses

Alcohols

Bactericidal, fungicidal, and virucidal against enveloped virues, not effective with fungal spores or bacterial endospores. Intermediate level. Ethanol and isopropanol most common. Denature proteins and disrupt cell membranes

If the In-use test is more realistic then why do we still use the Use-dilution?

Because In-use is time consuming.

What are the methods of microbial control using moist heat?

Boiling Autoclaving Pasteurization Ultrahigh-temperature sterilization

How does environmental conditions affect antimicrobial methods?

Change in Temp & pH affect microbial death rate and the efficacy of of antimicrobial methods. Works better with warmer (need chemical rxn) and more acidic disinfectants

Examples of degerming?

Chemicals play a secondary role to the mechanical removal of microbes Examples: handwashing, alcohol swabbing at site of injection

What are the effects of Moist Heat?

Commonly used to disinfect, sanitize, *sterilize* , and pasteurize, kills cells by denaturing proteins and destroying cytoplasmic membranes. More effective than dry heat.

Phenolics

Compounds derived from phenol molecules that have been chemically modified by the addition of halogens or organic functional groups. (Lister began using phenol in surgery) Intermediate to low level disinfectants that denature proteins and disrupt cell membranes in a wide variety of pathogens.

What is the D value?

D value is the time required at a given temperature to destroy 90% of the microbes in a sample

Aldehydes

Denature proteins. High. Disinfectant and embalming fluid. Cross-link functional groups to denature proteins and inactivate nucleic acids • Exert its effects through alkylation

What is sterilization?

Destruction of *all* microorganisms and viruses in oron an object. The preparation of microbiological culture media or canned food, i.e., incineration, steam under pressure, ethylene oxide gas

What is disinfection?

Destruction of *most* microorganisms and viruses on *nonliving* tissue use of physical or chemical agents known as disinfectants, including ultraviolet light, heat, alcohol, and bleach to inhibit or destroy microorganisms, especially pathogens. Does not guarantee that all pathogens are eliminated- CANNOT inhibit endospores or some viruses.

How is desiccation used as a microbial control method?

Drying, inhibits microbial growth because metabolism requires liquid water. Stops the spread of most pathogens, including the bacteria that cause syphilis, gonorrhea, and the more common forms of bacterial pneumonia and diarrhea.

What is Nonionizing Radiation?

Electromagnetic radiation with a wavelength greater than 1nm does not have enough energy to force electrons out of orbit. Does not contain enough energy to excite electrons and cause them to make new covalent bonds, which can affect the 3D structure of proteins and nucleic acids. UV light, infrared radiation, and radio waves.

What is the different between electron beams and gamma rays?

Electron beams - effective at killing but do not penetrate well Gamma rays - penetrate well but require hours to kill microbes

What is Ionizing Radiation?

Electron beams, gamma rays, and X rays (wavelengths shorter than 1nm) strike molecules and have sufficient energy to eject electrons from atoms, creating ions. Ions disrupt h-bonds, oxidize double covalent bonds, and create reactive hydroxide ions. These Ions denature molecules (DNA) causing fatal mutations and cell death. Medical and lab equipment and preserving food.

What types of bacteria can survive boiling?

Endospores, protozoan cysts, and some viruses can survive boiling

How can Phenol Coefficient be used to evaluate efficacy of disinfectants and antiseptics?

Evaluates efficacy of disinfectants and antiseptics by comparing a given agent's ability to control microbes to that of phenol under standardized conditions. A phenol coefficient > 1.0 indicates the agent is stronger and more effective than phenol. ex: Clorox 200x means it is 20 times better than phenol. Phenol is used in lysol

What can happen when there is damage to proteins and nucleic acids?

Extreme heat or certain chemicals denature proteins. Denatured proteins (broken bonds) cease to function, bringing cellular death. Chemicals, radiation, and heat can alter or destroy nucleic acids - Can produce fatal mutants - Can halt protein synthesis through action on RNA

What are the effects of Dry Heat?

For substances such as powders and oils that cannot be sterilized by boiling or with steam, or for materials that can be damaged by repeated exposure to steam (such as metal). Sterilization can be achieved by the use of dry heat. Higher temperatures for longer times because dry heat penetrates more slowly.

At what temperature is the fridge and freezer set?

Fridge: 4 degrees Celsius Freezer:-20 degrees Celsius

How are germicides classified?

Germicides classified as high, intermediate, or low effectiveness.

What is microbial Death Rate?

Given by a constant percentage of cells killed over time.

What does BSL-3 handle?

Handling of microbes in safety cabinets

What does BSL-4 handle?

Handling of microbes that cause severe or fatal disease ex: ebola

What does BSL-2 handle?

Handling of moderately hazardous agents ex: university lab

What does BSL-1 handle?

Handling pathogens that do not cause disease in healthy humans

How does the site affect antimicrobial methods?

Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects. Method of microbial control based on site of medical procedure.

Examples of Pasteurization?

Heat treatment is brief to reduce alteration of taste and nutrients, but microbes still remain and can still cause spoilage Examples: pasteurized milk and fruit juices

What are the effects of high temperatures?

High temp denatures proteins, interferes with integrity of cytoplasmic membranes and cell walls, and disrupt functions and structure of nucleic acids.

What are considered high-level disinfectants?

High-level kill all pathogens, including endospores. Really need to sterilize such as surgery.

How does hydrogen peroxide work?

Hydrogen peroxide can disinfect and sterilize surfaces • Kill most bacteria at concentrations of 3-6%; and kill all organisms (including spores) at higher concentrations (10-25%) • Not useful for treating open wounds due to catalase activity

What criteria is used when selecting microbial control agents?

Ideally, agents should be Inexpensive, Fast-acting, Stable during storage and capable of controlling microbial growth while being harmless to humans, animals, and objects

What can happen when there is alteration of cell walls and membranes?

If wall is disrupted by chemical or physical agents, it no longer protects cell from bursting. Damage to cell's membrane allows cellular contents to leak. Cells burst due to osmotic effects when damaged. Nonenveloped viruses have greater tolerance of harsh conditions

What is the ultimate means of sterilization?

Incineration is ultimate means of sterilization

What can ionizing radiation for?

Increase shelf life of food achieved by Radura Kill microbes and cells of fruit so that the fruit doesn't spoil.

What are considered Intermediate -level disinfectants?

Intermediate-level kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria. place in mucous membrane, such as respiratory, endospore, colonoscopy

How does oxidizing agents kill?

Kill by oxidation of microbial enzymes

What are considered low -level disinfectants?

Low-level kill vegetative bacteria, fungi, protozoa, and some viruses. Top of skin, electrodes of EKG.

Thermal Death Point

Lowest temp that kills all cells in a broth in 10 mins.

What is the relative susceptibility of microorganisms?

Microbes fall along a continuum from most susceptible to most resistant to antimicrobial agents.

Gaseous agents

Microbicidal and sporicidal gases used in closed chambers to sterilize items Denature proteins and DNA by cross-linking functional groups • Sporicidal activity is through alkylation of terminal hydroxyl, carboxyl, amino, and sulfhydryl groups Used in hospitals and dental offices to sterilized heat sensitive items

Ways to use alcohol?

More effective than soap in removing bacteria from hands - Swabbing of skin with 70% ethanol prior to injection

Least the agents from most resistant to most susceptible.

Most Resistant • Prions • Bacterial endospores • Mycobacteria • Cyst of protozoa • Active-stage protozoa (trophozoites) • Most Gram-negative bacteria • Fungi • Non-enveloped viruses • Most Gram-positive bacteria • Enveloped viruses Most Susceptible

How is refrigerating and Freezing used as a microbial control method?

Most convenient method in food preparation and storage (0-70C) Decrease microbial metabolism, growth, and reproduction because chemical reactions occur more slowly at low temperatures. Psychrophilic microbes can multiply and spoil food. Halts growth of mesophiles.

How can In-Use Test be used to evaluate efficacy of disinfectants and antiseptics?

Most realistic (Considering wide range of microbes at differing conditions) Swabs are taken from objects, before and after application of disinfectant or antiseptic. Swabs inoculated into growth media & examined for physical growth. Allows for more accurate determination of proper strength and application of agent in specific situations.

Halogens

NOT STERILIZING Iodine, chlorine, bromine, and fluorine. - Believed to damage enzymes via oxidation or by denaturation and to precipitate proteins Presumably denature proteins. Intermediate. Disinfectants, antiseptics, and water purification.

What factor does organic material play?

Organic material can interfere with the penetration of heat, chemicals, and some form of radiation Some organic material can also inactivate chemical disinfectants. Examples of organic material - Fat - Feces - Vomit - Blood - Biofilms

LIst the phenolics

Orthocresol (1 ring) Orthophenylphenol Triclosan Hexachlorophene

What phenolics are bispehnolics?

Orthophenylphenol Triclosan Hexachlorophene

What are possible sites?

Penetrate barriers: cause significant damage if not sterilized. Ex: needle, syringe , scalpel Surface of barriers: not sterilized. electrode of EKG, furniture, bed sheets.

What is peracetic good for?

Peracetic acid is effective sporocide used to sterilize equipment

What is the definition of microbial death?

Permanent loss of reproductive ability under ideal environmental conditions.

Oxidizing Agents

Peroxides, ozone, and peracetic acid. Denatures proteins by oxidation. High-level disinfectants and antiseptics for deep wounds, water purification, *sterilization* of food-processing and medical equipment.

What is sanitization?

Process of disinfecting places and utensils used by the public to reduce the number of pathogenic microbes to meet accepted public health standards. Public health term. Removal of pathogens from objects to meet public health standards

What is the effect of refrigeration?

Refrigeration halts growth of most pathogens (predominantly mesophiles); exceptions Listeria and Yersinia

What is degerming?

Removal of microbes from surface by scrubbing. Action of thoroughly scrubbing surface is more important than any chemical used (soap) for removing microbes. Mechanical means.

How can Use-Dilution Test be used to evaluate efficacy of disinfectants and antiseptics?

Researchers dip several metal cylinders into broth cultures of bacteria and briefly dries them at 37C. Immerses each contaminated cylinder into different disinfectants being evaluated for 10 mins. Placed into incubator for 48 hours. Most effective agent is the one that entirely prevents microbial growth at highest dilution. Current standard test, although developed decades ago and is *unrealistic*

What is most effective freezing method?

Slow freezing more effective than quick freezing in inhibiting microbial metabolism

Surfactants

Soaps and detergents. Decrease surface tension of water and disrupt cell membranes. Low. Soaps: degerming; Detergents: antiseptics.

Examples of sanitization?

Standards of sanitization varies among governmental jurisdictions Examples: washing tableware in scalding water in restaurants

What is Radiation?

Sterilizing method Particulate: high speed subatomic particles (electrons) that have been freed from atoms. Electromagnetic: energy without mass traveling in waves at the speed of light released from atoms that have undergone internal changes. Shorter the wave length, the more energy it carries and farther it can penetrate.

What can non-ionizing radiation be used for?

Suitable for disinfecting air, transparent fluids, and surfaces of objects

How is the efficacy of an antimicrobial agent to calculated?

The efficacy of an antimicrobial agent is evaluated by calculating the microbial death rate - Decimal Reduction Time (D value)

How is lyophilization used as a microbial control method?

combining freezing and drying, to preserve microbes and other cells for many years. Instantly freeze a culture in liquid nitrogen or frozen carbon dioxide and then vacuum which removes frozen water through process called sublimation (water transformed from solid to gas) Long term storage of bacterial cultures. Prevents formation of damaging ice crystals

What are the two best-known aldehydes?

formaldehyde and glutaraldehyde Can be used as *sterilants* or high-level disinfectants • Formalin used in embalming and disinfection of rooms and instruments - It is bacteriostatic at low concentrations and bactericidal at high concentrations • Glutaraldehyde is less toxic than formaldehyde

How is Osmotic Pressure used as a microbial control method?

high concentrations of salt of sugar in food to inhibit growth by osmotic pressure. (movement from higher water concentration to lower water concentration) Removal of water inhibits cellular metabolism because enzymes are fully functional only in aqueous environments.

How is boiling used as a microbial control method?

not sterile Kills vegetative cells of bacteria and fungi, prophozoites of protozoa, and most viruses within 10 minutes at sea level. Boiling time is a critical factor. Different elevations require different boiling times

How is pasteurization used as a microbial control method?

not sterilize Used for milk, ice cream, yogurt, and fruit juices Heat-tolerant microbes survive Pasteurization of milk » Batch method » Flash pasteurization » Ultrahigh-temperature pasteurization

How is filtration used as a microbial control method?

passage of a fluid (either liquid or gas) through a sieve designed to trap particles- cells or viruses and separate from the fluid.

9 major categories on antimicrobial chemicals

phenols, alcohols, halogens, oxidizing agents, surfactants, heavy metals, aldehydes, gaseous agents and antibiotics

What are the Four levels of safety?

• Biosafety Level 1 (BSL-1) • Biosafety Level 2 (BSL-2) • Biosafety Level 3 (BSL-3) • Biosafety Level 4 (BSL-4)

What are some disadvantages of gaseous agents?

• Can be hazardous to people • Damage viable tissues • Extremely poisonous • Potentially carcinogenic • Often highly explosive

What are the physical methods of microbial control?

• Extremes of heat • Extremes of cold • Desiccation • Filtration • Osmotic pressure • Radiation

How is Ultrahigh-temperature sterilization used as a microbial control method?

140ºC for 1 sec, then rapid cooling Treated liquids can be stored at room temperature

Why is mycobacteria so resistant?

60% mycolic acid which prevents dehydration. Blocks penetration of water based chemicals

Which alcohol is better 70% or 90%? and why?

70% is the better one because 90% evaporates too fast.

How can a straight line be observed when looking at microbial death rate?

A straight line is obtained when log survivors are plotted against time

How is autoclaving used as a microbial control method?

Achieving true sterilization using heat requires higher temperatures than that of boiling water. Pressure is applied to boiling water to prevent the escape of heat in steam. Applying pressure succeeds in achieving sterilization is that the temperature at which water boils and steam is formed increases as the pressure increases. Autoclave conditions - 121-132ºC, 15 psi, 15 min

What are Chemical Methods of Microbial Control?

Affect microbes' cell walls, cytoplasmic membranes, proteins, or DNA • Effect varies with differing environmental conditions • Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa

Heavy Metals

Arsenic, zinc, mercury, silver, copper etc. Denature proteins. Low-level bacteriostatic Fungistats in paints, silver nitrate creams: surgical dressings, burn creams, and catheters; Thimerosal (merthiolate) used to preserve vaccines copper: algicide in water reservoirs, swimming pools, and aquariums.

Why is bacterial endospores so resistant?

Bacterial endospores can survive 20 years in 70% alcohol dehydration.

What are the differences in susceptibility to antimicrobial agents?

Thermal death point - lowest temperature that kills all cells in broth in 10 minutes z value - temperature increase needed to change the D value by a factor of 10 Thermal death time - required time to sterilize volume of liquid at set temperature

What is unique about antisepsis agents?

These agents are also disinfectants, whose strength have been reduced to make them safe for living tissue, i.e., iodine, alcohol

Decimal Reduction Time

Time required to destroy 90% of the microbes in a sample, concept useful to food processors because they must heat foods to eliminate the endospores of Clostridium botulinum which could germinate to produce botulism toxin inside cans.

Thermal Death Time

Time that it takes to completely sterilize a particular volume of liquid at a set temperature.

What effect does UV have on radiation?

UV light causes pyrimidine dimers in DNA UV light does not penetrate well

What are the other UVs?

UV-A : 315 - 400nm UV-B : 280 - 315nm

Which UV is the most damaging?

UV-C with wavelength 100-280, 100% blocked by ozone layer. Can also be used a germicide

What is pasteurization?

Use of heat to destroy pathogens and reduce the number of spoilage microorganisms in food and beverages without ruining food. Not sterilization. Thermoduric and thermophilic prokaryotes survive. Only reduces amount of microorganisms so that food can be stored longer.

How halogens used in numerous applications?

Used extensively as disinfectants (iodine compounds are the most effective halogen disinfectants) • Iodine tablets, iodophores (iodine complexed with carriers), chlorine treatment, bleach, chloramines, and bromine disinfection • Activity of iodine is reduced by the presence of organic material

What is the only exception to when something must be sterilized?

When a person is immunocompromised.

What are Quats?

a type of surfactant • Low-level disinfectants • Ideal for many medical and industrial application

What is aseptic?

an *environment* or *procedure* that is *free* of contamination by pathogens resulting from sterilization. Standardized aseptic techniques, such as preparation of a surgical field, handwashing, flame sterilization of laboratory equipment