Appendix E Sterilization

Sterilization definition

complete elimination of all live microorganisms by physical or chemical means

How does dry heat sterilize

by dehydration and oxidation of microorganisms

Temperature for dry heat sterilization

require a much higher temperature than moist heat

What does dry heat sterilization kill

kills spores, mycobacteria, non-lipid viruses, fungi, and bacteria

Sterility check procedure for dry heat sterilization

checked at least once weekly

Dry heat sterilization biological indicators, what is and how

biological indicators containing spores of Bacillus subtilis (used for dry heat) are placed in the centre of the largest part of the load. Then, the spore strip is put in a broth, incubated at 35 degrees for 4-7 days. If there is no growth in the broth, then sterilization was successful

Methods for dry heat sterilization

• flame

• bacti-cinerators

• hot air ovens

How does moist heat sterilize

by coagulation of proteins (critical enzymes) of microorganisms

What does moist heat sterilization kill

kills most microorganisms but does not kill spores (unless steam or autoclave)

Sterility check procedure for moist heat sterilization

checked at least once weekly

Dry heat sterilization biological indicators, what is and how

use biological indicators containing spores of Bacillus subtilis. Once processed, the spore strip is put in broth, incubated in a 35 degree water bath for 4-7 days. If there is no growth, sterilization was successful

Methods for moist heat sterilization

• boiling in water

• steam

• tyndallization

• inspissation

How does steam under pressure (autoclave) sterilize

by coagulation of proteins (critical enzymes) of microorganisms

What does autoclaves kill

kills all microorganisms, including spores

Is it the temperature or pressure of an autoclave that sterilizes

the higher temperature

Sterility check procedure for moist heat sterilization

should be checked at least once weekly

What is the biological indicator for autoclaves

biological indicator containing spores of Geobacillus stearothermophilus (thermophile with spores that have a high heat tolerance)

What happens to the spore strip once autoclaved

the spore strip is put in a broth which is incubated in a 56 degree water bath for 4-7 days. No growth indicates that sterilization was successful

What can be done to each autoclave load, and what does it do

process/chemical indicators; these indicate that the process has occurred

Does process/chemical indicators indicate sterility in autoclaves

no, it is independent of steam, partially dependent on time

Examples of process/chemical indicators of autoclaves

autoclave tape, paper strips with colour change in proper temperatures, etc.

What materials can autoclaves sterilize

used to sterilize any materials that are not damaged by heat or moisture including swabs, rubber gloves, heat stable liquids, culture and most instruments

What are autclaves usually used for

mainly used for sterilization of basic nutrient broths and agar bases in media preparation and contaminated, cultured media prior to discard

Time, temperature, and pressure of autoclaves

• 20 min

• 121 degrees

• 106 kpa

In edmonton, what is the pressure required to raise the temperature to 121 degrees

120 kpa

What steam condition is required for the temperature to reach its max

the steam supplied to the autoclave must be dry and saturated not superheated (contains excess water molecules which lacks latent heat for sterilization). This cannot occur if air remains mixed with steam in the chamber so all air must be expelled from the autoclave

How must steam circulate in autoclave

must circulate around all materials in an autoclave load

How are autoclave efficiency monitored and placed

monitored with biological indicators (placed in the centre of load where sterilization is at least likely attained)

What are the main components of an autoclave

1. an external steam source

2. an outer jacket

3. a discharge line for air and condesate

4. a thermometer at the coolest point of the autoclave chamber

5. a vacuum system for drying of certain loads

6. a control system which performs the preselected cycle of sterilization including heat-up, holding, cooling, and drying

Sources of external steam source

either from a building steam-supply, or electrically generated in a “free-standing” autoclave

What happens if the steam comes from a building supply

a reducing valve and pressure regulator are required

Function of outer jacket

filled with steam, heating independently of the chamber, and thereby preventing excessive condensation. Jacket heat also facilitates drying following sterilization and exhausting of steam from the chamber

The vacuum system is used for what

used for instruments but not for fluids in loosely covered containers

What is filtration (membrane filters) used for

used to remove contaminating bacteria and fungi from solutions which cannot tolerate exposure to the high temperatures required for heat sterilization

Can viruses pass through filtration

yes, they can pass through bacterial filters which generally have pore diameters of 0.75 um or less

How do filters function

function by entrapping micro-organisms within the porous structure of the filter matrix

What kind of pressure is required to move the solution through the filter

negative or positive pressure

What is the most used filter systems in clinical and research labs

membrane filters

What are membrane filters composed of

composed of cellulose acetate or similar cellulose compounds (millipore) which are supported by plastic or glass filter holders

What is the sterility checking procedures for filtration

include incubating the filtrate on enriched media and observing for growth

Are fluids sterilized if filterd and why

not considered biologically sterile as viruses can remain in the solution

Is it a concern if viruses were to remain in the solution

viruses are not able to grow on bacterial media

What is things is filtration used to sterilize

bovine serum, sugars (carbohydrates), urea, toxins, tissue culture, and antibiotic preparations

What is ethylene oxide and how does sterilize

it is a colourless gas that inactivates microorganismms by replacing labile H atoms on hydroxyl, carboxyl or sulfhydryl groups (ex. guanine and adenine of DNA)

What can ethylene oxide effectively kill

all microbial vegetative cells and spores (require longer exposure)

What is the concentration sterilization range for ethylene oxide

250 - 1200 mg of ethylene oxide gas per litre of chamber space

What is the length of ethylene oxide sterilization process dependent on

depends upon the concentration of gas, temperatures, humidity, types of materials to be sterilized, and the degree of contamination

How often should autoclave efficiency with ethylene oxide be checked

at least once weekly

What biological indicator is used with ehtylene oxide and how does it work

Bacillus subtilis. Once autoclaved, the spore strip is put in a broth which is incubated at 35 degrees for 4-7 days Growth indicates that sterilization was not successful

What must happen to the materials to be sterilized with ethylene oxide

must be wrapped to prevent contamination upon removal from gas.

What kind of wrapping material is used for ethylene oxide

gas must be permeable to the wrapping material (foils and plastics not used)

Wht must ethylene oxide be used as a mixture

in its pure form it is explosive, therefore, it is used as a mixture of 10% ethylene oxide with 90% CO₂

What must happen to all items after sterilization with ETO

all items must be aerated after sterilization to remove residual vapours as ETO is toxic and can burn skin (cold burns)

How long are items aerated after sterilization with ETO

minimum of 24 hours and up to 1 week at RT

What happens when heated aerators is used with ETO

decreased time is required (takes 4-8 hours for aeration)

Is ETO cheap

it is a very expensive method for sterilization

When is ETO sterilization used

used in sterilization of items which cannot be sterilized by other means including fibre optic and rubber equipment, heart lung machines, delicate surgical instruments, electronics, mattresses, pillows, plastic toys, books, plastic petri dishes, etc.

What is the wavelength of ultraviolet light

100-400 nm and is not visible to human eye

Wht is ultraviolet irradiation bad for DNA

the shorter wavelengths are absorbed by DNA cells which damages them

What is the maximum absorbance and damage to DNA by ultraviolet irradiation

occurs at 265 nm (range 230-280 nm)

What nm of ultraviolet radiation is used for surface decontamination and to reduce airborne or surface contamination in hospitals

254 nm

Ultraviolet irradiation kills what

spores are killed as readily as vegetative cells

UV efficiency is checked with what biological indicator

Bacillus pumilus

How does the biological indicator for UV work

Bacillus pumilis endospores are resistant to gamma radiation. Once processed, the spore strip is put in a broth which is incubated at 35 degrees for 4-7 days. Growth indicates that sterilization was not successful

Why UV sterilization have limited use

organic matter, dust, or other protecting coatings shield microorganisms from UV radiation (poor penetrating power)

What factors are important in achieving a germicidal effect from UV radiation

distance from UV source and duration of exposure

What protective piece must be used with UV

protective eyewear as UV is damaging to the eye

Is UV harmful to human skin cells

yes, UV radiation is mutagenic and carcinogenic under certain conditions of exposure