STERILIZATION

Definition:

The freeing of an article from all living organisms, including viruses, bacteria , spores & fungi: Pathogenic & non-pathogenic.

Uses

Sterilization is used for :

1.Microbiology : Culture media, suspending fluids, reagents, containers & equipment

2.Hospital : medical & surgical instruments , surgical operations, I.V. infusions, hypodermic injections , diagnostic aspirations.

MethodsPhysical or chemical:

(1) Physical Methods:

• Heat • Filtration• Irradiation

(2) Chemical Methods:

@ Strong disinfectants:

* Formaldehyde * Ethylene oxide

Sterilization By Heat

Moist Heat:

@ Most effective and efficient. @ Kills organisms by denaturing their enzymes & proteins @ Spores are killed by exposure to moist heat at 121°C for 10-30 minutes.

Dry Heat:

@ Kills organisms by oxidative destruction of cell constituents. @ Spores killed at 160°C for 1 hour

Factors influencing Sterilization by Heat:

1.Temperature & exposure time: Higher temperature, shorter time

and vice versa.

2.No.of Vegetative cells + spores

@ Plenty of organisms, make sterilization less efficient.

@ So, clean before sterilizing

3. Nature of organism:

Vegetative cells & spores affect susceptibility of

organism to heat.

4. Nature of material containing organisms

@ Efficiency of sterilization is reduced by presence of organic matter that protect bacteria from lethal action of heat

@ Presence of a disinfectant enhances the kill.

@ Spores best killed in acidity or in alkalinity.

Holding Time:

Time required for killing organism. (Not including heating-up time).

Thermal Death Point:

Lowest temperature to give complete killing in aqueous suspension within 10 min. at standard conditions.

Thermal Death Time:

Shortest time for complete killing at a stated temperature under standard conditions.

Decimal Reduction Time (D-value):

Time in minutes required to achieve a ten fold reduction in viability of a bacterial population at a given temperature under standard conditions.

Methods of Sterilization By Dry Heat:

1.Red Heat:

Article is held on flame until red hot (inoculating wires, forceps, spatulas)

2.Flaming:

Burning an article in spirit or gas flame (scalpels, needles)

Method will not produce sterilization.

3. Hot-air oven:

@ Oven has a thermostat + fan to circulate hot air

@ Works at 160˚C for 1 hour, to sterilize glassware, metals , swabs, oils,

powder

4. Infra-red Radiation:

@ Given by an electrically heated element

@ To sterilize glass syringes at 160°C & surgical instruments above 200°C in vacuum chamber in which N2 is passed during cooling time to avoid oxidation.

Methods of Sterilization by Moist Heat:

@ Employed at a temp. below 100°C, at 100˚C, and above 100°C.

@ First two are disinfection methods, third is a sterilization method.

Temperature Below 100°C:

@ This is the pasteurization process.

@ Used for milk, vaccines & utensils.

Milk:

@ Pasteurized by 2 methods:-

1.Holder method: at 63°–66°C for 30 min.

2. Flash method: at 72°C for 20 seconds.

@ Both methods destroy only milk-borne organisms (Myco., Brucella, Salmonella)

Pasteurization is tested by :-

@ Phosphatase test : testing for presence of phosphatase enzyme found in milk and destroyed by pasteurization.

@ Methylene blue test : To indicate that bacteria present in milk is destroyed.

Coxiella burneti is destroyed by flash method.

Vaccines:

Inactivated in the vaccine water bath, at 60°C for 1 hr

House articles:

Utensils, clothing, bedding, are disinfected at 70°– 80°C for several minutes.

Temperature at 100°C:

Boiling at 100°C:

@ Kills non-sporing bacteria within 5–10 min.

@ Used to disinfect blades, syringes,

@ Dry up articles on removing from boiler (sterilizer) to prevent contamination

Steaming at 100°C:

@ This is done by the steamer

(Koch Steamer)

@ It uses steam of boiling water at 100°C and at atmospheric pressure.

@ Steamer is used in two ways:-

1. Single exposure at 100°C for 90 minutes.

@ Time includes heating-up time.

@ Thermophilic & mesophilic

spores will survive this treatment.

2. Tyndallization:

Exposure at 100°C for 20 – 45 minutes for 3 successive days.

@ Used for sterilizing sugars & gelatin

@ First steaming kills vegetative bacteria and spores germinating following day are killed by subsequent heating and so on.

@ Draw-back of tyndallization : spores not germinating in medium sterilized + thermophilic & anaerobic bacteria will escape killing.

Temperature above 100˚C:

@ Uses saturated steam - better than dry hot air because:-

1.Lethal action of moist heat is more

2. Quicker in :

• heating up exposed particles• penetrating cotton stoppers,

paper,wrappers, surgical linen & hollow

apparatus

How Does it Act?

@ Saturated steam when meets an

article, it condenses to a small

volume of water & liberates its

latent heat to article surface.

@ Avoid presence of air that

prevents steam penetration in article

What is the apparatus Used?

@ Autoclave : provides sterilization by dry saturated steam (steam at point of condensing to water)

@ Steam is under pressure higher than atmospheric.

Importance of Air Discharge:

@ Air is removed from autoclave: a) Mixture of steam + air lower

the temperature. b) Air hinders penetration of

steam in the load.

@ Air denser than steam, sinks down & makes a layer over load

@ Sealed bottles containing solutions

are autoclaved although air is

found in them – WHY?

Because water found in these solutions is heated up to steam temp. and so performing same work as moist heat.

Types of Autoclaves:

1. Simple Autoclave:

@ Pressure-cooker type, simple, not jacketed, & used in laboratory.

@ Consists of a cylinder for water to be heated, articles placed on a tray

@ Has got a discharge valve, + pressure & temperature gauges.

@ Autoclave is opened at right time because: # If opened still under pressure, an explosion occurs

# If opened below atmospheric pressure, evaporation of aqueous materials occurs.

Simple Autoclave Deficiencies :

1. Lacks control of air discharge

(nothing to show that discharge is complete)

2. Lacks means of drying load

after sterilization.

@ Suitable for load wrapped in paper to prevent contamination.

2. Steam-Jacketed Autoclave:

@ Has got an automatic air & condensate discharge.

@ Load is dried up by steam circulating in jacket, & vacuum in autoclave.

3. High Pre-Vacuum Sterilizers:

@ Have electric pumps creating a vacuum area in chamber.

@ First a vacuum is drawn, steam is admitted to chamber, load is heated very rapidly.

@ Temp. used 135˚C for 3 min. at 30 lb pressure & load dried by exhaustion of chamber

Advantages of high pre-vacuum sterilizers:

1.Operation time is shortened.

2. Damage to heat sensitive materials (sugars) is avoided.

Autoclave Control and Indicators:

1. Automatic process control, * Advantages are:-

@ Saving time of an operator.@ Safeguard against errors due

to negligence.

2. Recording Thermometer,

@ Draws graphic records of temperature changes inside chamber discharge channel.

@ Helps to avoid errors in timing the holding period.

3. Thermocouple load temp. measurement:

@ Thermocouple is inserted deeply inside load, its wire is carried to a potentiometer which reads temperature inside the load .

4. Chemical indicators:

a) Browne’s Tubes, containing a red fluid that turns green on heating:

* at 115˚C for 25 minutes (Type 1) * at 115˚C for 15 minutes (Type 2) * at 160˚ C for 60 minutes (Type 3)

@ Tubes are stored below 20˚C to avoid change of color .

@ Satisfactory for routine work .

b) Bowies-Dick tape,

@ Used to test efficiency of high pre-vacuum sterilizers & high-pressure autoclaves

@ An adhesive tape is applied around load in the shape of (X).

@ After sterilization, tape changes color all over .

5. Spore indicators,

@ Bacillus stearothermophilus spores are destroyed at 121˚C for 12 minutes.

@Spores placed within load and cultured after sterilizing

Disadvantages of Chemical and Spore Indicators:

@ Will not give a perfect efficiency of sterilization:

( heating may be inadequate in a site away from of indicator)

* To solve problem, autoclave is correctly operated & controlled by a thermometer & not by pressure gauge alone

Sterilization by Radiation

1. Ultra-violet Radiation:

@ U.V. rays induce thymine diamers in cell DNA, to destroy bacterial cell .

@ Produced by mercury vapor lamps, used to sterilize plastics

2. Ionizing Radiation: @ High-speed electrons, X-ray,

gamma rays, using cobalt 60

@ Produces free DNA radicals that destroy bacteria .

Indicator for Radiation:Micrococcus radiodurans that resistsradiation (has efficient DNA repair)

Sterilization by Filtration

@ Used to sterilize toxins, serum, antibiotics

@ Uses a filter of pore less than 0.75 µm for bacteria, & much smaller for viruses.

@ Some bacterial filters allow viruses, and Mycoplasma to pass through.

Test of Filter:

@ Retain Serratia marcescens from broth culture.

Types of Filters:

1. Asbestos pad (Seitz) filter:

@ Consists of an upper cylinder and a lower funnel with an asbestos pad in between.

Uses of asbestos filter pads :

1.Rapid clarification of fluids.

2. Clarifying viscous fluids.

3. General sterilization.

4. Removing pyrogenes.

5. Removing small organisms.

6. Sterilizing serum.

Disadvantage:

Asbestos pads absorb some of filtrate.

2. Sintered glass filters ;

Made up of ground glass, fused to make glass granules adhere together .

3. Cellulose membrane filters: @ Less absorptive, has got a greater rate of filtration than Seitz filter.

@ Used to separate viruses.

* 2 types of membrane filters: a) Cellulose nitrate( Gradocol membrane) .b) Cellulose acetate: commonly used now.

@ Membrane is 120 µm thick, placed in two layers.

@ Bacteria are trapped in upper layer : better than Seitz filter

Micro-filters:

@ To filter small fluid volumes @ May be membrane, or asbestos @ May be syringe or centrifuge filter.