STERILIZATION AND DISINFECTION I

Ms. M. Mwinga

CONTENTS

Terminologies Applications Factors affecting efficacy of sterilant /disinfectant Methods of sterilization and disinfectionPhysical methodsChemical methods

• Sterilization: is the freeing of an article from all living organisms including viable spores.

• Disinfection: Is a freeing of an article from harmful microorganisms that are capable of causing infection but does not destroy spores.

• Antiseptics: chemical disinfectants used on skin or mucus membrane; and prevents infection by inhibiting the growth of bacteria. Ex. Alcohol, boric acid, hydrogen peroxide.

• Asepsis: is a process where the chemical agents (antiseptics) are applied on to the body surface (skin), which kill or inhibit the microorganisms present on skin.

• Decontamination: Is the reduction of pathogenic microbial population to a level at which items are considered safe to handle without protective attire.

• Bactericidal agents or germicides: they are chemicals that kill bacteria.• Bacteriostatic agents: agents that prevent the multiplication of

bacteria.

Applications of sterilization

Prevents contamination by extraneous organisms.Used in surgery to maintain asepsis.In food and drug industry; to ensure safety from contaminating

organisms.

Methods of sterilization (classification)

Physical methods. Chemical methods.

Sunlight drying heat filtration radiation

Dry heat Moist Flaming

IncinerationMembrane filters

Depth filters

Hot air oven

Below 100°C

At 100 °C

Above 100 °C

Non-ionizing radiation

Ionizing radiation

Factors affecting the efficacy of sterilant/disinfectant

1. Organism load2. Nature of organism3. Concentration of chemical agents4. Nature of the sterilant/disinfectant5. Duration of exposure6. Temperature7. pH, organic matter and biofilm.

I. SUNLIGHTPossesses bactericidal/germicidal activity.Action is due to Ultraviolet rays and presence of the ozone layer in the

atmosphere.

II. DRYINGDrying has a deleterious effect on many bacteria.Many bacteria requires moisture for their growth.Spores are not affected by drying.

Solar-water purification

III. HEATKills microorganisms by denaturing their enzymes and other proteins.

Factors influencing sterilization by heatNature of heat -dry or moist.Temperature and time.Number of organisms present.Characteristics of the organisms, such as strain capacity.Type of material from which the organisms must be eradicated.

• Mechanism of Action of Heat1. Dry heat: used for glassware, instruments and paper-wrapped articles,

water impermeable oils, waxes and powders.

2. Moist heat: best at lower temperatures, used in autoclaves to sterilize media and other water containing materials.

• The time of sterilization is inversely proportional to the temperature of exposure.

• Thermal death time: is the minimum time required to kill a suspension of organisms at a predetermined temperature in a specific environment.

Dry heat methods• Flaming: used to sterilize items in a flame of a Bunsen burner-Red heat: applied to inoculation loops and wires, forceps and spatula; for a longer time.-Sterilization of the mouth of test tubes is applied for a shorter time.

Sterilizing an inoculating loop by flaming

• Incineration: used to sterilize anatomical wastes and microbiological wastes over high temperatures (870-1200°C) into ash and gas.• Hot air oven: removes all microbes including spores• used for glassware such as tubes, flasks, measuring cylinders, glass

syringes and glass pipettes, and metal instruments. -Requires a temperature of 160-180°C for 120 minutes.

Insulated wall

Trays

Temperature regulator

Indicators of hot air oven

• Physical indicators: thermocouples- measures the temperature.

• Biological indicators: uses spores (106) of non-toxigenic strains of Clostridium tetani or Bacillus subtilis.

• Chemical indicators: Browne’s tube- has a heat sensitive dye which turns green after exposure to certain temperature for a period of time.

Adv: -Do not require water. -less pressure build up. -very efficient & smaller. -more rapid and reaches higher temperatures.

-non corrosive.

Disad: -Time consuming (takes up to 30 to 120 minutes). -high temperatures are not suitable for most materials. -Ex. Prions.

Moist heat• Moist heat kills microorganisms at a lower temperature than dry heat.

Temperature for moist heat1. At a temperature below 100°C.2. At a temperature of 100°C.3. At a temperature above 100°C.

1. At a temperature below 100°Ca)Pasteurization-is a method to control microorganisms from beverages such as fruit and vegetable juices, beer, and dairy products such as milk.-it destroys all non-sporing pathogens. Ex: Mycobacteria, Brucellae, and Salmonellae, except for heat resistant Coxiella burnetii.

Methods of pasteurization• Holder method: done at 63°C for 30minutes.- Ex: HIV in serum at 56°C for 25 minutes.• Flash method: done at 72°C, followed by rapid cooling to 13°C or lower.

b) Water bath• Is a method used for disinfection of serum, body fluids (at 56°C) and

vaccines (at 60°C).

C) Inspissation /fractional sterilization• Process of heating an article on three successive days at 80-85°C for

30minutes by an inspissator. • The method kills all vegetative forms of spores.• It is used to sterilize egg-based media such as Lowenstein-Jensen (LJ)

medium and Dorset’s egg medium, and serum-based media such Loffler's serum slope.

2. Moist heat at 100°C• A) Boiling: kills vegetative bacteria, HBV, and some bacterial pores.

• B) Steaming: steamer is used to sterilize media which decomposes at high temperature of autoclave.• Method is carried out for 90minutes, killing most vegetative forms

except thermophiles and spores.

• C) Tyndallisation or Intermitted sterilization: sterilizes gelatine, egg, serum or sugar containing media for 20 minutes.

3. Moist heat above 100°C (Autoclave)

• Autoclave: is an equipment used to remove microorganisms and spores using high pressure and high temperature.

Principle of Autoclave:• When water boils, its vapour pressure equals that of the surrounding

atmosphere, so when the atm pressure is raised, the boiling temperature is also raised (Boyle’s Law).• Sterilization conditions: 121°C for 15 minutes at a pressure of 15lbs

per square inch (psi), 126°C for 10 minutes at pressure of 20 psi or 134°C at for 3 minutes.

.

Components of autoclave:a. Pressure chamber: is a large cylinder (vertical or horizontal) in

which materials to be sterilized are placed.

b. Steam jacket: it is where water is placed

c. The lid: fastened by screw clamp, rendered airtight by an asbestos washer.

• It has a discharge tap for air and steam discharge, a pressure gauge and a safety valve (to remove excess steam).

d. An electrical heater: heats the water to produce steam.

Mechanism of autoclave • Sufficient water is added to the cylinder, material to be sterilized is placed

on the tray and the autoclave is heated.

• The lid is screwed tight, discharge tap is left open until no more air bubbles come out in the pail. Safety valve is adjusted to the required pressure.

• Steam pressure rises and penetrates objects inside the autoclave

• Condensation creates negative pressure and draws additional steam

• Moist heat kills microorganisms via coagulation of proteins.

A schematic diagram of laboratory autoclave

Types of autoclaves

a. Gravity displacement type.• Cold air escapes through the bottom of the chamber as steam displaces

it from above.• Ex. Vertical type-small volume capacity• and horizontal autoclave-large volume capacity.

b. Positive pressure displacement type autoclave

c. Negative pressure (vacuum) displacement type.

Uses of autoclave

• Sterilization of surgical instruments• Culture media containing water that cannot be sterilized by dry heat• Autoclavable plastic containers• Plastic tubes and pipette tips• Solutions and water• Biohazardous wastes• Glassware

Advantages • Destroys all forms of microbes, including spores.

Disadvantages• Expensive.• Uses more electricity.• Unsuitable for powders and oils, injections and plastic which get

spoiled at autoclave temperatures.

Indicators of autoclave

• Biological indicators: uses spores of Geobacillus stearothermophilus.• Browne's tube and thermocouple.• Autoclave tapes.

IV. FILTRATION METHOD• Filtration: is the removal of microbes by passing of a liquid or gas

through a screen like material with small pores.• Method removes bacteria from heat labile liquids such as sera and

solutions of sugars or antibiotics used for preparation of culture media.

Types of filtersa. Depth filters. b. Membrane filter.

A. Depth filter B. Membrane filter

a. Depth filters• They are porous and retain particles throughout the depth of the filter.• Made of random mats of metallic polymeric or inorganic material.• They rely on the density and thickness of the filter to trap the particles.

Advantages:• Retain large mass of particles before becoming clogged• Have high flow rate of the fluid• Have low cost

Disadvantage: not suitable for filtration of bacterial solutions as some particles still come out in the filtrate.

Examples of Depth filtersi. Candle filtersii. Asbestos filters:iii. Sintered glass filters:

Applications: in industries for filtration of food, beverages and chemicals.

Asbestos filterMembrane filters

Glass filter

b. Membrane filters• Have uniform pore size, retain larger particles on the surface of their

pores.• Made of cellulose acetate, cellulose nitrate, polycarbonate,

polyvinylidene fluoride or other synthetic materials.

• Pore size: 0.22 µm-removes most bacteria, allows viruses to pass through; 0.45 µm-retains coliforms in water microbiology and 0.8 µm-removes airborne microbes in clean rooms and for production

of bacteria free gases.

Filtration of liquid• Is done to sterilize sera, sugar and antibiotic solutions.• Separates toxins and bacteriophages from bacteria.• Obtain bacteria free filtrates of clinical samples for virus isolation.• Water purification.

Filtration of air• Used to deliver bacteria-free air.• Ex. HEPA (High-efficiency particulate air) filters: removes 99.97% of particles

with size of 0.3 µm or more.• Used in ICU.• ULPA (Ultra-low particulate/penetration air) filters: removes from air atleast

99.999% dust, mould, bacteria and airborne particles with size of 0.12 µm or larger.

V. RADIATIONTwo typesa. Ionizing radiation.b. Non-ionizing radiation.

a. Ionizing radiation: includes x-rays, gamma rays and cosmic rays.• Cause breakdown of DNA without high temperature (cold sterilization).• Destroys bacterial endospores and vegetative cells, eukaryotic and prokaryotic

cells.• Ex. Gamma radiation: for sterilization of plastics, syringes, swabs, catheters,

animal feeds, cardboards, oils, fabric and metal foils.• Advantages: it has high penetrative power, rapidity of action and does not require

rise in temperature.

b. Non-ionizing radiation• Includes infra-red and ultraviolet rays.• They are lethal but do not penetrate glass, dirt films and water.

• Ex. Infrared radiation: used for rapid mass sterilization of prepacked items. Ex. Syringes and catheters.• Ultraviolet radiation: used for disinfecting enclosed areas, such as

entryways, operation theatres and laboratories.

THANK YOU