Sterilization and Disinfection

Sterilization vs. Disinfection

• Sterilization eliminates all living microorganisms, including vegetative and spore states.
• Disinfection kills or removes most vegetative microorganisms, but not spores, from non-living objects.

Related Terms

• Antiseptics are chemical agents safe for use on living tissue, preventing infection by inhibiting bacterial growth.
• Aseptic or Asepsis refers to preventing contamination of a person, object, wound, or tissue from microbes.
• Bactericidal agents kill bacteria, and can also be classified as germicides, fungicides (kill fungi), and virucides (kill viruses).
• Bacteriostatic agents prevent or stop bacterial growth and can also be fungistatic (prevents fungal growth) or virustatic (prevents viral growth).
• Contamination is the presence of living microbes on an object.
• Decontamination is the process that frees an article or area from microorganism contamination.

Objective of Sterilization

• The goal of sterilization is to remove or kill microorganisms.

How to Kill Bacteria

• Bacteria can be killed by:
  • Denaturing proteins using dry heat or ethylene oxide.
  • Oxidation using dry heat or hydrogen peroxide.
  • Taking them out via filtration.
  • Interrupting DNA synthesis using radiation.
  • Interfering with protein synthesis using bleach.
  • Disrupting cell membranes using phenols.

Historical Context

• 1862: Louis Pasteur developed pasteurization.
• 1867: Joseph Lister used carbolic solution spray on patient wounds.
• 1876: Charles Chamberland developed the first pressure steam sterilizer (autoclave).

Survival of Pathogens on Surfaces

• Pathogen survival times on surfaces vary:
  • MRSA can survive for 7 days to 7 months.
  • Acinetobacter can survive for 3 days to 5 months.
  • Mtb can survive for 1 day to 4 months.
  • C. difficile spores can survive for 5 months.
  • Norovirus can survive for 12-28 days.
  • HIV can survive for minutes to hours.
  • HBV can survive for 7 days.
  • HCV can survive for 16 hours to 4 days.
  • Covid-19 can survive for 3 hours to 7 days.

Order of Resistance

• Microorganisms' resistance to disinfection and sterilization ranges from easiest to hardest to kill:
  • Enveloped viruses
  • Gram- bacteria
  • Gram+ bacteria
  • Fungi
  • Non-enveloped viruses
  • Mycobacteria
  • Cysts of parasites
  • Spores
  • Prions

Sterilization and Disinfection Methods

• Physical Methods:
  • Sunlight
  • Drying
  • Dry heat
  • Moist heat
  • Filtration
  • Radiation
  • Ultrasonic and sonic vibrations
• Chemical Methods:
  • Alcohols
  • Aldehydes
  • Dyes
  • Halogens
  • Phenols
  • Surface-active agents
  • Metallic salts
  • Gases (ethylene oxide, formaldehyde, beta-propiolactone).

Choice of Method

• Method selection depends on:
  • Device's intended use.
  • Risk of infection.
  • Degree of soiling.
  • Ensuring the process does not damage the device.

Physical Methods of Sterilization

• Heat (dry and moist)
• Radiation
• UV light
• Ionizing and non-ionizing radiation
• Filtration

Dry Heat Sterilization

• Considered the most reliable sterilization method.
• Mechanism: protein denaturation, oxidative damage, and toxic effects from elevated electrolyte levels.
• Types:
  • Flaming: Bunsen flame used until the object is red hot.
  • Uses: sterilization of inoculation loops and wires.
  • Incineration: very high temperature (600 to 1000°C) used for surgical and tissue waste.
  • Dry Heat Sterilization (Hot air):
    • Requires hot-air ovens.
    • Used for glassware, metallic items, powders, and oil/grease.
    • Two hours at 160°C, one hour at 170°C, or 30 minutes at 180°C.
    • Plastics, rubber, paper, and cloth cannot be used due to fire risk.
  • Advantages:
    • Suitable for powders and anhydrous oils.
    • Inexpensive.
    • No corrosive effect.
  • Disadvantages:
    • High temperatures can damage some items.
    • Slow, uneven heat penetration.

Moist Heat Sterilization

• Employs hot water.
• Kills microorganisms by denaturing and coagulating proteins, offering better penetration than dry heat.
• Temperature should be below 100°C for some applications.
• Pasteurization:
  • Inactivates harmful organisms in milk but doesn't achieve sterilization.
  • Kills only vegetative bacteria, not spores.
  • Coxiella burnetiid may survive.
  • Holder method: 63°C for 30 minutes; flash method: 71.7°C for 15-20 seconds.

Moist Heat - Temperature at 100°C

• Boiling:
  • Common in domestic settings.
  • Kills most vegetative bacteria in minutes, but spores need prolonged time.
• Tyndallisation:
  • Named after John Tyndall.
  • Reduces activity of sporulating bacteria left after boiling.
  • Involves first exposure to kill vegetative bacteria, followed by favorable incubation and re-boiling.
• Water bath and vaccine bath:
  • Water bath uses 56°C for 1 hour, and vaccine bath uses 60°C for 1 hour.
  • Used for serum and vaccine preparation.
• Temperature above 100°C:
  • Steam under pressure: a common and effective sterilization method.

Moist Heat - Steam Under Pressure (Autoclaving)

• Standard sterilization method in hospitals.
• Works on the same principle as a pressure cooker, boiling water at increased atmospheric pressure.
• Increased pressure raises the boiling point of water above 100°C.
• The autoclave is a double-walled chamber where air is replaced by saturated steam under pressure.
• Air is evacuated, chambers are filled with saturated steam, tightly closed and pressure gradually increases.
• Applications:
  • Sterilizes materials not damaged by steam.
  • All solid and liquid media.
  • Distilled water, saline solution.
  • Lab coats, swabs, syringes, needles, surgical instruments, and dressing material.

Moist Heat - Autoclaving (Temperature and Pressure)

• Typically achieves 121°C at 15 pascal (Pa) pressure with 15-20 minute exposure time.
• Increasing the temperature further reduces sterilization time.

Monitoring of Autoclaves

• Physical Monitoring: use of thermocouples for accurate temperature measurement.
• Chemical Monitoring: uses heat-sensitive chemicals that change color at the correct temperature and exposure time (e.g., Autoclave tape, Browne's tube).
• Biological Monitoring: Bacillus stearothermophilus spores are added during sterilization and cultured afterward to confirm they have been killed.

Radiation Sterilization

• Non-ionizing Radiation:
  • Includes infrared and UV light.
  • Limited sterilizing power due to poor penetration, and is non-sporicidal.
  • Used in air irradiation in areas like operating rooms and TB labs.
• Ionizing Radiation (Cold Sterilization):
  • Includes X-rays, cosmic rays, and gamma radiation (Cobalt 60 source).
  • Has greater energy than UV light and is sporicidal, making it more effective.
  • Mainly used in industrial facilities for sterilizing cannulas, cardiac implants, pacemakers, disposable plastic syringes, gloves, specimen containers, and Petri dishes.

Filtration

• Helps remove bacteria from heat-sensitive liquids like sera, sugar solutions, or antibiotics.
• Can be done under negative or positive pressure.
• Types of Filters:
  • Candle filters, asbestos filters, sintered glass filters, and membrane filters.
  • Membrane filters made from cellulose acetate are considered the best.
• Removes most bacteria, but viruses and small bacteria may pass through.
• Used mainly for heat-labile substances like sera and antibiotics.
• Recommended filter size to exclude the smallest bacterial cells: 0.22 micron.

Chemical Agents

• Includes both antiseptics and disinfectants.
• Ideal properties:
  • Wide spectrum activity.
  • Effective in both acidic and alkaline mediums.
  • Speedy action.
  • High penetration power.
  • Stable.
  • Non-corrosive to metals.
  • Does not cause local irritation.
  • Non-toxic.
  • Inexpensive, easily available, safe, and easy to use.
• Activity depends on:
  • Contamination level.
  • Disinfectant concentration.
  • Temperature.
  • Time.
  • Range of action.

Alcohols

• Commonly Used: ethyl (ethanol) and isopropyl alcohol.
• Primarily used as skin antiseptics.
• Action: denaturing bacterial proteins.
• Not sporicidal.
• Effective concentration: 60-90%, mostly 70% use.
• Isopropyl alcohol is used for disinfecting clinical thermometers.
• Methyl alcohol is effective against fungi and used for treating cabinets and incubators.
• Methyl alcohol vapors are toxic and inflammable.

Aldehydes

• Action: combines with nucleic acids and proteins, inactivating them.
• Sporicidal activity and are also lethal for viruses.
• Formaldehyde and glutaraldehyde are used to preserve anatomical specimens.
• Can destroy anthrax spores in hair and wool.
• Formaldehyde gas: used for sterilizing instruments and heat-sensitive catheters and for fumigating wards and labs.
• It is an irritant and toxic when inhaled.

Dyes

• Aniline and acridine dyes are used as skin and wound antiseptics.
• Aniline Dyes (brilliant green, malachite green, and crystal violet):
  • More effective against gram-positive bacteria.
  • Ineffective against tubercle bacilli.
  • Non-toxic and non-irritant.
• Acridine Dyes: More active against gram-negative bacteria.
• Halogens: Includes iodine and chlorine.
• Iodine: used in aqueous and alcoholic solutions as a skin disinfectant, bactericidal, and effective against spores, Mtb, and viruses.
• Chlorine: used to disinfect water supplies, swimming pools, and in the food industry.
• Other chemical agents: Phenol (Lysol and cresol), hydrogen peroxide, and potassium permanganate.

Gases

• Common Gases: ethylene oxide, formaldehyde gas, beta-propiolactone (BPL), and hydrogen peroxide fogging.
• Ethylene Oxide:
  • Highly penetrating, inflammable, and explosive at normal temperature and pressure.
  • Mixing with inert gases (CO2 or nitrogen) to 10% concentration eliminates its explosive tendency.
  • Used for sterilizing heart-lung machines and respirators.

Effectiveness of Antiseptics and Disinfectants

• High-Level Germicides: kill vegetative cells, fungi, viruses, and endospores, leading to sterilization with extended use.
• Intermediate-Level Germicides: less effective against endospores and certain viruses.
• Low-Level Germicides: kill only vegetative cells and certain enveloped viruses, and are ineffective against endospores.

Classification of Disinfectants

• Sterilants: Destroy all microorganisms, including high numbers of bacterial spores (e.g., glutaraldehyde, hydrogen peroxide). Use: Heat-sensitive reusable items, immersion only.
• High-Level Disinfectants: Destroy all microorganisms but not necessarily high numbers of bacterial spores (e.g., glutaraldehyde, hydrogen peroxide). Use: Heat-sensitive reusable items, immersion only.
• Intermediate-Level Disinfectants: Destroy vegetative bacteria, most fungi, and most viruses; inactivates Mycobacterium tuberculosis var. bovis (e.g., chlorine-based products, phenolics, iodophors, quaternary ammonium compounds with alcohol). Use: Clinical contact surfaces, noncritical surfaces with visible blood.
• Low-Level Disinfectants: Destroy vegetative bacteria, some fungi, and some viruses; do not inactivate Mycobacterium tuberculosis var. bovis (e.g., quaternary ammonium compounds). Use: Housekeeping surfaces, noncritical surfaces without visible blood, clinical contact surfaces.

Testing Disinfectant Effectiveness

• Rideal-Walker Test: Disinfectant effectiveness is compared to phenol's ability to kill Staphylococcus aureus and Salmonella enterica serovar Typhi, calculating a phenol coefficient.
• Disk-Diffusion Method: Tests a chemical disinfectant’s effectiveness against a specific microbe.
• Use-Dilution Test: Determines a disinfectant’s effectiveness on a surface.
• In-Use or Kelsey-Sykes Tests: Determines if disinfectant solutions are used correctly in clinical settings.

Spaulding's Classification

• The Spaulding classification serves as the main component of infection control in healthcare settings, classifying devices used on patients.
• It determines the appropriate method for disinfection or sterilization.
• Categories:
  • Critical items
  • Semi-critical items
  • Non-critical items

Management of Contaminated Reusable Items

• Should be handled as little as possible.
• Staff should wear appropriate PPE.
• Gross debris should be removed at the point of use.
• Soiled items should be removed immediately after use.

Cleaning Instruments

• Soak in enzymatic or nonenzymatic detergent.
• Wear appropriate PPE.
• Keep instruments submerged in solution and scrub with a brush.
• Thoroughly rinse the instruments.
• Allow instruments to dry.

Fumigation

• For rooms contaminated with pathogens like MRSA and Clostridium difficile.
• Release of hydrogen peroxide, chlorine dioxide gas, or ozone in sealed rooms.
• Requires special equipment.
• Presents a risk of damage to sensitive items.

Plasma Sterilization

• New method for heat-sensitive articles.
• Plasma is the fourth state of matter, consisting of ions, electrons, or neutral particles.
• Radio-frequency energy applied creates an electromagnetic field.
• Highly reactive/charged particles from hydrogen peroxide are generated under vacuum.
• Can be used to sterilize heat- and moisture-sensitive items:
  • Some plastics
  • Electrical/electronic devices
  • Corrosion-susceptible metal alloys
• Special wrapping is required.