Sterilization and Disinfection Techniques

Sterilization, Disinfection & Antiseptic

• Microorganisms cause contamination, infection and decay
• Early civilizations used methods like salting, smoking, pickling and sunlight exposure to reduce microorganisms
• In the mid-1800s, Lister developed aseptic techniques to prevent contamination of surgical wounds
• Before this development, nosocomial infections caused death in 10% of surgeries and up to 25% of mothers delivering in hospitals died due to infection
• Methods of microbial growth control include alteration of membrane permeability, damage to proteins and damage to nucleic acids
• Different microorganisms have varying resistance to different methods

Definition of Sterilization

• Sterilization is the process of removing all microorganisms, including spores, from an item
• Sterilization is necessary before surgery

Methods of Sterilization

• Sterilization is usually done by autoclaving, which involves exposing materials to steam at 121°C under a pressure of 15 lb/in² for 15 minutes
• Surgical instruments that are damaged by heat can be sterilized by ethylene oxide gas
• Many intravenous solutions are sterilized by filtration

Definition of Disinfection

• Disinfection kills many, but not all, microorganisms
• Chemicals used to kill microorganisms on skin and mucous membranes are called antiseptics

Terminology

• Sterilization destroys all viable microbes, including viruses and endospores
• Disinfection destroys vegetative pathogens, not endospores (in inanimate objects)
• Antiseptics are disinfectants applied directly to exposed body surfaces
• Sanitization is a cleansing technique that mechanically removes microbes
• Degermation is the mechanical removal of microbes from a surface
• Aseptic refers to the absence of significant contamination
• A bactericidal agent kills bacteria, whilst a bacteriostatic agent inhibits their growth and multiplication

Bacteriostatic Agent and Bactericide

• A bacteriostatic agent inhibits bacterial growth but doesn't necessarily kill them
• A bactericide agent kills bacteria. Most do not kill endospores

Aim of Sterilization

• Asepsis is a practice that minimizes or eliminates contaminants like bacteria, viruses, fungi, and parasites in surgical or medical procedures to prevent infection
• Ideally, a sterile field is free of contaminants but difficult to achieve
• The goal of sterilization is to eliminate infection, not necessarily sterility

Methods of Sterilization

• Methods of sterilization include physical agents (e.g., heat, radiation) and chemical agents (e.g., gases, liquids) as well as mechanical methods of removal

Physical Agents

• Heat methods: incineration, dry oven, autoclaving (steam under pressure) , boiling water, pasteurization
• Radiation methods: ionizing (gamma rays, X-rays), non-ionizing radiation (UV)

Chemical Agents

• Gases: Ethylene oxide, Formaldehyde
• Liquids: Chemotherapy agents , Antiseptics

Mechanical Removal Methods

• Filtration (air, liquids)

Physical Methods of Sterilization (Examples)

• Hot Air Oven: Sterilizes items using oxidation, operated between 160°C and 250/300°C, with a holding period of 160°C for 1 hour for optimal results.
• Flaming: Used to sterilize inoculation loops and forceps through the use of a Bunsen burner

Incineration

• A method for destroying contaminated materials, animal carcasses, or pathological materials

Moist Heat Sterilization

• Kills microorganisms by coagulating their proteins.

Moist Heat Sterilization Methods

• Methods include: Temperature below 100° C (pasteurization, inspissator), 100° C (boiling), Steam at atmospheric pressure (Koch/Arnold's steamer), and Steam under pressure (autoclave).

Pasteurization

• Killing of pathogens in milk, not necessarily sterilizing it. Methods involve heating (Holder method) at 63°C for 30 minutes or (flash process) at 72°C for 15-20 seconds, followed by rapid cooling to 13°C

Water Bath

• Used to inactivate non-spore-forming bacteria.
• Sterilizing serum or body fluids with coagulable proteins.

Inspissator

• Sterilizes by heating to 80-85°C for half an hour for 3 consecutive days.
• Used to sterilize media, such as Lowenstein-Jensen and Loeffler's serum

Boiling

• Kills vegetative forms of bacterial pathogens (at 100°C).
• Some viruses can survive up to 30 minutes, and endospores can survive up to 20 hours.

Steam at Atmospheric Pressure

• Uses a steamer (e.g., Koch/Arnold), with a tin cabinet, conical lid for condensate drainage, and perforated tray to surround materials in steam
• Routine sterilization involves 90 minutes of exposure

Tyndallisation/Intermittent Sterilization

• Used for media containing sugar and gelatin
• 100°C exposure for 20 minutes for 3 successive days

Steam under Pressure (Autoclave)

• Invented by Charles Chamberland in 1879
• It works by utilizing steam under pressure.
• Essential parts include: exhaust valve, steam chamber, safety valve, pressure gauge, and operating valve

Radiation

• Two types are used (ionizing, non-ionizing)

Non-Ionizing Radiation

• Absorbed as heat
• Useful for rapid mass sterilization
• Examples include UV rays

Ionizing Radiation

• High penetrative power
• Does not cause an appreciable temperature increase
• Used for sterilizing plastics, syringes, catheters, fabrics, or metal foils

Ultrasonic and Sonic Vibration

• Microorganisms have varying sensitivity

Chemical Agents

• Act by protein coagulation, disrupting cell membranes, removing sulfhydryl groups, or substrate competition

Principles of Effective Disinfection

• Disinfectant concentration, organic matter removal, pH, and time

Alcohols

• Ethanol/Isopropyl alcohol; frequently used as disinfectants
• No action on spores
• Concentration is typically 60-90% in water
• Used for clinical thermometer disinfection and skin disinfection (venipuncture)
• 20 minutes application is enough for most viruses

Aldehydes

• Formaldehyde & Glutaraldehyde
• Formaldehyde is bactericidal, sporicidal, and lethal to viruses
• Glutaraldehyde is effective against mycobacteria, fungi, and viruses

Dyes

• Aniline dyes (e.g., Brilliant green, Malachite green, Crystal violet) are active against gram-positive bacteria
• Acridine dyes (e.g., Proflavine) are effective against gram-positive bacteria

Halogens

• Iodine (in aqueous and alcoholic solutions): widely used as a skin disinfectant
• Actively bactericidal with moderate spore activity
• Chlorine: used as a disinfectant in water

Phenols

• Obtained by coal tar distillation
• Powerful microbicidal agents
• Used in many hospital disinfectants (e.g., Lysol, cresol, Detol)

Uses

• Various combinations used in the control of pyogenic cocci in surgical neonatal units and aqueous solutions for wound treatment

Hydrogen Peroxide

• Used as an antiseptic to clean wounds and disinfect contact lenses
• Effectiveness limited by the organism's ability to produce catalase
• Oxidizing agent that attacks sulfhydryl groups, inhibiting enzymatic activity

Gases

• Ethylene Oxide: colorless, penetrating gas with a sweet, ethereal odor
• Effective against all microorganisms –viruses and spores
• Used in sterilizing heart-lung machines/ respirators and dental equipment.

Formaldehyde Gas

• Widely employed for the fumigation of operation theaters

Beta Propiolactone

• Used in fumigation.
• 0.2% BPL for sterilization and for rapid biocidal activity
• Very effective against viruses

Sterilization by Filtration

• Used to remove bacteria from heat-labile liquids such as sera and solutions of sugar, antibiotics
• Methods include: Candle filters (unglazed ceramic filter, diatomaceous earth filter), asbestos filter, sintered glass filter, membrane filters

References

• Principles and Methods of Sterilization in Health Sciences (2nd Edition).
• Jawetz Melnick & Adelbergs Medical Microbiology
• Review of Medical Microbiology and Immunology (Fourteenth Edition)