Microbial Control Methods Quiz

Sterilization, Disinfection, and Antisepsis

• Microorganisms are the agents of contamination, infection, and decay
• Early civilizations practiced methods like salting, smoking, pickling, and sunlight exposure to remove microorganisms
• In the mid-1800s, Lister developed aseptic techniques to prevent contamination of surgical wounds
• Prior to this, nosocomial infections caused death in 10% of surgeries and up to 25% of mothers delivering in hospitals died due to infections
• Methods for controlling microbial growth include altering membrane permeability, damaging proteins, and damaging nucleic acids

Learning Outcomes

• Students should be able to define sterilization, disinfection, and antisepsis
• Students should understand methods of sterilization and disinfection (physical, chemical, and mechanical removal)
• Students should be able to explain the role of sterilization and disinfection in disease control

Introduction

• Microorganisms are the agents of contamination, infection, and decay
• Hence it's necessary to remove them from materials and areas
• Early civilizations practiced salting, smoking, pickling, and exposure to sunlight

Aseptic Techniques

• In the mid-1800s, Lister developed aseptic techniques to prevent contamination of surgical wounds
• Prior to this, nosocomial infections caused death in 10% of surgeries
• Up to 25% of mothers delivering in hospitals died due to infection

Methods of Microbial Growth Control

• Alteration of membrane permeability
• Damage to proteins
• Damage to nucleic acids
• Order of resistance (most to least): Prions, Endospores of bacteria, Mycobacteria, Cysts of protozoa, Vegetative protozoa, Gram-negative bacteria, Fungi, Viruses without envelopes, Gram-positive bacteria, Viruses with lipid envelopes

Definition of Sterilization

• The process of freeing an article from microorganisms, including their spores

Sterilization

• Sterilization is the killing or removal of all microorganisms, including bacterial spores, which are highly resistant.
• Sterilization is usually carried out by autoclaving, which consists of exposure to steam at 121°C under a pressure of 15 lb/in² for 15 minutes.
• Surgical instruments that can be damaged by moist heat are usually sterilized by exposure to ethylene oxide gas, and most intravenous solutions are sterilized by filtration.

Methods of Sterilization

• Table 3-1 (Physical Sterilants): Steam under pressure (121°C or 132°C for various time intervals), Filtration (0.22- to 0.45-μm pore size; HEPA filters), UV radiation (variable exposure to 254-nm wavelength), Ionizing radiation (variable exposure to microwave or gamma radiation)
• Table 3-1 (Gas Vapor Sterilants): Ethylene oxide (450-1200 mg/L at 29°C to 65°C for 2-5 hr), Hydrogen peroxide vapor (30% at 55°C to 60°C), Plasma gas (Highly ionized hydrogen peroxide gas)
• Table 3-1 (Chemical Sterilants): Peracetic acid (0.2%), Glutaraldehyde (2%)

Disinfection

• Disinfection is the killing of many, but not all, microorganisms
• For adequate disinfection, pathogens must be killed, but some organisms and bacterial spores may survive
• Disinfectants: corrosive phenol-containing
• Antiseptics: less toxic materials (such as ethanol and iodine) used on skin surfaces

Methods of Disinfection

• Table 3-2 (Heat): Moist heat (75°C to 100°C for 30 min)
• Table 3-2 (Liquid): Glutaraldehyde (2%-3.2%), Hydrogen peroxide (3%-25%), Chlorine compounds (100-1000 ppm of free chlorine), Alcohol (70%-95%), Phenolic compounds (0.4%-5.0%), lodophor compounds (30-50 ppm of free iodine/L), Quaternary ammonium compounds (0.4%-1.6%)

Don't Forget

• Sterilization: Destroys all viable microbes, including viruses and endospores
• Disinfection: Destroys vegetative pathogens, not endospores; for inanimate objects
• Antiseptic: Disinfectants applied directly to exposed body surfaces
• Sanitization: Any cleansing technique that mechanically removes microbes

Antiseptic Agents

• Table 3-3: Alcohol (70%-90%), lodophors (1-2 mg of free iodine/L; 1%-2% available iodine), Chlorhexidine (0.5%-4.0%), Parachlorometaxylenol (0.50%-3.75%), Triclosan (0.3%-2.0%)

Terminology

• Degermation: Mechanically removing microbes
• Sepsis: Bacterial contamination
• Asepsis: Absence of significant contamination
• Bactericidal (microbicidal): Kills microbes
• Bacteriostatic (microbiostatic): Inhibits growth and multiplication

Terminology (continued)

• Bacteriostatic Agent: inhibits bacteria growth but does not necessarily kill them.
• Bactericide: kills bacteria, most do not kill endospores.
• Sporicidal: kills spores.
• High, intermediate, and low-level agents are different levels of germicides.

Box 3-1 Definitions

• Antisepsis (Use of chemical agents on the skin or other living tissue to inhibit or eliminate microbes; no sporicidal action is implied)
• Disinfection (Use of physical procedures or chemical agents to destroy most microbial forms; bacterial spores and other relatively resistant organisms may remain viable; disinfectants are sub-divided into high-, intermediate-, and low-level agents)
• Germicide (Chemical agent capable of killing microbes; includes virucide, bactericide, sporicide, tuberculocide, and fungicide)
• High-level disinfectant (A germicide that kills all microbial pathogens except large numbers of bacterial spores)
• Intermediate-level disinfectant (A germicide that kills all microbial pathogens except bacterial endospores)
• Low-level disinfectant (A germicide that kills most vegetative bacteria and lipid-enveloped and medium-size viruses)
• Sterilization (Use of physical procedures or chemical agents to destroy all microbial forms, including bacterial spores) Order of resistance (most to least): Prions, Endospores of bacteria, Mycobacteria, Cysts of protozoa, Vegetative protozoa, Gram-negative bacteria, Fungi, Viruses without envelopes, Gram-positive bacteria, Viruses with lipid envelopes

Aim of Sterilization & Asepsis

• Asepsis: Practice to reduce or eliminate contaminants.
• Ideal situation: contaminant-free field ("sterile"). This is difficult to achieve, with the goal being elimination of infection rather than absolute sterility.

Methods of Sterilization

• Physical agents
• Chemical agents
• Mechanical removal methods

Physical Agents

• Heat (Dry, Moist)
• Radiation (Ionizing, Non-ionizing)

Dry Heat

• Kills by oxidation effects
• Operates between 50°C to 250/300°C
• A holding period of 160°C for 2 hrs is desirable
• Temperature controlled by thermostat
• Double-walled insulation keeps heat in and conserves energy
• Uses: to sterilize forceps, scissors, scalpels, swabs, pharmaceuticals (liquid paraffin, dusting powder, fats, and grease)

Flaming/Incineration

• Method of destroying materials: contaminated cloth, animal carcasses, and pathological materials

Moist Heat Sterilization

• Kills microorganisms by coagulating their proteins (changes proteins structure).
• Methods:
  • Temp below 100°C ("Pasteurization")
  • Temperature at 100°C: Boiling
  • Steam at atmospheric pressure: Koch/Arnold's steamer
  • Steam under pressure: Autoclave

Pasteurization

• Process of killing pathogens in milk but does not sterilize it
• Milk heating at 63°C for 30 minutes (Holder method).
• Heating milk at 72°C for 15-20 seconds and rapidly cooling to 13°C (Flash process)

Hot Water Bath

• To inactivate non-sporing bacteria for vaccines.
• Bath at 60°C/one hour.
• Serum or body fluids containing coagulable proteins can be sterilized by heating for 1 hr at 56°C in a water bath (several successive days)

Temperature at 100°C

• Kills vegetative forms of bacterial pathogens/
• Hepatitis virus can survive up to 30 minutes of boiling.
• Endospores can survive up to 20 hours or more of boiling.

Steam Under Pressure - Autoclave

• Works on the principle of steam under pressure.
• Invented by Charles Chamberland in 1879
• Autoclave consists of a vertical or horizontal cylinder with a lid for keeping materials to be sterilized.
• Pressure gauge to measure Pressure, Safety valve to permit steam escape.
• Sterilization 121°C for 15 minutes or 132°C for 3 minutes.
• Quality control is important

Chemical Agents

• Protein coagulation
• Disruption of the cell membrane
• Removal of sulfhydryl groups
• Substrate competition

Principles of Effective Disinfection

• Concentration of disinfectant
• Organic matter
• pH
• Time

Alcohols

• Ethanol/isopropyl alcohol are frequently used.
• No action on spores.
• Concentration is recommended at 60-90% in water.
• Uses: disinfectant of clinical thermometers, skin disinfectant (venupuncture)

Aldehydes

• Formaldehyde & Glutaraldehyde are frequently used.
• Formaldehyde is bactericidal, sporicidal, and lethal to viruses.
• Glutaraldehyde is effective against tubercle bacilli, fungi, and viruses.
• Formaldehyde uses: Preserves anatomical specimens, destroys Anthrax spores, sterilizes metal instruments (10% formalin + 0.5% sodium tetraborate).
• Glutaraldehyde uses: Treatments for corrugated rubber tubes, facemasks, plastic endotracheal tubes, and polythene tubing

Halogens

• Iodine in aqueous and alcoholic solutions for skin disinfection.
• Actively bactericidal with moderate activity against spores.
• Chlorine and its compounds: disinfectant in water supplies and swimming pools.

Phenols

• Obtained by coal tar distillation.
• Phenols are powerful microbicidal substances.
• Phenol derivatives are widely used as disinfectants in hospitals (e.g., Lysol, cresol).

Hydrogen Peroxide

• Hydrogen peroxide is used as an antiseptic to clean wounds and disinfect contact lenses.
• Effectiveness is limited by the organism's ability to produce catalase (enzyme degrading H2O2).
• Hydrogen peroxide is an oxidizing agent that attacks sulfhydryl groups, thereby inhibiting enzymatic activity.

Gases

• Ethylene oxide: Colorless, ethereal-smelling gas with high penetration.
• Effective against all microbes, including viruses and spores.
• Used to sterilize heart-lung machines, respirators, sutures, dental equipment, books and clothing.
• Sterilizes glass, metal, paper surfaces, plastics, oil, some foods and tobacco.

Metallic Salts

• Salts of silver, copper, and mercury are used as disinfectants.
• Act by coagulating proteins.
• Marked bacteriostatic, weak bactericidal, and limited fungicidal activity.

Sterilization by Filtration

• Filtration removes bacteria from heat-labile liquids (sera and solutions of sugar, antibiotics).
• Filters used: Candle filters, Asbestos filters, Sintered glass filters, Membrane filters

Radiation

• Non-ionizing radiation (e.g., UV rays): Absorbed as heat, used in rapid mass sterilization of prepacked syringes and catheters
• Ionizing radiation (e.g., X-rays, gamma rays, cosmic rays): High penetrative power, cold sterilization, sterilizing plastics, syringes, catheters, grease fabrics, metal foils.

Ultrasonic and Sonic Vibration

• Bactericidal activity
• Microorganisms vary in sensitivity, limited practical value for sterilization/disinfection.

Table 3-1: Methods of Sterilization

Table 3-2: Methods of Disinfection

Table 3-3: Antiseptic Agents

Table 3-4: Germicidal properties (properties of disinfectants & antiseptic agents)

References

• Murray, Patrick R., MEDICAL MICROBIOLOGY, EIGHTH EDITION ISBN: 978-0-323-29956-5
• Lippincott's Illustrated Reviews Microbiology, 3rd Edition (Richard A. Harvey, Cynthia Nau Cornelissen, Ph.D)
• Jawetz Melnick & Adelbergs Medical Microbiology
• Review of Medical Microbiology and Immunology, Fourteenth Edition (Warren Levinson, MD, PhD)
• www.cdc.com