Microbial Death & Sterilisation – Key Concepts

Microbial Death

Introduction

• This lecture focuses on microbial death and various sterilization techniques used to eliminate or inhibit microbial growth.

• Includes matching questions to provide practical application of sterilization techniques.

• The learning outcomes include differentiating between gram-positive and gram-negative bacteria, understanding the principles of sterilization, and knowing various sterilization methods.

Bacterial Structure

• The primary focus is on the structural differences between gram-positive and gram-negative bacteria, particularly the cell wall.

• The cell wall maintains cell shape and prevents osmotic rupture.

• Gram-positive bacteria:

  • Have a thick peptidoglycan layer, typically 20-80 nm thick, providing significant rigidity and protection.

  • Possess a single cell membrane (a bilipid layer) containing proteins and carbohydrates that regulate the movement of ions and substances in and out of the cell.

  • Contain teichoic acid, unique polysaccharides that are species-specific and play roles in cell wall maintenance, cell division, and adhesion.

• Gram-negative bacteria:

  • Have a thin peptidoglycan layer, usually 5-10 nm thick, located in the periplasmic space.

  • Have two membranes: an inner membrane (cytoplasmic membrane) and an outer membrane, separated by the periplasmic space.

  • The periplasm contains the peptidoglycan layer and various enzymes and transport proteins.

  • The outer membrane contains lipopolysaccharide (LPS), a potent endotoxin that is antigenic, stimulating a strong immune response.

  • LPS consists of:

    • Lipid A: a hydrophobic component responsible for the endotoxic activity, causing fever (pyrogenic effect) and inflammation.

    • Core oligosaccharide: a short chain of sugars linked to lipid A.

    • O-specific polysaccharide: the outermost component, which is highly variable and antigenic, used for serotyping bacteria.

• Gram-negative bacteria are generally more resistant to disinfectants and sterilization methods due to the protective outer membrane, which restricts the entry of many antimicrobial agents.

Definition

• Sterilization: The complete destruction or removal of all microorganisms, including bacteria, viruses, fungi, and endospores.

• Disinfection: The elimination of harmful microorganisms, but not necessarily all microorganisms or endospores.

  • Endospores are highly resistant, dormant structures that can survive extreme conditions for extended periods.

  • Sterilization is capable of killing endospores, while disinfection might not.

• Sanitization: Reducing the microbial load to a level considered safe by public health standards, typically involving cleaning and disinfecting surfaces.

• -cidal: A suffix indicating the death of a cell (e.g., bacteriocidal, fungicidal).

• -static: A suffix indicating inhibition of growth or reproduction (e.g., bacteriostatic, fungistatic).

• Antiseptics: Chemical agents applied to living tissue to prevent infection, generally less toxic than disinfectants.

• Antimicrobial: A substance that inhibits the growth of or kills microbes, including antibiotics, antifungals, and antivirals.

Reasons for Sterilization

• Diarrheal diseases remain a significant cause of mortality worldwide, especially in low-income countries where access to proper sanitation and medical care is limited.

  • Diarrheal diseases rank as the 8th leading cause of death globally but are the 5th leading cause in low-income countries.

• In high-income countries, the prevalence of diarrheal diseases is lower due to better sanitation, hygiene practices, and availability of disinfectants.

• Sterilization plays a crucial role in maintaining public health by preventing the spread of infectious diseases.

• Healthcare-associated infections (HAIs), also known as nosocomial infections, are infections acquired by patients during their stay in healthcare facilities.

• HAIs lead to increased mortality rates, prolonged hospital stays, and higher healthcare costs.

• Hospitals are strongly incentivized to implement and maintain sterile environments to minimize the occurrence of HAIs.

Sterilization Methods

• Various methods are employed to achieve sterilization, each with specific applications and limitations:

  • Membrane filtration

  • Heat (dry and moist)

  • Irradiation

  • Chemical agents

Membrane Filtration

• Utilizes filters with tiny pores to mechanically trap microorganisms from liquids or gases.

• Pore sizes for bacteria sterilization are typically around 0.22 \mu m, while smaller pore sizes are required to capture viruses.

• Not always effective for complete virus removal due to the potential for filter clogging or the presence of very small viruses.

• Limitations and considerations:

  • Applicability: limited to liquids and gases that can pass through the filter.

  • Selectivity: primarily effective for removing bacteria but less reliable for viruses.

  • Certainty: may not guarantee complete sterilization in all cases.

  • Scalability: filter costs can be high, limiting scalability for large volumes.

  • Advantage: suitable for heat-sensitive fluids that cannot be sterilized by heat-based methods.

Dry Heat

• Achieves sterilization through complete oxidation and burning of organic molecules.

• Temperature requirements depend on the duration of exposure; higher temperatures require shorter exposure times.

• Heat is transferred layer by layer, requiring sufficient time for the core of the object to reach the minimum sterilization temperature.

• A relatively time-consuming process compared to moist heat sterilization.

• Dry heat sterilization is typically conducted at temperatures between 150 and 170 degrees Celsius.

  • 170 degrees Celsius requires 30 minutes of exposure.

  • 160 degrees Celsius requires 60 minutes of exposure.

  • 150 degrees Celsius requires 150 minutes of exposure.

• Many materials can be damaged by high temperatures, limiting the applicability of dry heat.

• Examples:

  • Passing wire loops through a Bunsen burner flame during aseptic procedures.

• Limitations and considerations:

  • Applicability: broad, but not suitable for heat-sensitive materials.

Moist Heat

• Sterilization with moist heat is faster and more effective than dry heat because it denatures proteins more efficiently.

• Boiling water (limited to 100^\circ C at normal atmospheric pressure) can be used to disinfect but may not achieve complete sterilization.

• Autoclaves use high-pressure steam to achieve sterilization by increasing the temperature above the boiling point of water.

• Effective for sterilizing biohazardous waste, culture media, aqueous solutions, and surgical dressings.

• Moist heat irreversibly denatures proteins and destroys cellular structures, leading to microbial death.

• Examples

  • Cooking an egg

• Advantages:

  • Nontoxic, easily controllable, and relatively quick.

• Limitations:

  • Not suitable for heat-sensitive or moisture-damaged items, such as certain metallic or electronic objects.

• Some endospores can survive boiling, but are often rendered non-reproductive (static state).

• Cold temperatures are generally not useful for sterilization, but can slow microbial growth.

Irradiation

• Sterilization by irradiation involves using electromagnetic radiation to damage microbial DNA and other essential cellular components.

• Two main types:

  • Ionizing radiation

  • Non-ionizing radiation

Ionizing Radiation

• Example: Gamma radiation, X-rays, and electron beams.

  • High-energy radiation that creates ions, electrons, and free radicals, leading to the degradation of enzymes and breakage of DNA strands.

• Effective against endospores and vegetative cells, but less effective against certain viruses.

• Offers rapid action and high penetrating power, making it suitable for sterilizing heat-sensitive materials.

• Can be used on objects in any phase (solid, liquid, or gas).

• Limitations and considerations:

  • Costly due to the need for specialized equipment and facilities.

  • Potential hazards associated with radioactive materials if not handled properly.

Non-ionizing Radiation

• Lower energy radiation that does not cause ionization but can still damage microbes.

• Examples:

  • Infrared radiation:

  • Causes molecules to vibrate intensely, generating heat and leading to sterilization.

  • Commonly used for sterilizing syringes, catheters, and other medical devices.

  • Ultraviolet (UV) radiation:

  • Damages DNA by causing the formation of thymine dimers, which inhibit DNA replication and transcription.

  • Not effective for sterilizing dense objects due to poor penetration, but suitable for surface sterilization of laboratory benchtops, cabinets, and water.

  • Microwave radiation:

  • Less effective than infrared and ultraviolet radiation for sterilization.

Chemical Agents

• Chemical agents can be used to sterilize or disinfect materials by disrupting microbial cell structures or interfering with metabolic processes.

• Bacteria can grow in a range of pH levels (acidic, alkaline, neutral), influencing the effectiveness of chemical agents.

• Examples:

  • Formaldehyde:

  • A strong sterilizing agent used for surgical and dental instruments and air ducts.

  • Highly toxic to humans and classified as a carcinogen, limiting its use.

  • Peracetic acid:

  • A safer alternative to formaldehyde, used for sterilizing surgical gowns and drapes.

  • Effective against a broad range of microorganisms, including endospores.

  • Alcohol:

  • Causes cell dehydration, membrane disruption, and protein denaturation.

  • Ethanol and isopropanol are commonly used as antiseptics and disinfectants.

  • Easily mass-produced and has relatively low toxicity, but does not kill all microorganisms.

  • Antibiotics:

  • Selective toxicity, targeting specific microbial pathways or structures.

  • Microbial resistance is a significant concern, limiting the long-term effectiveness of many antibiotics.

  • Not broadly used.

Examples of Sterilization Methods and Materials

• Powder: Dry heat (first choice) or ionizing radiation

• Ophthalmic drops: Membrane filtration (preferred) or UV radiation

• Blood bags: Ionizing radiation to kill pathogens without heating

• Surgical instruments:

  • Steam under pressure (autoclave)

  • Dry heat

  • Chemical sterilization/high-level disinfection (depending on use)

  • Ethylene oxide (EtO) sterilization for heat-sensitive instruments

• Sutures: Radiation-sterilized because heat can damage them

• Equipment with electronic components: Low-temperature sterilization methods like EtO or hydrogen peroxide gas plasma

• Swabs:

  • Autoclaving if heat-stable

  • Gamma irradiation as it is typically a single-use item

  • Dry heat

Conclusion

• Review of various sterilization techniques, including their applications, advantages, and limitations.

• Understanding the rationale behind sterilization choices in laboratory and healthcare settings.