L9L

Medical Microbiology I: Aseptic Microbial Techniques

Lecture Overview

Course Information

• Course Code: 2.34813: Medical Microbiology I

• Course Code: 2.34903: Pharmaceutical Med Micro I

• Program: BOH/BDS

• Lecture: 09 Aseptic Microbial Techniques

• Instructor: ANLangarap-Kilepak, Medical Lab Science, SMHS-UPNG, Semester 01-2021

Lecture Objectives

At the end of these lectures, students should be able to understand and know the following:

• Different means of eliminating potentially harmful microbes.

• Physical methods of controlling unwanted microbial growth.

• Chemical agents used for the control of unwanted microbes.

• Evaluation tests for sterility.

• Evaluation of disinfectants, antiseptics, and preservative activity.

Protecting From Detrimental Microbes

Key Considerations

• Methods of controlling pathogens that threaten health and environmental organisms that damage products and contaminate water supplies.

• Importance of physical and chemical control procedures to reduce and eliminate microorganisms.

• Applications in hospitals, safe food preparation, water purification, pharmaceutical safety, cosmetics, and products applied to or ingested by humans.

Antimicrobial Effects

Definition and Mechanisms

• Establishing conditions that microbes cannot tolerate.

• Achieved through bacteriocidal or bacteriostatic agents:

  • Bacteriocidal Agents (cide = kill): Agents that kill microorganisms.

  • Bacteriostatic Agents (static = stand still): Agents that inhibit microbial growth and multiplication.

Means of Eliminating Potentially Harmful Microbes

Sterilization

• Definition: Eliminating all forms of life, including vegetative cells, spores, and viruses.

• Mechanism: Destroys potentially infectious nucleic acids such as viroids.

• Methods: Organisms are killed or physically removed from treated objects or substances.

• Note: Some harmful microbes, such as endotoxins and other bacterial products, cannot be removed by sterilization but reduced through washing and rinsing.

Disinfection

• Definition: Eliminates vegetative forms of most potentially hazardous and pathogenic organisms but does not ensure all microbes are eliminated.

• Resistance: Bacterial spores, tubercle bacilli, and many viruses are resistant to common disinfectants.

• Purpose: Employed when sterilization is impossible or unnecessary, its main goal is to minimize infection or product spoilage by reducing the microbial load, particularly pathogens.

Sanitization

• Definition: Supplements disinfection with cleaning to ensure the absence of dirt or organic debris alongside infectious microbes.

• Application: Typically employed for equipment used in food preparation or reusable instruments in hospitals.

Antisepsis

• Definition: Inhibition or destruction of microorganisms on the surface of living tissue to prevent infections.

• Characteristics: Antiseptics, the chemical agents used, must not harm living tissue, making them milder than disinfectants.

Physical Methods of Sterilization

Common Methods

1. Heat

   • Moist Heat

   • Dry Heat

2. Radiation

3. Filtration

4. Mechanical Scrub

Moist Heat

General Characteristics

• Effectiveness depends on heat resistance of organisms and environmental moisture content.

• Moist heat, especially steam, kills cells by coagulating proteins, particularly critical enzymes. Lack of water inhibits protein coagulation.

Three Antimicrobial Processes

1. Pasteurization

   • Developed by Louis Pasteur, involves controlled temperature and duration to kill most heat-resistant pathogens without damaging product quality.

   • Example: Coxiella burnetti, responsible for Q fever.

   • Note: Does not kill all microbes but eliminates common pathogens.

2. Boiling

   • Heating water to a temperature of 100ºC at normal atmospheric pressure.

   • Vegetative cells are eliminated with 10 minutes of boiling, but some endospores survive (e.g., $Clostridium botulinum$, the causative agent of botulism).

   • Considered for disinfection rather than sterilization, often used with surgical instruments or fractional sterilization.

3. Autoclaving

   • The most common method of sterilizing heat-stable materials using steam under pressure.

   • Standard conditions: 15 lb/in² above atmospheric pressure allowing temperatures to reach 121ºC for 15 minutes. Higher pressures (27-33 lb/in²) can reach temperatures of 132-136ºC but for shorter periods (3-10 minutes).

   • Principle: Air is displaced by saturated steam which condenses, creating a partial vacuum that enhances steam penetration for effective sterilization. Longer periods are necessary for large items. Saturated steam heats objects approximately 2500 times more efficiently than hot air at the same temperature.

Dry Heat

General Types

1. Flaming

   • Used to sterilize loops, scalpel blades, and needles to transfer microorganisms without contamination.

   • Mouths of test tubes and containers are flamed routinely to prevent contamination.

2. Incineration

   • Useful for destroying heavily contaminated materials or tissues in hospitals and lab cultures.

3. Baking (Hot Air Ovens)

   • Prolonged exposure to temperatures between 150ºC and 180ºC.

   • Length of exposure depends on heat penetration; higher temperatures require shorter exposure times. Used for sterilizing glassware and metal instruments.

Radiation

Description

• Effective for sterilization or reducing microbes through electromagnetic radiation including microwaves, ultraviolet (UV), X-rays, gamma rays, and electrons.

• Sterilization is often termed “cold sterilization” as it does not rely on heat.

• Ideal for heat-sensitive materials such as disposable plastics.

Ionizing Radiation

• Higher energy rays cause biologically active molecules to lose electrons, leading to loss of critical cellular functions.

• Gamma rays from radioactive elements like cobalt-60 sterilize items without damaging them, such as petri dishes for shipping, but require safety precautions.

• High energy electrons penetrate less efficiently, useful for small individually wrapped items.

Ultraviolet Radiation

• Causes thymine dimers that distort cellular DNA, impairing replication and gene expression.

• Effectiveness is dose-dependent; longer exposures increase the number of dead cells.

• Limitations include poor penetration power and some bacterial spores' protection, making UV light a less effective sterilizant. Useful in disinfecting air and surfaces in surgical areas.

Filtration

• A process for sterilizing liquids and gases by passing through filters that trap larger microbes. Common materials for standard filters include nitrocellulose with a pore size of 0.2 µm, which stops most bacteria while allowing liquid/gas passage.

• Applications include: cotton and gauze masks, lamina flow hoods for labs, and air filtration systems in surgical rooms.

Mechanical Scrub

• Primary source of nosocomial infections is unwashed hands of medical staff. Hand washing is crucial for reducing pathogen transmission.

• Hands often carry microbes residing in sweat ducts and hair follicles, thus reducing transient microbes requires effective hand washing.

Chemical Agents

Classification

• Chemical agents used for microbial control range from household detergents and antiseptics to toxic sterilizing agents in hospitals, characterized as:

  • Antiseptics

  • Disinfectants

  • Sterilants

Chemical Sterilization Agents

• Effective chemical sterilizers include:

  • Ethylene oxide

  • Hydrogen peroxide

  • Formaldehyde

  • Glutaraldehyde

• Mechanism: These agents kill microbes by damaging proteins and nucleic acids, representing methods of cold sterilization.

• Example: Vaporized hydrogen peroxide is effective for sterilizing incubators and clean rooms.

Germicidal Activity Levels

• High-level germicides: Kill all microbes including spores.

• Intermediate-level germicides: Kill vegetative cells and most viruses but may not destroy all endospores (e.g., glutaraldehyde).

• Low-level germicides: Effective against some bacteria, fungi, and enveloped viruses, often used in antiseptics.

Evaluation Tests for Sterility

Importance

• Though many sterilization methods theoretically kill all microbes, failures can occur through improper use.

• Monitoring sterilization procedures is critical to ensure sterility.

Methods of Evaluation

1. Recording Devices: Measure temperature, moisture, pressure, gas content.

2. Random Sampling: Identify contamination by evaluating random items.

3. Chemical Indicators: Include tapes that change color under specific conditions indicating sterilization success.

4. Biological Indicators: Use spore strips placed in the sterilization load that are incubated to check for growth post-cycle.

Evaluation of Antiseptic and Disinfectant Activity

Evaluative Techniques

1. Laboratory Tests

   • Determine antimicrobial activity under controlled laboratory conditions against specific test organisms.

   • Use-dilution method assesses germicide effectiveness by measuring survival rates after exposure to disinfectants.

   • Preservative effectiveness tests evaluate how long products protect against microbial growth.

2. In-Use Tests

   • Designed to assess disinfectant effectiveness under actual conditions.

   • Employ techniques for microbial counts following routine treatments, including:

     1. Direct Sampling: Use agar plates for smooth surfaces.

     2. Swabbing: For uneven surfaces, use swabs for sampling.

     3. Viable Microbial Counts: Analyze rinsing with sterile water to count viable microbes.

Hand Evaluation Techniques

• Antiseptics evaluated similarly, focusing on determining organisms on hands after scrubbing through various methods including swabbing, direct pressing, rinsing, and using sterile tape on skin surfaces and gloves after use.

These comprehensive study notes cover significant topics in the lecture on Aseptic Microbial Techniques, providing sufficient detail for an effective understanding of microbial control methods, their significance, and applications.