Microbial Control Methods in Microbiology

BIOL 2260K: Microbiology for Allied Health Professions

Department of Biology

University of North Georgia

Dr. Paul Johnson

Microbial Control Methods

Concepts in Antimicrobial Control

• Definition of Sterilization:

  • The process that destroys or removes all viable microorganisms, including viruses.

  • Important Note: The term "sterile" should only be used in the strictest sense. There is no such thing as "slightly sterile."

  • Common Uses:

  • Surgical instruments

  • Syringes

  • Commercially packaged food

  • Method:

  • Heat (autoclave)

  • Sterilants (chemical agents capable of destroying spores)

• Definition of Disinfection:

  • A process that destroys or removes vegetative pathogens but not bacterial endospores.

  • Common Uses:

  • Boiling food utensils

  • Applying a 5% bleach solution to an examining table

  • Immersing thermometers in iodine solution between uses

• Decontamination/Sanitization:

  • A cleansing technique to mechanically remove microorganisms along with other debris to reduce contamination to safe levels.

  • Common Uses:

  • Cooking utensils

  • Dishes

  • Bottles and cans

• Antisepsis/Degermation:

  • A process that reduces the number of microbes on human skin.

  • Involves scrubbing with soap (mechanical friction) or immersing in chemicals (or both).

Comparative Definitions of Terms Related to Microbial Control

Resistance of Microbial Types to Microbial Control Agents

• List of Microbial Resistance:

  • Most Resistant:

  • Prions

  • Bacterial endospores

  • Mycobacterium

  • Staphylococcus and Pseudomonas

  • Protozoan cysts

  • Less Resistant:

  • Protozoan trophozoites

  • Most gram-negative bacteria

  • Fungi and fungal spores

  • Most gram-positive bacteria

  • Enveloped viruses

Key Terms Related to Microbial Control

• Sepsis: The growth of microorganisms in blood and other tissues.

• Aseptic Techniques: Practices that prevent the entry of infectious agents into sterile tissues.

Agents vs Processes of Microbial Control

• Antiseptics: Chemical agents applied to body surfaces to prevent vegetative pathogens.

• Static Agents:

  • Bacteristatic: Chemicals that inhibit bacterial growth.

  • Fungistatic: Chemicals that inhibit fungal growth.

• -cidal Agents: Even a -cidal agent does not necessarily result in sterilization, and -static agents at high concentrations can become -cidal.

Definition of Agents

Practical Matters in Microbial Control

• Critical Questions:

  • Does the application require sterilization, or is disinfection adequate?

  • Is the item to be reused or permanently discarded?

  • Can the item withstand heat, pressure, radiation, or chemicals if reused?

  • Is the control method appropriate for a given application? (e.g., will it leave undesirable residue?)

  • Will the agent penetrate to the necessary extent?

  • Is the method cost-effective, labor-efficient, and safe?

Factors Affecting Death Rate

• Factors include:

  • The number of microbes present

  • The nature of the microorganisms in the population

  • Temperature and pH of the environment

  • The concentration (dose, intensity) of the agent used

  • The mode of action of the agent

  • Presence of solvents, or interfering organic matter which can inhibit disinfectants (e.g., saliva, blood, feces).

Understanding Microbial Death

• The death of a microbial population is not instantaneous; it begins once a threshold of antimicrobial agent is achieved.

• Population death is generally modeled as continuing in a logarithmic manner as time/concentration increases.

• Actively growing cells tend to die more quickly than less metabolically active cells.

• A point is reached at which the survival of any cells is highly unlikely, equivalent to sterilization.

Actions of Physical and Chemical Agents Upon the Cell

• Cellular Targets:

  • Cell Wall:

  • Agents can block synthesis or digest the cell wall.

  • Cell Membrane:

  • Agents bind to lipid layers opening the membrane allowing harmful chemicals to enter and result in ion loss.

  • Cellular Synthesis:

  • Agents interrupt protein synthesis at ribosomes, preventing growth and metabolism.

  • Proteins:

  • Agents denature proteins which causes breakdown of protein structure.

  • Some attach to active sites, preventing interactions with chemical substrates.

Examples of Agents Used

Physical Control

Heat as Physical Control

• Types of Heat:

  • Moist Heat:

  • Microbicidal effect involves the coagulation and denaturation of proteins.

  • Effective between ~60°C and 135°C.

  • Dry Heat:

  • Dehydrates cells and denatures proteins, effective from ~160°C to thousands of degrees Celsius.

  • Requires higher temperatures than moist heat for the same effect.

Key Measurements

• Thermal Death Time (TDT): The shortest time required to kill all microbes at a specified temperature.

• Thermal Death Point (TDP): The lowest temperature required to kill all microbes in a sample in 10 minutes.

Resistance to Heat

• Bacterial Endospores: Most heat-resistant pathogens; require high temperatures for destruction.

• Vegetative Cells: Vary in heat sensitivity; death times range from 50°C for 3 minutes to 60°C for 60 minutes.

• Pathogens and Non-Pathogens: Similar susceptibility to heat; fungi, protozoa, and worms exhibit similar sensitivity.

• Viruses: Can resist heat; examples include 55°C for 2-5 minutes, and 60°C for 600 minutes.

Cold and Desiccation as Physical Control

• Cold:

  • Slows microbial growth in food during processing/storage; most microorganisms are not adversely affected by gradual cooling.

  • Extremely low temperatures (~ -70°C) are effective for culture storage.

  • Pathogens can survive several months in the refrigerator, including Staphylococcus aureus, Clostridium species, and others.

• Desiccation:

  • A state of extreme dryness can dehydrate vegetative cells; various pathogens have differing resistance to drying.

  • Lyophilization: A method of preserving microorganisms by freezing and removing water under vacuum.

Radiation Methods

Ionizing Radiation

• Types:

  • Gamma rays and X-rays; effective in sterilizing materials sensitive to heat or chemicals.

  • Items are irradiated with a chosen dosage, with exposure typically ranging from 5 to 50 kiloGrays (kGray).

  • Effective for disinfecting various foods and medical materials.

Non-Ionizing Radiation

• Ultraviolet (UV) Rays:

  • Effective against many types of microorganisms, requiring direct exposure for effectiveness, typically applied in water purification and air disinfection.

  • The most lethal wavelength is from 240 nm to 280 nm, peaking at 260 nm.

Mechanical Control

Filtration as Mechanical Control

• Definition: A method to remove microbes from liquids or air by straining through filters.

• Types of Filters:

  • Made of materials such as cellulose acetate, polycarbonate, and plastics, with pore sizes controlled for effective sterilization.

• Applications:

  • Used in the preparation of liquids that cannot withstand heat, such as blood products, vaccines, and others.

Chemical Control

Germicidal Effects

• Evaluation: Germicides are assessed based on effectiveness in medical and dental settings.

  • Categories of Germicides:

  • High-level: Kill endospores, can be used as sterilants.

  • Intermediate-level: Kill fungal viruses, resistant pathogens.

  • Low-level: Eliminate vegetative bacteria and some viruses.

Factors Affecting the Germicidal Activity of Chemicals

• Length of Exposure: Adequate contact time is necessary for effectiveness.

• Composition of Material: Smooth, solid objects are more reliably disinfected than porous ones.

• Degree of Contamination: The presence and amount of organic matter can interfere with germicidal activity.

Required Concentrations and Times for Chemical Destruction

Table of Chemicals

Germicidal Categories According to Chemical Group

• Chemical Agent Group:

  • Phenol/Phenolics: Disrupt plasma membranes and denature enzymes; rarely used except as a standard.

  • Biguanides (Chlorhexidine): Disrupt plasma membranes; used in skin disinfection and surgical scrubs.

  • Halogens (Iodine and Chlorine): Iodine inhibits protein function, chlorine acts as a strong oxidizing agent; used in water disinfection and sanitization.

Other Antimicrobial Agents

Dyes

• Active against certain gram-positive bacteria and fungi, used in ointments for skin infections; limited application due to staining.

Acids and Alkalis

• Very low or very high pH can destroy or inhibit activity; hazardous in application.