TK

Control of Microorganisms by Physical and Chemical Agents

7.1 DEFINITIONS OF FREQUENTLY USED TERMS

  • Terminology is crucial in microbial control because words like disinfectant and antiseptic are often used loosely and a given treatment can either inhibit growth or kill depending on conditions.

  • Important terms and their definitions:

    • Sterilization: the complete removal or destruction of all viable microorganisms (including spores) and acellular entities; a sterile object is totally free of viable microorganisms.

    • Sterilant: a chemical agent that achieves sterilization.

    • Disinfection: the killing, inhibition, or removal of microorganisms that may cause disease; normally used on inanimate objects; does not necessarily sterilize (viable spores may remain).

    • Disinfectant: a chemical agent used to carry out disinfection.

    • Sanitization: reduction of microbial populations to levels considered safe by public health standards; inanimate objects are usually cleaned and partially disinfected.

    • Antisepsis: chemicals applied to body surfaces to destroy or inhibit vegetative pathogens; must be less toxic to host tissues than disinfectants.

    • Antiseptic: a chemical agent used for antisepsis (applied to living tissue).

    • Chemotherapy: chemicals used internally to kill or inhibit growth of microorganisms within host tissues.

    • Germicide (and suffixes): substances that kill organisms;

    • -cide denotes killing (e.g., bactericide, fungicide, viricide). A germicide kills pathogens (and many non-pathogens) but may not kill endospores.

    • -static denotes inhibition of growth without necessarily killing (e.g., bacteriostatic, fungistatic).

  • Note on endospores: many chemical agents do not reliably destroy bacterial endospores; sterilization often requires physical methods or sterilants capable of destroying endospores.

  • The types of control agents and their uses are summarized conceptually in Figure 7.1 (not reproduced here).

  • Connections to broader context:

    • The chapter emphasizes two practical goals: (1) destroy pathogens and prevent transmission, and (2) reduce/eliminate microorganisms that contaminate water, food, and other substances.

    • Historical context: disinfection and sterilization have ancient roots (Egyptians, Greeks, Mosaic law) and remain essential for aseptic techniques, food preservation, disease prevention, and personal safety in labs/hospitals.

7.2 THE PATTERN OF MICROBIAL DEATH

  • A microbial population exposed to an antimicrobial agent dies in a non-instantaneous, generally exponential (logarithmic) manner: the same fraction of organisms is killed in each equal time interval.

  • If the logarithm of the surviving population is plotted versus time, the result is a straight line during the main phase of killing (Figure 7.2 in the text).

  • As killing proceeds and the population is greatly reduced, regression may slow due to the survival of more resistant subpopulations or strains.

  • Key concepts introduced:

    • Decimal reduction time (D value): the time required at a given temperature to reduce the microbial population by one log cycle (i.e., to kill 90% of the organisms in the population).

    • Thermal death time (TDT): the shortest time required to kill all organisms in a suspension at a specified temperature.

    • Thermal death point (TDP): the lowest temperature at which a microbial suspension is killed in 10 minutes (older concept; largely replaced by TDT).

    • F value: the time (in minutes) at a specified temperature needed to achieve a given level of kill (often used in canning/minimum processing calculations at a reference temperature).

    • z value: the increase in temperature required to reduce the D value by one log cycle (i.e., to a tenfold reduction in D). This is used to compare heating resistance across temperatures.

  • VBNC state (viable but nonculturable): bacteria can be alive but not culturable under standard methods; conventional killing tests may misclassify VBNC cells as dead, posing a risk of later recovery and infection.

  • Example interpretation (from the text): The pattern of microbial death can be illustrated by a theoretical scenario where 90% of organisms are killed per minute at a fixed temperature; the remaining survivors drop by a factor of 10 each minute, as depicted in the provided table/graph and the figure showing a straight-line semilog plot of survivors versus time.

7.3 CONDITIONS INFLUENCING THE EFFECTIVENESS OF ANTIMICROBIAL AGENTS

Destruction or inhibition of microorganisms by antimicrobials is influenced by at least six factors:

  • 1) Population size

    • Equal fractions are killed in each interval; larger populations require longer times to achieve the same level of kill.

    • Example: in the heating example (Fig. 7.2 and Table 7.1), a larger starting population takes longer to reduce to a given fraction.

  • 2) Population composition

    • Susceptibility varies with organism type. Endospores are highly resistant; vegetative cells are more susceptible.

    • Mycobacterium tuberculosis is notably more resistant than many other bacteria.

  • 3) Concentration or intensity of the antimicrobial

    • More concentrated or intense treatments often kill more rapidly, but the relationship is not always direct or linear.

    • Within a short range, a small increase in concentration can cause an exponential rise in effectiveness; beyond a certain point, increases yield diminishing returns.

    • Water content can affect activity: 70% ethanol can be more effective than 95% ethanol because the presence of water enhances activity.

  • 4) Duration of exposure

    • Longer exposure generally increases kill.

    • For sterilization, exposure must be sufficient to reach a probability of survival of 10^-6 or less.

  • 5) Temperature

    • Higher temperatures typically enhance antimicrobial activity; often a lower concentration can be used at higher temperatures.

  • 6) Local environment (organic matter, pH, biofilms, etc.)

    • Organic matter can protect microorganisms and shield them from disinfectants; biofilms provide a protective environment and can alter microbial physiology, reducing susceptibility to antimicrobials.

    • Environmental context matters (e.g., heat kills more readily at acidic pH; foods with different pH levels respond differently to pasteurization).

    • Cleaning surfaces or devices to remove organic matter before disinfection is important (e.g., syringes, dental instruments, water treatment).

  • Practical questions raised in this section (study prompts):

    • Explain how effectiveness varies with population size, composition, concentration/intensity, exposure duration, temperature, and environmental conditions.

    • Describe the impact of being in a biofilm on susceptibility to antimicrobials.

    • Propose two factors that would most affect disinfectant efficacy when cleaning showerheads in patient rooms, and explain the expected impact.

7.4 THE USE OF PHYSICAL METHODS IN CONTROL

Heat and other physical agents are used to control microbial growth and sterilize objects. The four most commonly used physical agents are heat, low temperature, filtration, and radiation.

Heat

  • Moist heat vs. dry heat:

    • Moist heat readily kills viruses, bacteria, and fungi by mechanisms such as degradation of nucleic acids, denaturation of enzymes and proteins, and disruption of membranes.

    • Boiling water (100°C / 212°F at sea level) for 10 minutes kills vegetative cells but does not destroy bacterial endospores; boiling is a disinfection method, not sterilization.

    • Moist heat sterilization requires temperatures above 100°C; this is achieved with saturated steam under pressure using an autoclave.

  • Autoclave (steam sterilizer):

    • Standard condition: 121°C and 15 psi.

    • Effective for destroying vegetative cells and endospores in liquids within 10–12 minutes; total cycle is usually at least 15 minutes to ensure safety margin.

    • Proper autoclave operation requires:

    • (a) Adequate air removal to allow steam contact with all materials (air flush out).

    • (b) Avoid overpacking: ensure steam can circulate and contact everything.

    • (c) Verification using biological indicators (e.g., Geobacillus stearothermophilus spores) or chemical indicators (tapes or color-changing strips) to confirm adequate sterilization.

    • Larger volumes require longer times for heat to penetrate to the center of the load (e.g., ~70 minutes for 5 L of liquid).

  • Pasteurization:

    • Controlled heating at ~55-60^ ext{°C} to destroy specific pathogens while preserving much of the product.

    • History: Pasteur’s work on wine spoilage and the adaptation by Soxhlet chemists for milk preservation in the 1860s–1880s; milk pasteurization introduced in the US in 1889.

    • Pasteurization does not sterilize; it kills pathogens and slows spoilage by reducing nonpathogenic spoilage microorganisms.

  • Dry heat sterilization: incineration and dry oven

    • Dry heat is used for materials that withstand oxidation and is slower than moist heat.

    • Inoculating loops can be sterilized in a bench-top incinerator; glassware and some instruments are often sterilized in ovens at 160–170°C for 2–3 hours.

    • Mechanism: oxidation of cell constituents and denaturation of proteins. Dry heat does not corrode glassware as moist heat does, and is suitable for powders, oils, and similar items.

    • Comparison: ∙ Clostridium botulinum spores require 5 minutes at 121°C with moist heat, but 2 hours at 160°C with dry heat to achieve sterilization for spore-killing purposes.

  • Summary of heat-related parameters:

    • Moist heat is generally faster and more effective for sterilization than dry heat for many materials, but not all materials tolerate moist heat (e.g., heat-sensitive plastics).

    • Key heat metrics include:

    • Thermal Death Point (TDP): historically used, now less common.

    • Thermal Death Time (TDT): shortest time to kill all organisms at a given temperature.

    • D value: time to reduce population by one log at a given temperature.

    • z value: temperature change needed to change the D value by a factor of 10.

    • F value: time at a specified temperature to achieve a defined kill (often used in food processing).

  • Practical driving metrics (examples from the text):

    • For Clostridium botulinum spores at 121°C: D{121} = 0.204 ext{ min}; to achieve a 12D reduction (i.e., to reduce from 10^{12} spores to 1 spore) requires approximately 12 imes D{121}
      ightarrow 12 imes 0.204 ext{ min} \

7.1 DEFINITIONS OF FREQUENTLY USED TERMS

  • Terminology is crucial in microbial control because words like disinfectant and antiseptic are often used loosely and a given treatment can either inhibit growth or kill depending on conditions.
  • Important terms and their definitions:
    • Sterilization: the complete removal or destruction of all viable microorganisms (including spores) and acellular entities; a sterile object is totally free of viable microorganisms.
    • Sterilant: a chemical agent that achieves sterilization.
    • Disinfection: the killing, inhibition, or removal of microorganisms that may cause disease; normally used on inanimate objects; does not necessarily sterilize (viable spores may remain).
    • Disinfectant: a chemical agent used to carry out disinfection.
    • Sanitization: reduction of microbial populations to levels considered safe by public health standards; inanimate objects are usually cleaned and partially disinfected.
    • Antisepsis: chemicals applied to body surfaces to destroy or inhibit vegetative pathogens; must be less toxic to host tissues than disinfectants.
    • Antiseptic: a chemical agent used for antisepsis (applied to living tissue).
    • Chemotherapy: chemicals used internally to kill or inhibit growth of microorganisms within host tissues.
    • Germicide (and suffixes): substances that kill organisms;
    • -cide denotes killing (e.g., bactericide, fungicide, viricide). A germicide kills pathogens (and many non-pathogens) but may not kill endospores.
    • -static denotes inhibition of growth without necessarily killing (e.g., bacteriostatic, fungistatic).
  • Note on endospores: many chemical agents do not reliably destroy bacterial endospores; sterilization often requires physical methods or sterilants capable of destroying endospores.
  • The types of control agents and their uses are summarized conceptually in Figure 7.1 (not reproduced here).
  • Connections to broader context:
    • The chapter emphasizes two practical goals: (1) destroy pathogens and prevent transmission, and (2) reduce/eliminate microorganisms that contaminate water, food, and other substances.
    • Historical context: disinfection and sterilization have ancient roots (Egyptians, Greeks, Mosaic law) and remain essential for aseptic techniques, food preservation, disease prevention, and personal safety in labs/hospitals.

7.2 THE PATTERN OF MICROBIAL DEATH

  • A microbial population exposed to an antimicrobial agent dies in a non-instantaneous, generally exponential (logarithmic) manner: the same fraction of organisms is killed in each equal time interval.
  • If the logarithm of the surviving population is plotted versus time, the result is a straight line during the main phase of killing (Figure 7.2 in the text).
  • As killing proceeds and the population is greatly reduced, regression may slow due to the survival of more resistant subpopulations or strains.
  • Key concepts introduced:
    • Decimal reduction time (D value): the time required at a given temperature to reduce the microbial population by one log cycle (i.e., to kill 90% of the organisms in the population).
    • Thermal death time (TDT): the shortest time required to kill all organisms in a suspension at a specified temperature.
    • Thermal death point (TDP): the lowest temperature at which a microbial suspension is killed in 10 minutes (older concept; largely replaced by TDT).
    • F value: the time (in minutes) at a specified temperature needed to achieve a given level of kill (often used in canning/minimum processing calculations at a reference temperature).
    • z value: the increase in temperature required to reduce the D value by one log cycle (i.e., to a tenfold reduction in D). This is used to compare heating resistance across temperatures.
    • VBNC state (viable but nonculturable): bacteria can be alive but not culturable under standard methods; conventional killing tests may misclassify VBNC cells as dead, posing a risk of later recovery and infection.
  • Example interpretation (from the text): The pattern of microbial death can be illustrated by a theoretical scenario where 90% of organisms are killed per minute at a fixed temperature; the remaining survivors drop by a factor of 10 each minute, as depicted in the provided table/graph and the figure showing a straight-line semilog plot of survivors versus time.

7.3 CONDITIONS INFLUENCING THE EFFECTIVENESS OF ANTIMICROBIAL AGENTS

Destruction or inhibition of microorganisms by antimicrobials is influenced by at least six factors:

  • 1) Population size
    • Equal fractions are killed in each interval; larger populations require longer times to achieve the same level of kill.
    • Example: in the heating example (Fig. 7.2 and Table 7.1), a larger starting population takes longer to reduce to a given fraction.
  • 2) Population composition
    • Susceptibility varies with organism type. Endospores are highly resistant; vegetative cells are more susceptible.
    • Mycobacterium tuberculosis is notably more resistant than many other bacteria.
  • 3) Concentration or intensity of the antimicrobial
    • More concentrated or intense treatments often kill more rapidly, but the relationship is not always direct or linear.
    • Within a short range, a small increase in concentration can cause an exponential rise in effectiveness; beyond a certain point, increases yield diminishing returns.
    • Water content can affect activity: 70% ethanol can be more effective than 95% ethanol because the presence of water enhances activity.
  • 4) Duration of exposure
    • Longer exposure generally increases kill.
    • For sterilization, exposure must be sufficient to reach a probability of survival of 10^{-6} or less.
  • 5) Temperature
    • Higher temperatures typically enhance antimicrobial activity; often a lower concentration can be used at higher temperatures.
  • 6) Local environment (organic matter, pH, biofilms, etc.)
    • Organic matter can protect microorganisms and shield them from disinfectants; biofilms provide a protective environment and can alter microbial physiology, reducing susceptibility to antimicrobials.
    • Environmental context matters (e.g., heat kills more readily at acidic pH; foods with different pH levels respond differently to pasteurization).
    • Cleaning surfaces or devices to remove organic matter before disinfection is important (e.g., syringes, dental instruments, water treatment).
  • Practical questions raised in this section (study prompts):
    • Explain how effectiveness varies with population size, composition, concentration/intensity, exposure duration, temperature, and environmental conditions.
    • Describe the impact of being in a biofilm on susceptibility to antimicrobials.
    • Propose two factors that would most affect disinfectant efficacy when cleaning showerheads in patient rooms, and explain the expected impact.

7.4 THE USE OF PHYSICAL METHODS IN CONTROL

Heat and other physical agents are used to control microbial growth and sterilize objects. The four most commonly used physical agents are heat, low temperature, filtration, and radiation.
Heat
  • Moist heat vs. dry heat:
    • Moist heat readily kills viruses, bacteria, and fungi by mechanisms such as degradation of nucleic acids, denaturation of enzymes and proteins, and disruption of membranes.
    • Boiling water (100^\text{°C} / 212^\text{°F} at sea level) for 10 minutes kills vegetative cells but does not destroy bacterial endospores; boiling is a disinfection method, not sterilization.
    • Moist heat sterilization requires temperatures above 100^\text{°C}; this is achieved with saturated steam under pressure using an autoclave.
  • Autoclave (steam sterilizer):
    • Principle: Uses saturated steam under pressure to achieve temperatures above 100^\text{°C}, which is necessary to effectively kill endospores and achieve sterilization. The pressure itself does not kill the microbes but allows the water to boil at higher temperatures.
    • Standard condition: 121^\text{°C} and 15 psi (103 kPa above atmospheric pressure), for a minimum of 15-20 minutes depending on the load.
    • Mechanism: Moist heat from saturated steam rapidly degrades nucleic acids, denatures enzymes and structural proteins, and disrupts cell membranes. The latent heat of condensation released by steam efficiently transfers heat to the items being sterilized.
    • Applications: Widely used in laboratories for sterilizing culture media, glassware, and waste; in medical and dental settings for surgical instruments and medical devices; and for sterilizing certain pharmaceuticals.
    • Effectiveness: Effective for destroying all vegetative cells and bacterial endospores. Typical cycle times for liquids range from 10–12 minutes at 121^\text{°C} for small volumes, with total cycle times often extended to at least 15 minutes to ensure a safety margin.
    • Proper autoclave operation requires:
    • (a) Adequate air removal to allow steam contact with all materials (air flush out). Incomplete air removal can create 'cold spots' where sterilization is not achieved.
    • (b) Avoid overpacking: ensure steam can circulate freely and contact all surfaces. Overpacking can hinder steam penetration.
    • (c) Verification using biological indicators (e.g., heat-resistant Geobacillus stearothermophilus spores) or chemical indicators (tapes or color-changing strips which change color upon exposure to sufficient temperature and time) to confirm adequate sterilization and proper functioning of the equipment.
    • Load considerations: Larger volumes or dense loads require longer exposure times for heat to penetrate to the center of the load (e.g., ~70 minutes for 5 L of liquid), as heat transfer takes time.
  • Pasteurization:
    • Principle: A process of controlled heating, typically below 100^\text{°C} for a specific duration, designed to destroy specific spoilage organisms and disease-causing pathogens (e.g., Mycobacterium tuberculosis, Salmonella, Listeria) while minimizing the impact on the flavor, nutritional value, and physical properties of the product.
    • History: Originated from Louis Pasteur's work in the 1860s to prevent spoilage in wine and beer. Later adapted by Soxhlet chemists for milk preservation in the 1880s, with milk pasteurization being introduced in the US in 1889.
    • Methods:
    • High-Temperature Short-Time (HTST) Pasteurization: Most common method for milk, involves heating milk to 72^\text{°C} for 15 seconds. This method is efficient and continuous.
    • Ultra-High-Temperature (UHT) Pasteurization: Involves heating milk to 138^\text{°C} for 2 seconds. This process, when combined with aseptic packaging, results in "shelf-stable" products that do not require refrigeration until opened, as almost all microorganisms are killed.
    • Batch Pasteurization (Low-Temperature Long-Time, LTLT): Older method, involves heating milk to 63^\text{°C} for 30 minutes, commonly used for smaller batches or unique products.
    • Effect: Pasteurization significantly reduces the total microbial population, including all spoilage organisms and human pathogens that are not spore-formers. It does not sterilize the product, as it does not reliably destroy bacterial endospores (e.g., Clostridium spores can survive).
    • Applications: Primarily associated with dairy products (milk, yogurt, ice cream mixes), but also used for fruit juices, beer, wine, and certain liquid egg products.
  • Dry heat sterilization: incineration and dry oven
    • Dry heat is used for materials that withstand oxidation and is slower than moist heat.
    • Inoculating loops can be sterilized in a bench-top incinerator; glassware and some instruments are often sterilized in ovens at 160–170^\text{°C} for 2–3 hours.
    • Mechanism: oxidation of cell constituents and denaturation of proteins. Dry heat does not corrode glassware as moist heat does, and is suitable for powders, oils, and similar items.
    • Comparison: ∙ Clostridium botulinum spores require 5 minutes at 121^\text{°C} with moist heat, but 2 hours at 160^\text{°C} with dry heat to achieve sterilization for spore-killing purposes.
  • Summary of heat-related parameters:
    • Moist heat is generally faster and more effective for sterilization than dry heat for many materials, but not all materials tolerate moist heat (e.g., heat-sensitive plastics).
    • Key heat metrics include:
    • Thermal Death Point (TDP): historically used, now less common.
    • Thermal Death Time (TDT): shortest time to kill all organisms at a given temperature.
    • D value: time to reduce population by one log at a given temperature.
    • z value: temperature change needed to change the D value by a factor of 10.
    • F value: time at a specified temperature to achieve a defined kill (often used in food processing).
    • Practical driving metrics (examples from the text):
    • For Clostridium botulinum spores at 121^\text{°C}}: D{121} = 0.204\ ext{ min}; to achieve a 12D reduction (i.e., to reduce from 10^{12} spores to 1 spore) requires approximately 12 \times D{121} \rightarrow 12 \times 0.204\ ext{ min}$$.