Chapter 9 – controlling microbial growth in the environment

Know these terms – terminology of microbial control

• Antisepsis - reduction in the number of microorganisms’ and viruses, particularly potential pathogens, on living tissue

• Aseptic - refers to an environment or procedure free of pathogenic contaminants

• -cide(-cidal) – suffixes indicating destruction of a type of microbe

• Degerming – removal of microbes by mechanical means

• Disinfection – destruction of most microorganisms and viruses on nonliving tissues

• Pasteurization – use of heat to destroy pathogens and reduce the number of spoilage microorganisms in foods and beverages

• Sanitization – removal of pathogens from objects to meet public health standards

• -stasis (-static) – suffixes indicating inhibition but not complete destruction of a type of microbe

• Sterilization – destruction of all microorganisms and viruses in or an object

Basic Principles of Microbial Control

• Action of Antimicrobial Agents

  • 1. Alteration of cell walls and membranes

    • Cell wall maintains integrity of cell

      • Cells burst due to osmotic effects when damaged

  • Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell

    • Cellular contents leak out when damaged

• 2. Damage to proteins and nucleic acids

  • Extreme heat or certain chemicals denature proteins

  • Chemicals, radiation, and heat can destroy nucleic acids

The Selection of Microbial Control Methods

• Ideally, agents for the control of microbes
  should be:

  • Inexpensive

  • Fast-acting

  • Stable during storage

  • Capable of

    • 1. controlling microbial growth while being

    • 2. harmless (not toxic) to humans, animals, and objects

• Factors Affecting the Efficacy of Antimicrobial Methods

  • Environmental conditions can affect the ability of disinfectants

    • Temperature (works better at higher temp

    • and pH (better at neutral pH

    • Organic materials (vomit, feces, etc)

      • Interfere with the penetration of heat, chemicals, and some forms of radiation

      • May inactivate chemical disinfectants

• Biosafety Levels

  • Four levels of safety in labs dealing with pathogens

    • Biosafety Level 1 (BSL-1) (We work with BSL-1 microbes

      • Handling pathogens that do not cause disease in healthy humans

    • Biosafety Level 2 (BSL-2)

      • Handling moderately hazardous agents

    • Biosafety Level 3 (BSL-3)

      • Handling microbes in safety cabinets

    • Biosafety Level 4 (BSL-4)

      • Handling microbes that cause severe or fatal disease

Figure 9.2 Relative susceptibilities of microbes to antimicrobial agents.

• We often choose our method of control depending upon what we need to kill.

• Prions have no treatment, besides avoidance.

• Mycobacteria (TB) have a resistant, mycolic acid filled cell wall and take more disinfectant for a longer time.

• Enveloped viruses can be broken apart with detergent type chemicals that break up the envelope.

Physical Methods of Microbial Control

• Heat-Related Methods

  • Effects of high temperatures:

    • Denature proteins

    • Interfere with integrity of cytoplasmic membrane and cell wall

    • Disrupt structure and function of nucleic acids

  • Moist heat

    • Used to disinfect, sanitize, sterilize, and pasteurize

    • Denatures proteins and destroys cytoplasmic membranes

    • More effective than dry heat

    • Methods of microbial control using moist heat:

      • Boiling

        • Kills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses

        • Boiling time is critical

        • Endospores, protozoan cysts, and some viruses can survive boiling

      • Autoclaving

        • Pressure applied to boiling water prevents steam from escaping (higher pressure increase steam temperature)

        • Boiling temperature increases as pressure increases

        • Autoclave conditions: 121°C, 15 psi, 15 minutes

      • Pasteurization

        • Used for milk, ice cream, yogurt, and fruit juices

        • Not sterilization

Heat-tolerant microbes survive

• Pasteurization of milk (3 types—preserve flavor)

Batch method

Flash pasteurization

Ultra-high-temperature pasteurization

• Ultra-high-temperature sterilization

  • 140°C for 1 to 3 seconds, then rapid cooling

  • Treated liquids can be stored at room temperature

  • Tiny creamer cups, Chocolate milk in juice boxes on shelves

  • Dry heat

    • Used for materials that cannot be sterilized with
      moist heat

    • Denatures proteins and oxidizes metabolic and structural chemicals

    • Requires higher temperatures for longer time than moist heat

    • Incineration is ultimate means of sterilization, (burning trash, flaming loop)

  • Refrigeration and Freezing

    • Decrease microbial metabolism, growth, and reproduction

    • Refrigeration halts growth of most pathogens

    • Some microbes can multiply in refrigerated foods (Listeria)

    • Organisms vary in susceptibility to freezing

  • Desiccation and Lyophilization

    • Desiccation (drying) inhibits growth due to removal of water

    • Lyophilization (freeze-drying) used for long-term preservation of microbial cultures

      • Prevents formation of damaging ice crystals

  • Osmotic Pressure

    • High concentrations of salt or sugar (jelly, jam) in foods to inhibit growth

    • Cells in hypertonic solution of salt or sugar lose water

    • Fungi have greater ability than bacteria to survive hypertonic environments

  • Radiation

    • Ionizing radiation

      • Wavelengths shorter than 1 nm

        • gamma rays, some X rays

      • Ejects electrons from atoms to create ions

      • Ions disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxyl radicals

      • Gamma rays penetrate well but require hours to kill microbes

      • X rays require long time to kill microbes

        • Not practical for microbial control

    • Nonionizing radiation (UV light)

      • Wavelengths greater than 1 nm

      • Excites electrons, causing them to make new
        covalent bonds

        • Affects 3-D structure of proteins and nucleic acids

      • UV light causes pyrimidine dimers in DNA

      • UV light does not penetrate well

      • Suitable for disinfecting air, transparent fluids, and surfaces of objects

Figure 9.11 The roles of high-efficiency particulate air (HEPA) filters in biological safety cabinets.

• Filtration is used to physically remove bacteria from the air. HEPA filters are used.

• Liquid media is also filter—sterilized and the membrane filter removes the bacteria from the liquid.

Figure 9.10 Filtration equipment used for microbial control.

• Liquid media is also filter—sterilized and the membrane filter removes the bacteria from the liquid.

Chemical Methods of Microbial Control

• Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA

• Effect varies with differing environmental conditions

• Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa

• Phenol and Phenolics

  • Denature proteins and disrupt cell membranes

  • Effective in presence of organic matter

  • Remain active for prolonged time

  • Commonly used in health care settings, labs,
    and homes

  • Have disagreeable odor and possible toxicity issues

• Alcohols

  • Denature proteins and disrupt cytoplasmic membranes

  • More effective than soap in removing bacteria
    from hands

  • Swabbing of skin (antiseptic) with alcohol prior to injection removes most microbes

• Halogens

  • Damage enzymes by denaturation

  • Widely used in numerous applications

  • Iodine tablets, iodophors, chlorine treatment, bleach, chloramines, and bromine disinfection

• Oxidizing Agents

  • Peroxides, ozone, and peracetic acid

  • Kill by oxidation of microbial enzymes

  • Hydrogen peroxide can disinfect and sterilize surfaces

    • Not useful for treating open wounds due to catalase activity

  • Ozone treatment of drinking water

• Surfactants

  • “Surface active” chemicals

    • Reduce surface tension of solvents

  • Soaps and detergents

    • Soaps have hydrophilic and hydrophobic ends

      • Good degerming agents but not antimicrobial

    • Detergents are positively charged organic surfactants

  • Quaternary ammonium compounds (quats)

    • Low-level disinfectants

    • Disrupt cellular membranes

    • Ideal for many medical and industrial applications

• Heavy Metals

  • Heavy-metal ions denature proteins

  • Low-level bacteriostatic and fungistatic agents

  • 1% silver nitrate to prevent blindness caused by Neisseria gonorrhoeae

  • Thimerosal used to preserve vaccines

  • Copper controls algal growth

  • Silver thread in wound coverings

• Gaseous Agents DANGER!!

  • Microbicidal and sporicidal gases used in closed chambers to sterilize items

  • Denature proteins and DNA by cross-linking functional groups

  • Used in hospitals and dental offices

  • Disadvantages:

    • Can be hazardous to people

    • Often highly explosive

    • Extremely poisonous

    • Potentially carcinogenic

• Enzymes

  • Antimicrobial enzymes act against microorganisms

  • Human tears contain lysozyme

    • Digests peptidoglycan cell wall of bacteria

  • Use enzymes to control microbes in the environment

    • Lysozyme used to reduce the number of bacteria
      in cheese

    • Prionzyme can remove prions on medical instruments

      • Used in European Union

• Antimicrobial Drugs

  • Antibiotics, semisynthetic, and synthetic chemicals

  • Typically used for treatment of disease

  • Some used for antimicrobial control outside the body

• Methods for Evaluating Disinfectants and Antiseptics

  • Use-dilution test

    • Metal cylinders dipped into broth cultures of bacteria

    • Contaminated cylinder immersed into dilution of disinfectant

    • Cylinders removed, washed, and placed into tube of medium

    • Most effective agents entirely prevent growth at highest dilution

    • Current standard test in the United States

    • New standard procedure being developed

  • Kelsey-Sykes capacity test

    • Alternative assessment approved by the European Union

    • Bacterial suspensions added to the chemical being tested

    • Samples removed at predetermined times and incubated

    • Lack of bacterial reproduction reveals minimum time required for the disinfectant to be effective

  • In-use test

    • Swabs taken from objects before and after application of disinfectant or antiseptic

    • Swabs inoculated into growth medium and incubated

    • Medium monitored for growth

    • Accurate determination of proper strength and application procedure for each specific situation