Techniques

• Control of microorganisms is essential in order to prevent the transmission of diseases and infection, stop decomposition and spoilage, and prevent unwanted microbial contamination
• Microbial population growth is exponential, and the effectiveness of an agent is not fixed but influenced by many environmental factors
• Sterilization


  • Process by which all living cells, viable spores, viruses and viroids are either destroyed or removed from an object or habitat
  • Achieved by physical means and chemical means
• Disinfection


  Killing, inhibition, or removal of microorganisms that may cause disease
• Disinfectants


  Agents, usually chemical, used to carry out disinfection and are normally used only on inanimate objects
• Germicide


  Substances that can kill organisms, often have the suffix -cide (Latin cida, to kill)
• Bactericide, fungicide, algaecide, viricide


  Disinfectant or antiseptic that is particularly effective against a specific group
• Bacteriostatic, fungistatic


  Chemicals that do not kill, but prevent growth. If removed, growth will resume
• A bacterium is defined as dead if it does not grow and reproduce when inoculated into culture medium that would normally support its growth. In like manner an inactive virus cannot infect a suitable host
• Factors influencing effectiveness of antimicrobial agents

  • Population size
  • Population composition
  • Concentration or intensity of antimicrobial agent
  • Duration of exposure
  • Temperature
  • Local environment
• Population size


  • A larger population requires a longer time to die than a smaller one
• Population composition


  • Effectiveness varies greatly with the nature of the organisms
  • Bacterial endospores vs vegetative forms
  • Younger cells are usually more readily destroyed than mature organisms
  • Some species are able to withstand adverse conditions better than others
• Concentration or intensity of antimicrobial agent


  • Often, but not always, the more concentrated a chemical agent or intense a physical agent, the more rapidly organisms are destroyed
  • Sometimes an agent is more effective at lower concentrations
• Duration of exposure


  • The longer a population is exposed to a microbicidal agent, the more organisms are killed
• Temperature


  • An increase in the temperature at which a chemical acts often enhances its activity
• Local environment


  • The population to be controlled is not isolated but surrounded by environmental factors that may either offer protection or aid in its destruction
• Physical methods of sterilization

  • Heating (moist heat, dry heat)
  • Desiccation
  • Freezing
  • Filtration
• Moist heat sterilization


  1. Pasteurization (flash, UHT)

  2. Vaccine bath

  3. Serum bath

  4. Inspissation

  5. Boiling

  6. Fractional sterilization

  7. Autoclave
• Dry heat sterilization


  1. Red flame

  2. Open flame

  3. Incineration

  4. Hot air oven

  5. Infrared rays
• Desiccation


  Depriving microorganisms of moisture, destroys vegetative forms but endospores are resistant
• Freezing


  Inhibits microbial growth and reproduction, does not destroy contaminating microbes
• Filtration


  Removes microorganisms rather than destroying them, used for heat-sensitive materials
• Filtration


  • HEPA filters (air)
  • Membrane filters (liquids)
• Drying


  • Microorganisms are resistant to drying
• Freezing


  1. Inhibit growth and reproduction

  2. Use freezing or refrigeration

  3. Freezing at -20°C or lower stops microbial growth

  4. Freezing does not destroy contaminating microbes

  5. Frozen food can contain many microorganisms, should be prepared and consumed after thawing
• Filtration


  Removes contaminating microorganisms rather than destroying them
• Filtration examples


  • HEPA Filters (Air) - used in operating rooms and burn units to remove bacteria from air
  • Membrane Filters (Liquids) - porous membranes to remove most vegetative cells, but not viruses, from solutions
• Ultraviolet Light (UVL)


  • Effective wavelength is 200 nm - 280 nm, with 260 nm as the most effective
  • Induce formation of thymine-thymine dimers resulting in mutations
  • Can inactivate microorganisms like bacteria, viruses, and yeasts within seconds
  • Not sporicidal
  • Frequently used for surface disinfection
  • Disadvantages: low penetrance, limited lifespan of UV bulb, some bacteria have DNA repair systems that counteract the UV rays, harmful to skin and eyes
• Ionizing Radiation


  • Greater penetrance than UV rays
  • Causes formation of free radicals that chemically interact with proteins and nucleic acids, resulting in cell death
  • Not routinely used because of potential harm on human tissues
• Ionizing Radiation examples


  • Electron Beams - used to sterilize syringes, gloves, dressing packs, food and some pharmaceuticals
  • Electromagnetic (Gamma Rays) - produced from nuclear disintegration of selected radioactive isotopes, greater penetrance than electron beams but require longer exposure time, bactericidal, fungicidal, virucidal, and sporicidal
• Sonic and Ultrasonic Vibrations

  • Can kill some bacteria and viruses after exposure to certain frequency of sound waves
  • High frequency sound waves act by disrupting cells
  • Used to disinfect and clean instruments and to reduce microbial load
• Osmotic Pressure


  • Creating a hypertonic environment with high concentration of salts and sugar causes bacterial cell to collapse
  • Used for preservation of fruits in syrup and meats
• Factors affecting the efficacy of a chemical agent

  • Concentration and potency of the chemical agent
  • Duration of exposure
  • Temperature
  • Nature of the surrounding medium
  • Nature of the organisms
  • Number of organisms/size of inoculum
• Characteristics of a good chemical agent

  • Broad spectrum
  • Fast-acting
  • Active in the presence of organic matter
  • Active in any pH
  • Stable
  • Non-toxic, non-allergic, non-irritative, and non-corrosive
  • Soluble in water and easy to apply
  • Leave residual antimicrobial film on the treated surface
  • High penetrating power
  • Must not be expensive; readily available
  • Safe under storage and shipping for reasonable periods of time
  • Not have a bad odor
• Classification of chemical disinfectants

  • Consistency (liquid or gaseous)
  • Spectrum activity (high level, intermediate level, low level)
  • Mechanism of action
• Damage to the cell membrane


  Can cause smaller molecules to leak out of the bacterial cell and interfere with the active transport and energy metabolism within the cell
• Surface active agents (surfactants)


  • Long chain hydrocarbons that are fat-soluble and charged ions that are water soluble
  • Disrupt membranes resulting in leakage of cell membranes
  • Active against vegetative microbial forms including Mycobacteria as well as enveloped viruses
• Cationic agents


  • Detergents with fat soluble portion positively charged due to combination with a quaternary nitrogen atom
  • Effective at alkaline pH
• Anionic agents


  • Negatively charged agents with long chain hydrocarbons
  • Remove dirt through the process of emulsification
  • Effective at acidic pH
• Phenolic compounds


  • Disrupt cell membranes and cause precipitation of proteins and inactivation of enzymes
  • Bactericidal and fungicidal, good against Mycobacteria but poor activity against spores and most viruses
  • Phenols no longer used as disinfectant because of toxic effect to human cells, cresols are phenol derivatives more potent and safer