Microbial Growth Control

• Microbial growth is controlled to prevent contamination and infection.

Microbial Control Terminology

• Sterilization is the removal/destruction of all microbial life forms, including viruses and bacterial endospores.
  • Sterilization is used for culture media, reagents, containers, loops, glassware, medical/surgical equipment, and materials entering sterile areas of the body.
• Disinfection uses physical and chemical agents (disinfectants) to destroy pathogens or unwanted microbes when sterility is unnecessary/impractical.
• Sanitization reduces pathogenic microbe levels on places/eating utensils to meet public health standards.
• Sepsis refers to microbial contamination.
• Asepsis is the absence of significant contamination.
• Antisepsis removes pathogens from living tissue by using an antimicrobial agent (antiseptic), whereas disinfectants are antimicrobial agents used on inanimate objects.
• Degerming removes microbes from a limited area.
• Germicides/biocides kill microorganisms.
• Bacteriostats stop bacterial growth/multiplication.

Methods of Microbial Control

• Physical methods include heat, refrigeration, freezing, desiccation, lyophilization, filtration, osmotic pressure, and radiation.
• Chemical methods use phenol, phenolics, alcohols, halogens, oxidizing agents, surfactants, heavy metals, aldehydes, gaseous agents, and antimicrobials.
• Microbial control methods can affect things other than microbes.
  • These include inactivation of vitamins and drugs as well as possible heat damage to medical equipment.

Modes of Action

• Damage to the plasma membrane and cell walls causes cells to lose integrity, affecting osmosis.
  • The plasma membrane contains the cytoplasm and controls the passage of chemicals.
  • Enveloped viruses require proteins/phospholipids for target cell attachment
• Damage to proteins and nucleic acids changes protein shape/denaturation, which stops function.
  • Nucleic acid damage can produce mutations that stop translation

Heat

• Moist heat is often used to disinfect, sanitize, sterilize, and pasteurize.
  • Moist heat kills cells by denaturing proteins and destroying membranes, making it more effective for microbial control than dry heat because water is a better heat conductor than air.
• Boiling kills vegetative bacterial cells and some viruses, but not endospores.
• Autoclaves achieve sterilization by applying heat under pressure.
• Dry heat includes hot air sterilization and flaming.

Autoclave

• An autoclave uses moist heat with applied pressure in an enclosed space to prevent steam from escaping.
  • Boiling temperature increases as pressure increases.
• Autoclave components include a steam chamber, pipes to introduce/remove steam, and valves to remove air and control pressure.
  • Steam enters, forcing air out, raising the temperature and pressure until set parameters are met.
  • Standard autoclave conditions are 121° C at 15 psi for 15 minutes.
• Uses for autoclaves include sterilizing culture media, surgical instruments, dressings, and IV equipment.

Pasteurization

• Pasteurization uses mild heating to destroy spoilage microbes in heat-sensitive foods like milk and beer.
• Pasteurization is not a method of sterilization.
• Pasteurization process occurs at 63 °C for 30 minutes.

Other Physical Methods

• Refrigeration and Freexing decrease microbial metabolism, growth, and reproduction by:
  • Slowing chemical reactions at low temperatures.
  • Halting pathogen growth.
  • Causing liquid water to be unavailable if frozen.
  • Psychrophiles such as Listeria and Yersinia can still multiply in refrigerated foods.
• Dessication is microbiostatic because it inhibits microbial growth, where metabolism requires H2O.
• Lyophilization, or freeze-drying, combines freezing with drying to preserve microbial cultures, preventing damaging ice crystal formation.
• Osmotic Pressure was previously discussed.
• Filtration separates fluids using a screen-like sieve filter.
  • This excludes particles of a specific size, with filters available with pore sizes of 0.01µm.
• HEPA (high-efficiency particulate air) filters can reduce airborne allergens and various particulates.

Radiation

• Ionizing radiation includes gamma rays, X-rays, and electron beams.
  • Ionizing radiation creates ions by ejecting electrons from atoms when striking molecules.
• Non-ionizing radiation does not force electrons out.
  • UV light is the only non-ionizing source with enough energy to be antimicrobial.
  • Visible light and microwaves have little direct effect on microorganisms.

Chemical Methods of Microbial Control

• Many disinfectants are available.
  • Few chemicals can achieve sterilization.
• Quats are quaternary ammonium compounds.
• Effectiveness of various chemical agents:
  • Phenolics are poor against endospores but good against mycobacteria.
  • Quats are ineffective.
  • Chlorines are fair against endospores and mycobacteria.
  • Alcohols are poor against endospores but good against mycobacteria.
  • Glutaraldehyde is effective against endospores and mycobacteria

Characteristics of an Ideal Chemical Antimicrobial Agent

• An ideal chemical antimicrobial agent should:
  • Kill many different microbes.
  • Be fast acting with short contact time.
  • Be inexpensive and easy to prepare.
  • Be odorless.
  • Not be affected by organic material.
• An ideal chemical antimicrobial agent must be:
  • Nontoxic to human tissues.
  • Noncorrosive and nondestructive on materials.
  • Water soluble and easy to apply.
  • Stable as both a concentrate and a working dilution.

Phenols, Phenolics & Bisphenols

• Intermediate to low level disinfectants disrupt the lipid-containing plasma membrane and denature proteins.
  • They can irritate the skin and have a disagreeable odor, although they remain active with pus, feces, vomit, saliva.
• An example is Lysol (cresol).
• Bisphenols are chemically altered phenols that are effective against Gram+ bacteria, Staphyloccoci, and Streptococci and used in hospital nurseries.
  • Overuse can cause neurological damage.

Alcohols

• Intermediate-level disinfectants dissolve lipids and denature proteins, and work against vegetative bacteria, mycobacteria, enveloped viruses, and fungi.
  • Alcohols are not effective against endospores.
• They are good for "de-germing" prior to injection because they evaporate quickly; are fast acting.
• A 70-90% alcohol in H2O solution is more effective than pure alcohol for protein denaturation because denaturation requires H2O.
• Isopropanol is an example (rubbing alcohol).

Halogens

• Intermediate-level antimicrobial chemicals damage enzymes via oxidation or denaturation.
• Iodine is a skin antiseptic active against mycobacteria and spore-forming bacteria as well.
  • An example is Betadine.
  • Povidone iodine is iodine complexed with polyvinylpyrrolidone.
• Chlorine includes Chlorox (household bleach) and is used to treat drinking water.
• Aqueous chlorine solutions are bactericidal.
  • Three forms of chlorine present in water include elemental chlorine (Cl2), hypochlorous acid (HOCl), and hypochlorite ion (OCl2).
  • Chlorine exerts its effect by the irreversible oxidation of sulfhydryl (SH) groups of essential enzymes.
• Bromine is used for the disinfection of bathtubs
  • Also used as an alternative to disinfect swimming pools, colling towers and other water containers.

Quaternary Ammonium Compounds (Quats)

• Quats consist of four organic groups covalently linked to a nitrogen with germicidal activity determined by the nature of these organic groups.
  • An example is benzalkonium chloride.
  • These compounds denature cell membranes to release intracellular components.
• Quats are bacteriostatic at low concentrations, bactericidal at high concentrations.
  • Pseudomonas, Mycobacterium, and the fungus Trichophyton are resistant to these compounds.

Oxidizing Agents/Peroxygens

• High level disinfectants produce oxygen, which damages anaerobic microbes.
  • Hydrogen peroxide is sporicidal on inanimate objects, effectively killing most bacteria at a concentration of 3%- 6% and killing all organisms, spores included, at 10%-25%.
  • Benzoyl peroxide is used to treat acne.
  • Peracetic acid is a very effective sporicidal.

Surfactants

• Soaps and detergents have little value as antiseptics but they are good for mechanical removal of microbes through scrubbing.
• Quaternary ammonium compounds are popular synthetic detergents with a positive charge.
  • They are active against gram+, fungi, enveloped viruses, and protozoa.
  • They are not active against mycobacteria or endospores.
  • Some bacteria, such as Pseudomonas, can actively grow in quats.
  • Mode of action is unknown.
  • Harmless to humans.
  • Example: Cepacol mouthwash.

Heavy Metals

• Ions are antimicrobial because they alter the 3-D shape of proteins and inhibit or eliminate their function.
  • Heavy metals interfere with proteins needed for cellular processes.
  • These include silver, mercury, copper, and gold.
• Examples:
  • Topical cream for burns.
  • 1% silver nitrate prevents blindness caused by N. gonorrhoeae.
  • Thimerosal (mercury based) is used to preserve vaccines.

Aldehydes

• Aldehydes are the most effective of all chemical antimicrobial disinfectants.
• They function by cross-linking amino, hydroxyl, and other organic functional groups, which denatures proteins and inactivates nucleic acids.
• Aldehydes are active against Mycobacteria and spores.
• Aldehydes achieve sterilization.
• Examples:
  • Glutaraldehyde sterilizes heat-sensitive hospital instruments.
  • Formaldehyde preserves biological specimens and is used for embalming.

Gaseous Chemosterilizers

• Chemicals that sterilize or kill in a closed chamber.
• Similar to autoclave.
• They use highly reactive microbicidal and sporicidal gases.
  • Example: ethylene oxide.
  • This is active against all microbes and spores.
• Problems:
  • Long exposure time, explosive, toxic, suspected carcinogen.

Relative Susceptibilities of Microbes to Antimicrobial Agents

• Microbes, ordered from most- to least resistant, are prions, bacterial endospores, mycobacteria, cysts of protozoa, active-stage protozoa (trophozoites), Gram-negative bacteria, fungi, nonenveloped viruses, Gram-positive bacteria, enveloped viruses.

Q&A

• A Bacteriostatic agent inhibits bacterial growth, but does not kill bacteria.
• Antiseptics are generally less toxic compared to disinfectants.
• An agent that specifically kills fungi but not other kinds of microorganisms is also knows as a fungicidal agent.
• Sterilization involves killing OR removing all viable microorganisms.
• Destruction/removal of all viable organisms is called sterilization.
• The reduction of the microbial population to levels that are considered safe by public health standards is called sanitization.
• The prevention of infection caused by microorganisms is antisepsis.