Control of Microbial Growth Notes

Control of Microbial Growth

Ch. 13.1 Controlling Microbial Growth

  • Cleanliness is relative, and the main goal is to reduce microbial load, thereby reducing infection or contamination.

  • Different situations require different levels of cleanliness.

  • Sterilization: Removal or killing of ALL microbes.

  • Disinfection: Inactivation of microbes.

  • Sanitization: Decreasing microbial load.

Ch. 13.1 Controlling Microbial Growth - Biological Safety Level

  • Biological Safety Levels (BSL) are cleanliness levels assigned to labs.

  • The CDC, NIH, and WHO have established 4 levels.

  • UTA micro labs are BSL-2.

Ch. 13.1 Controlling Microbial Growth - Requirements

  • BSL-1: Sink for handwashing & door to close off lab (e.g., freshmen bio labs).

  • BSL-2: BSL-1 plus PPE, self-closing doors, eyewash station, autoclave or sterilization method (e.g., Micro labs).

  • BSL-3: BSL-2 plus respirator, bio safety cabinets, hands-free wash sink, two sets of doors (none at UTA).

  • BSL-4: BSL-3 plus full biohazard suit, shower on exit, decontaminate all material on exit, lab must have own air supply (only 13 in USA).

Ch. 13.1 Controlling Microbial Growth - Level of Clean in the Clinic

  • Critical: Must be sterile; items contact sterile tissue (i.e., blood).

  • Semicritical: Does not require high-level sterilization; items might contact non-sterile tissue (i.e., gut).

  • Noncritical: Do not require sterilization; items do not penetrate tissue (i.e., stethoscopes on skin).

Ch. 13.1 Controlling Microbial Growth - Sterilization

  • Sterilization involves the complete removal or killing of all microbes from a fomite or organism.

  • Methods include:

    • Heat

    • Pressure

    • Filtration

    • Chemical (sterilants)

  • Aseptic technique is used to prevent a sterile environment from becoming contaminated.

Ch. 13.1 Controlling Microbial Growth - Disinfection

  • Disinfection involves the inactivation or killing of microbes on fomites.

  • Antiseptic acts on microbes but not the organism/tissue.

  • Some microbes may not be inactivated; disinfection ≠ sterile!

  • Most disinfectants do not kill endospores.

  • Examples: hydrogen peroxide, rubbing alcohol.

Ch. 13.1 Controlling Microbial Growth - Sanitization & Degerming

  • Sanitization and degerming involve the decrease of microbial load.

  • Commonly mechanical – washing hands, wiping with a paper towel, etc.

  • May be used in combination with disinfectant to maximize microbial reduction.

Protocol For Use on Fomites

  • Disinfection: Reduces or destroys the microbial load of an inanimate item through the application of heat or antimicrobial chemicals. Common application: Cleaning surfaces like laboratory benches, clinical surfaces, and bathrooms. Common agents: Chlorine bleach, phenols (e.g., Lysol), glutaraldehyde.

  • Sanitization: Reduces microbial load of an inanimate item to safe public health levels through the application of heat or antimicrobial chemicals. Common application: Commercial dishwashing of eating utensils, cleaning public restrooms. Common agents: Detergents containing phosphates (e.g., Finish), industrial-strength cleaners containing quaternary ammonium compounds

  • Sterilization: Completely eliminates all vegetative cells, endospores, and viruses from an inanimate item. Common application: Preparation of surgical equipment and needles used for injection. Common agents: Pressurized steam (autoclave), chemicals, radiation.

Protocol For Use on Living Tissue

  • Antisepsis: Reduces microbial load on the skin or tissue through application of an antimicrobial chemical. Common application: Cleaning skin broken due to injury; cleaning skin before surgery. Common agents: Boric acid, isopropyl alcohol, hydrogen peroxide, iodine (betadine).

  • Degerming: Reduces microbial load on skin or tissue through gentle to firm scrubbing and the use of mild chemicals. Common application: Handwashing. Common agents: Soap, alcohol swab.

Ch. 13.2 – Physical Means of Control Measuring Success of Control

  • Methods can partially or fully kill various microbes.

  • -cides: kill.

  • -static: stop growth.

  • Examples:

    • Bacteriocidal vs. Bacteriostatic

    • Viricidal vs. Viristatic

    • Fungicidal vs. Fungistatic

  • The degree of control can be observed with a microbial death curve.

  • Many factors affect the success of control, including length of exposure, concentration of agent, and population level.

Ch. 13.2 – Physical Means of Control - Microbial Death Curve

  • Measure of percentage of kill.

  • Decimal reduction time (DRT) – how much time it takes to kill 90% (1 log reduction) of the population.

  • Example: 1 x 10^6 -> 1 x 10^5

Ch. 13.2 – Physical Means of Control

  • Most are applied to non-living items (i.e., fomites).

  • Types:

    • Temperature

    • Radiation

    • Filtration

    • Desiccation

    • Pressure

Ch. 13.2 – Physical Means of Control - Heat Sterilization

  • Oldest and most common method.

  • Alters membranes and/or denatures proteins.

  • Thermal Death Point – the lowest temperature that will kill in 10 min.

  • Thermal Death Time – the length of time to kill at a certain temperature.

Ch. 13.2 – Physical Means of Control - Heat Sterilization

  • Dry Heat – aka incineration; direct application of high heat (>250^\circ C).

    • Example: Bunsen burner.

  • Moist Heat – application of high-temperature liquid/vapor. Beneficial because it penetrates cells better than dry heat.

    • Example: autoclave.

Ch. 13.2 – Physical Means of Control - Autoclave

  • Autoclave – raises the temperature of water above boiling point (\approx 121^\circ C) by raising pressure to 15 psi.

  • Kills viruses & endospores.

Ch. 13.2 – Physical Means of Control - Pasteurization

  • Pasteurization – semi-sterilizes food but does not ruin food quality.

  • Many methods rely on “flash” heating foods to kill most microbes.

Ch. 13.2 – Physical Means of Control - Refrigeration & Freezing

  • Usually not a sterilization method but static.

  • Slows metabolism but will grow when temps are raised (reason why USDA recommends thawing at temps lower than optimal).

  • Ultra-low temps (-80^\circ C) can be used for preservation.

Ch. 13.2 – Physical Means of Control - Pressure Sterilization

  • Pascalization – high pressure used in the food industry to kill microbes.

  • Hyperbaric chambers can be used to treat infections. Induces hypoxia to saturate the infection site with oxygen.

  • Used in combination with temperature in autoclaves Treatment of C. botulinum with high pressure of pressure cooker.

Ch. 13.2 – Physical Means of Control - Desiccation

  • AKA dehydration.

  • Creates a difference in osmotic pressure through salt or lyophilization to remove water.

Ch. 13.2 – Physical Means of Control - Radiation

  • High energy radiation - used to kill or inhibit microbes.

  • Ionizing radiation – enters cells and disrupts molecular structures such as DNA.

    • X-rays & gamma rays.

    • Can be used to sterilize non-autoclavable items.

    • Can be an alternative to pasteurization in canned foods.

  • Non-ionizing radiation – does not penetrate glass, plastics, etc. but can damage cells with direct exposure.

    • UV irradiation – forms thymine dimers in DNA causing lethal mutations.

Ch. 13.2 – Physical Means of Control - Sonication

  • High-frequency sound waves disrupt cell structure.

  • Causes bubbles to form inside cells and induce lysis.

Ch. 13.2 – Physical Means of Control - Filtration

  • Use of barrier to physically separate microbes.

  • Useful when media cannot be autoclaved (e.g., Urea broth).

  • Filters usually have a pore size of 0.2 µm (or smaller for viruses).

Physical Methods of Control

  • Heat:

    • Boiling: 100 °C at sea level, denatures proteins and alters membranes. Example uses: Cooking, personal use, preparing certain laboratory media.

    • Dry-heat oven: 170 °C for 2 hours, denatures proteins and alters membranes, dehydration, desiccation. Example uses: Sterilization of heat-stable medical and laboratory equipment and glassware.

    • Incineration: Exposure to flame, destroy by burning. Example uses: Flaming loop, microincinerator

    • Autoclave: Typical settings: 121 °C for 15 minutes at 15 pounds per square inch (psi), denatures proteins and alters membranes. Example uses: Sterilization of microbiological media, heat-stable medical and laboratory equipment, and other heat-stable items

    • Pasteurization: Can vary. One type is 72 °C for 15 seconds (HTST), denatures proteins and alters membranes. Example uses: Prevents spoilage of milk, apple juice, honey, and other ingestible liquids

  • Cold:

    • Refrigeration: 0 °C to 7 °C, inhibits metabolism (slows or arrests cell division). Example uses: Preservation of food or laboratory materials (solutions, cultures)

    • Freezing: Below -2 °C, stops metabolism, may kill microbes. Example uses: Long-term storage of food, laboratory cultures, or medical specimens

  • Pressure:

    • High-pressure processing: 100-800 MPa, denatures proteins and can cause cell lysis. Example uses: Preservation of food

    • Hyberbaric oxygen therapy: Air pressure three times higher than normal, inhibits metabolism and growth of anaerobic microbes. Example uses: Treatment of certain infections (e.g., gas gangrene)

  • Desiccation:

    • Simple desiccation: Drying, inhibits metabolism. Example uses: Dried fruits, jerky

    • Addition of salt or water: Reduce water activity, inhibits metabolism and can cause lysis. Example uses: Salted meats and fish, honey, jams and jellies

    • Lyophilization: Rapid freezing under vacuum, inhibits metabolism. Example uses: Preservation of food, laboratory cultures, or reagents

  • Radiation:

    • Ionizing radiation: Exposure to X-rays or gamma rays, alters molecular structures, introduces double-strand breaks into DNA. Example uses: Sterilization of spices and heat-sensitive laboratory and medical items; used for food sterilization in Europe but not widely accepted in US

    • Nonionizing radiation: Exposure to ultraviolet light, introduces thymine dimers, leading to mutations. Example uses: Surface sterilization of laboratory materials, water purification

Chemical Means of Control

  • Depending on safety, can be used on living tissues or fomites.

  • Types:

    • Phenolics

    • Heavy metals

    • Halogens

    • Alcohols

    • Surfactants

    • Bisbiguanides

    • Alkylating agents

    • Peroxygens

    • Supercritical fluid

    • Food, cosmetic, & pharma preservatives

Phenols

  • A carbon molecule with a benzene ring and –OH group.

  • MOA: Denature proteins & membranes.

  • Examples:

    • Carbolic acid - first used by Joseph Lister for surgical wounds.

    • Lysol - original formulations (now is quaternary compound).

    • Triclosan - commonly used in hand soaps; banned by FDA in 2017.

Heavy Metals

  • MOA: Binds to & inhibits proteins (not exclusive to microbes).

  • Mercury – treated syphilis but banned b/c of neural toxicity effects.

  • Silver – used today to treat burn wounds, pediatric ophthalmia neonatorum, and in antibiotics.

  • Copper sulfate – used as algicide to treat pools.

  • Zinc oxide – calamine lotion, baby powder.

Halogens

  • Iodine – oxidizes cellular components; commonly used as an iodophor (complex with organic molecule).

  • Chlorine

    • Hypochlorous acid – Cl + H_2O; used to treat water.

    • Sodium hypochlorite – bleach.

    • Chloramine – Cl + NH_3; very stable “swimming pool smell”.

  • Fluorine

    • Most recognizable with dental products.

    • Deposits in tooth enamel and provides disruption in microbial fermentation and processes.

Alcohols

  • MOA: Disrupts membranes and denatures cytoplasmic proteins.

  • Used as 70% to allow better cell penetration.

  • Only viricidal for enveloped viruses.

  • Can be used in combo with iodine.

Surfactants

  • Chemicals that lower the surface tension of water.

  • In most soaps and detergents.

  • Soaps – fatty acid salts Not –cidal or –static but means of mechanical removal.

  • Detergents – synthetic polar & non-polar molecules.

    • Anionic – neg. anion on chain.

    • Cationic – pos. anion on chain.

Surfactants - Quaternary ammonium salts

  • Quaternary ammonium salts - cationic detergents.

  • Mimics phospholipids and can insert into the lipid bilayer.

  • Common day Lysol.

Bisbiguanides

  • Cationic molecules that disrupt the membrane.

  • Not active against naked viruses, Mycobacteria tuberculosis, etc.

  • Chlorhexidine – common surgical scrub and longer lasting than iodophors.

  • Alexidine – faster-acting surgical scrub “up and coming”.

Alkylating Agents

  • Agents replace a hydrogen atom with an alkyl group.

  • MOA: Inactivates enzymes and nucleic acids.

  • Formaldehyde – fixes specimens by cross-linking proteins.

  • Glutaraldehyde – acts faster than formaldehyde; a common disinfectant of surgical equipment.

  • Ethylene oxide – gaseous sterilizer that has high penetrating ability.

Peroxygens

  • Oxidizing agents that produce radical oxygen to disrupt macromolecules.

  • Hydrogen peroxide – common & cheap disinfectant.

  • Benzoyl Peroxide – present in acne medications; very effective against Propionibacterium acnes.

  • Carbamide peroxide – agent in toothpaste that combats biofilms.

  • Ozone gas – used to clean air and water supply.

Supercritical Fluids

  • Pressure and temp are increased in molecules to have properties between liquid and gas Ex. Supercritical CO_2.

  • Allows for easier cell penetration and formation of carbonic acid and increase acidity.

  • Non-reactive, non-toxic, & non- flammable.

  • Good for many vulnerable materials such as foods and some tissues.

Preservatives

  • Most inhibit microbial growth in food products.

  • In foods, important to be non-toxic.

  • Examples:

    • Sorbic acid

    • Benzoic acid

    • Propionic acid

    • Sulfur dioxide

    • Nitrites

Disinfectant/Preservative Effectiveness Testing

  • Mandated by many agencies (FDA, EU, etc.).

    • What level of kill can the agent have?

    • How long does it last?

    • What microbes does it work on?

  • Methods:

    • Phenol coefficient – how strong is the agent relative to phenol.

    • Disk diffusion – measures degree of inhibition.

    • In-use test