General Consideration for Microbial Growth
- Decontamination Methods
- Relative Resistance of Microbial Forms
- Highest Resistance
- Endospores of bacteria
- Moderate Resistance
- Mycobacteria
- Cysts of protozoa
- Vegetative protozoa
- Gram- bacteria
- Fungi, including most fungal spores.
- Least Resistance
- Viruses without envelopes.
- Gram+ bacteria.
- Viruses with lipid envelopes. (least resistance)
- General Terms
- Sterilization: a process that kills all life, including viruses & endospores.
- Disinfection: a process of possibly killing or reducing growth of microbes on a non-living surface.
- Sanitization: any cleaning technique that removes dirt and usually microbes.
- Antisepsis: a process of killing or reducing growth of microbes on living tissue.
- Chemotherapy: process of treating a disease with chemicals inside the host.
- Bacteriocidal: a substance that kills bacteria.
- Bacteriostatic: a substance that kills the growth of bacteria. If the substance is removed, bacteria resumes growth.
- Antibiotic: a natural substance produced by one organism (usually a microbe) in tiny quantities to kill or inhibit another.
- Aseptic: microbe-free, asepsis prevents entry of pathogens into host.
- Pasteurized: heat-treated to reduce numbers of microbes and destroy pathogens (eg liquids).
- Sanitary: safe and clean, pathogen-free non-living objects (eg water).
- Fungicidal/static, Virucidal/static, aglicidal/static, pesticidal/static.
- Microbial Death
- The permanent loss of reproductive capability, even under optimum growth conditions, has become the accepted microbiological definition of death.
- Most practical way to detect damage/cell death for microorganisms is by exposing it to a suitable environment and determine if it can reproduce.
- Most susceptible: cells to microbicidal agent are younger, actively growing cells.
- Least susceptible: older, inactive cells.
- Factors of Microbial Death
- Temperature: if temp goes up or down , so does metabolism. Diffusion rates!
- Cold & slow growing cells are going to be hard to kill.
- Environment:
- pH , Chemical interference.
- Organic matter: chemical interference and may protect microbes.
- Presence of solvents and other molecules.
- Number of Microbes
- Age & Stage in Life Cycle of Microbe: actively growing/dormant.
- Cell Structure & function: bacerial cell wall type.
- Gram+ or Gram- or wall-less: Mycobacteria (mycolic acid wall), endospores, cysts, spores/eucaryotes.
How Microbial Agents Work: Their Modes of Action
- Mode (or mechanism) of action: antimicrobial agent's adverse effect on cells.
- Agents on the Cell Wall
- Chemical agents used to damage cells wall by blocking its synthesis, digesting it, or breaking down its surface.
- Result in fragile cells which later lyses very easily.
- Anitibial drugs (penicillin): interfere with synthesis of cell wall in some bacteria.
- Detergents and alcohols can disrupt cell walls especially gram-negative bacteria.
- Agents on the Cell Membrane
- Surfactant: detergents that work by lowering the surface tension of cell membranes.
- Surfactants are polar molecules with hydrophilic regions that can bind to membrane and penetrate internal hydrophobic regions.
- Similar structure to lipids in membrane, but they act as replacements with lower tension.
- Agents on Protein and Nucleic Acid Synthesis
- Some agents used to inhibit ability to reproduce and metabolize by inhibiting protein synthesis.
- Chlororamphenicol: a microbiotic that binds to the ribosomes of bacteria in a way that stops peptide bonds from forming.
- Some agents impede transcription or translation.
- An agent can bind to DNA and prevent both transcription and and translation.
- Others are mutagenic.
- X-radiation causes mutation that result in permanent inactivation of DNA.
- Formaldehyde and ethylene oxide interfere with DNA and RNA function.
- Agents That Alter Protein Function
- Denature: disrupt proteins by bonds of secondary or tertiary structure are broken.
- Example: coagulation by moist heat.
- Chemicals: organic solvents (alcohol, acids) or phenolics.
- Metalic Ions
- Methods of inhibiting metabolism.
- Practical Concerns
- 1. Must spores be destroyed or only vegetative pathogens?
- 2. Re-usability?
- If reusable, it must be a quick and least expensive method.
- Can it withstand head, pressure, radiation, or chemicals?
- 3. Practical application?
- 4. Penetrate to the necessary extent?
- 5. Safe?
Physical Control
- Physical agents: physical means to destroy or remove contaminant.
- Heat
- Elevated temperatures are microbicidal where lowered temperatures are microbistatic.
- Moist Heat (hot water, boiling water, steam)
- Range: 60-135 degrees Celsius
- Most heat works faster than dry heat; at the same length of time, moist heat kills cells at lower temp.
- Moist heat denatures cell proteins.
- Membranes, ribosomes, RNA, and DNA are also damaged by moist heat.
- Dry Heat (flame or electric coil)
- Range: 160-several thousand degrees Celsius.
- Oxidizes cells.
- Dehydrates cell components.
- Can denatures protein and DNA, but requires more heat than moist heat due to proteins more stable in dry heat.
- Heat Resistance
- Endospore
- Endospores have greatest resistance to heat.
- Spores of B. antrhacis requires boiling water at 100 degrees Celsius for minutes.
- Thermophilic spores can take hours.
- Vegetative Cells (including yeasts, molds, and other spores)
- Moist heat: 50-60 degrees Celsius for 60 minutes.
- All non-heat resistant forms of bacteria, yeast, molds, protozoa, worms, and viruses destroyed by exposure to 80 degrees Celsius for 20 minutes.
- Viruses
- Relatively resistant to heat.
- Thermal Death Time (TDT): shortest length of time required to kill all microbes at a specified temperature.
- Common Methods of Moist Heat
- Autoclave: pressure-temperature method that can subject pure steam to pressures greater than 1atm.
- Most efficient pressure-temperature is 15psi which results in 121 degree Celsius.
- Great for glassware, cloth (surgical dressings), rubber (gloves), metallic instruments, liquids, paper, some media, and some heat-resistant plastics.
- Ineffective for sterilizing substances that repel moisture (oils, waxes, powders).
- Intermittent Sterilization (Tyndallization)
- Items in chamber are exposed to free-flowing steam for 30 to 60 minutes, incubated for 23-24 hours, then repeat the process 3 days in a row.
- Used for culture media with sera, egg, or carbohydrates and some canned foods.
- Pasteurization
- Technique in which heat is applied to liquids to kill potential agents of infection and spoilage.
- Flash Method: 71.6 degrees Celsius for 15 seconds.
- Batch Method: 63- 66 degrees Celsius for 30 minutes
- Ultrahigh Temperature (UHT) Method: 134 degrees Celsius for 1 to 2 seconds.
- Used to produce sterile milk which has shelf-life of 3 months.
- Do not kill endospores, Coxiella, Mycobacterium, or thermoduric species (lactobacilli, micrococci, and yeasts).
- Boiling Water
- Exposing items to boiling water (100 degrees Celsius) for 30 minutes can kill most non-spore forming pathogens.
- Can quickly decontaminate items.
- Items can easily be recontaminated when removed from water.
- Common Methods of Dry Heat
- Incineration: heat treatment from flame or heating coil.
- Flame of Bunsen burner is 1,870 degrees Celsius.
- Furnaces/incinerators burn from 800-6,500 degrees Celsius.
- Dry oven: heat is circulated in an enclosed compartment.
- Sterilization requires 150-180 degrees Celsius for 2 to 4 hours (destroys spores).
- Used for glass-ware, metallic instruments, powders, and oils.
- Method not suitable for plastics, cotton, and paper, which may burn at high temp.
- Cold and Desiccation
- Cold merely retards the activities of most microbes.
- Vegetative cells exposed to normal room air gradually become dehydrate (desiccated).
- Many viruses and fungal spores can also withstand long periods of desiccation.
- Lyophilization: freezing and drying is a common method to preserve microorganisms and other cells in a viable state for many years.
- Radiation: energy emitted from atomic activities and dispensed at high velocity through matter or space.
- Electromagnetic Radiation
- Microbial controlled done with gamma ray, X-ray, and ultraviolet ray levels.
- Particle Radiation
- High-speed electron (beta-particle or cathode ray)
- Ionizing Radiation Versus Non-Ionizing Radiation
- Ionizing radiation: radiation ejects orbital electrons from an atom and causes ions to form.
- Causes chemical changes in organelles and the production of toxic substances.
- Gamma rays, X-rays, and high-speed electrons are all ionizing in their effects.
- Method of cold sterilization (absence of heat).
- Dosage measured in rads (radiation absorbed dose).
- Gamma ray most penetrating, then X-rays, then finally cathode rays.
- Non-ionizing: radiation that excites atoms by raising them to a higher energy state.
- Results in abnormal linkages within molecules such as DNA and results in mutations.
- UV rays (100nm-400nm)
- Most lethal dose from 240nm to 280nm, but peaks at 260nm.
- UV rays are absorbed by DNA and damages pyrimidine bases (thymine and cytosine) to form pyrimidine dimers.
- UV rays also disrupt cells by generating toxic photochemical products (free radicals).
- Destroys fungal cells and spores, bacterial vegetative cells, protozoa, and viruses.
- Vegetative spores are 10Xs more resistant to radiation, but longer exposure time can kill them.
- Disinfectant technique but can also be used to treat water.
- Poor penetrating power through solid material.
- Sound Waves
- Frequencies from 15,000 to 200,000 cycles per seconds (supersonic to ultrasonic) disrupt cells.
- Gram negative rods most sensitive to ultrasonic vibrations.
- Gram positive cocci, fungal spores, and bacterial spores are most resistant to them.
- Heat is also generated from sonic waves (up to 80 degrees Celsuis).
- Used in dental and some medical centers to clear debris and saliva from instruments before sterilization and to clean dental restorations.
- Also used for removing plaque and calculus from teeth.
- Sterilization by Filtration: Techniques for Removing Microbes
- Effective for removing microbes from air and liquids.
- Most made from thin membranes of cellulose acetate, polycarbonate, and a variety of plastic materials (Teflon, nylon).
Chemical Agents in Microbial Control
- Chemical agents: chemical means to destroy or remove contaminants.
- Three Levels of Chemical Decontamination
- High-level germicides kill endospores, and if properly used, are sterilants.
- Intermediate germicides kill fungal (not bacterial) spores, resistant pathogens such as tubercle bacillus, and viruses (used for non-invasive equipment).
- Low-level germicides kill vegetative bacteria and fungal cells, and some viruses (used for materials that may touch the skin, not mucous membrane).
- Dilutions
- Chemical (solute) is added to water (solution), aqueous.
- Noted at (solute):(solution) --> For 1 part Lysol there are 100 parts water in 1:100.
- Penetration
- Smooth, solid objects are easier to disinfect due to rough objects can collect dirt or contaminants in crevices that the disinfectant can't penetrate.
- Germicidal Categories According to Chemical Group
- Halogens: non-metallic elements that commonly occur in minerals, seawater, and salts
- Fluorine, bromine, chlorine, and iodine.
- Fluorine and bromine difficult and dangerous to handle.
- Microbicidal (sporicidal with longer exposure time)
- Chlorine (CL2), hypochlorite (Clorox), chloramines.
- Denaturation of proteins.
- (disrupts disulfide bonds)
- Can be sporicidal.
- Formula: 8 drops of solid bleach per gallon, if looks cloudy, double. Boil for 10 minutes after.
- Iodine (I2), iodophors (betaiodine)
- Denatures proteins.
- Can be sporicidal.
- Milder medical & dental degerming agents, disinfectants, ointments.
- Kills protozoans.
- Phenol (Phenolics)
- Damage cell membrane & precipitate proteins; bactericidal, fungicidal, virucidal, not sporicidal.
- Active in organic matter.
- Stable & Persistent
- Lysol
- Triclosan: soap antibacterial additive.
- Chlorhexidine
- Hibiclens , Hibitane.
- Halogen & phenol compound.
- A surfactant & protein denatured with broad microbicidal properties.
- Not sporicidal.
- Low toxicity.
- Used as skin degerming agents for scrubs.
- Alcohol
- Ethyl alcohol, isopropyl.
- Solutions of 70-95%
- 70% better than 100% due to more dilutive more effective.
- Dissolve membrane lipids and coagulating proteins of vegetative bacterial cells and fungi.
- Swabs
- Not sporicidal.
- Action depends on concentration, but generally microbistatic.
- Hydrogen Peroxide
- Weak (3%) to strong (35%)
- Produce highly reactive hydroxyls: (free radicals) that damage protein & DNA while also decomposing to O2 gas, toxic to anaerobes.
- Strong solutions are sporicidal.
- Detergents & soaps <--QUATS
- Quaternary ammonium compounds are surfactants that alter membrane proteins of some bacteria and fungi. Hospital cleaners.
- Not sporicidal.
- Smells nice.
- Soaps: mechanically remove dirt/soil and grease containing microbes.
- Heavy metals
- Solutions of silver & mercury kill vegetative (wont kill spores) cells in low concentrations by inactivating proteins.
- Oligodynamic action.
- Not sporicidal.
- Eg. silver nitrate, thimerosol.
- Aldehyde
- Glutaraldehyde & Formaldehyde kill everything by crosslinking/alkylating proteins & DNA.
- Glutaraldehyde in 2% solution (T.B.) used as sterilant for heat sensitive instruments.
- Formaldehyde: disinfectant, preservative, toxicity limits use.
- Formaldehyde sources: common in household.
- Gases and Aerosols
- Ethylene oxide, propylene oxide, betapropiolactone, and chlorine dioxide.
- Strong alkylating agents, sporicidal.
- Equipment, bedding, disposables, sterilization.
- Very toxic (eyes, skin, mucus, membranes). Carcinogenic?
- Organic Acids & Food Preservatives
- Used in foods to inhibit microbial growth.
- Sulfur dioxide
- Sodium benzoate/benzoic acid
- Sorbic acid
- Propionic acid/Ca propionate
- Nitrates and Nitrites