General Consideration for Microbial Growth
Physical Control
AgentCategory Level of Activity Target Microbes Chlorine IntermediateSporicidal (slowly) Iodine IntermediateSporicidal (slowly) Phenolics Intermediate to Low Some bacteria, viruses, fungi Alcohols IntermediateMost bacteria, viruses, fungi Hydrogen peroxide, stabalized High Sporicidal Quaternary Ammonia Compounds Low Some bactericidal, virucidal, fungicidal activity. Soaps Very Low Certain very sensitive species. Mercurials Low Weakly microbistatic. Silver Nitrate Low Bactericide Glutaraldehyde High Sporicidal Formaldehyde Low to Intermediate Sporicidal Ethylene Oxide Gas High Sporicidal Dyes Low Weakly bactericidal, fungicidal. Chlorhexidine Low to Intermediate Most bacteria, some viruses, fungi
REFERENCES:
https://sites.google.com/site/rccmicrobiology/chapter-7-elements-of-microbial-nutrition-ecology-and-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.
- 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: 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
- Germicidal Categories Summary
AgentCategory Level of Activity Target Microbes Chlorine IntermediateSporicidal (slowly) Iodine IntermediateSporicidal (slowly) Phenolics Intermediate to Low Some bacteria, viruses, fungi Alcohols IntermediateMost bacteria, viruses, fungi Hydrogen peroxide, stabalized High Sporicidal Quaternary Ammonia Compounds Low Some bactericidal, virucidal, fungicidal activity. Soaps Very Low Certain very sensitive species. Mercurials Low Weakly microbistatic. Silver Nitrate Low Bactericide Glutaraldehyde High Sporicidal Formaldehyde Low to Intermediate Sporicidal Ethylene Oxide Gas High Sporicidal Dyes Low Weakly bactericidal, fungicidal. Chlorhexidine Low to Intermediate Most bacteria, some viruses, fungi
REFERENCES:
https://sites.google.com/site/rccmicrobiology/chapter-7-elements-of-microbial-nutrition-ecology-and-growth