Chapter 7 - Control of Microbial Growth Flashcards
What is sterilization, its common application, and common agents?
Destruction or removal of all forms of microbial life, including endospores and viruses from an inanimate item
Application: Preparation of surgical equipment and of needles used for injection
Agents: Pressurized steam (autoclave), chemicals, and radiation
What is commercial sterilization?
Sufficient heat treatment to kill endospores of C. botulinum in canned food
More resistant endospores of thermophilic bacteria may survive, but they will not germinate and grow under normal storage conditions
What is disinfection, its common application, and common agents?
Destruction or reduction of vegetative pathogens on inanimate objects through application of heat or antimicrobial chemicals
Application: Cleaning surfaces like laboratory benches, clinical surfaces, and bathrooms
Agents: Chlorine bleach, phenols (Lysol), glutaraldehyde
What is antisepsis, its common application, and common agents?
Destruction or reduction of vegetative pathogens on living tissue through application of an antimicrobial chemical
Application: Cleaning skin broken due to injury; cleaning skin before surgery
Agents: Boric acid, isopropyl alcohol, hydrogen peroxide, iodine (betadine)
What is degerming, its common application, and common agents?
Reduces microbial load on skin or tissue through gentle to firm scrubbing and the use of mild chemicals
Application: Handwashing
Agents: Soap, alcohol swab
What is sanitization, its common application, and common agents?
Reduces microbial load of an inanimate item to safe public health levels through application of heat or antimicrobial chemicals
Application: Commercial dishwashing of eating utensils; cleaning public restrooms
Agents: Detergents containing phosphates, industrial-strength cleaners containing quaternary ammonium compounds
What does bacteriostatic mean?
Inhibit the growth and multiplication of bacteria
Once bacteriostatic is removed, growth could resume
What does bactericidal mean?
Kills bacteria
What is decimal reduction time (DRT)?
Time it takes to kill 90% of the population (1 - log decrease in total population) when exposed to a specific microbial control protocol at a specified temperature
How is decimal reduction time used in sterilization?
Determines effectiveness - Lower DRT indicates a faster reduction of microbial population, meaning it is more efficient
What 4 factors influence the effectiveness of antimicrobial treatments?
Number of microbes
- The more microbes there are to begin with, the longer it takes to eliminate the entire population
Environmental influences (organic matter, temperature, biofilms)
- Concentration of disinfecting agent or intensity of exposure is important
- Higher temperature and higher concentrations of disinfectants kill microbes more quickly and effectively
- Presence of bodily fluids, tissue, organic debris, or biofilms limit contact between the agent and targeted cells, which increases the cleaning time or intensity of the microbial control protocol required to reach the desired level of cleanliness
Time of exposure
- Longer exposure times kill more microbes
Microbial characteristics
- Susceptibility of the microbe to that disinfecting agent or protocol
What should be considered when choosing a microbial control agent?
Cheap, fast, stable, and not harmful
Consider:
- Site to be treated
- Relative susceptibility
- Environmental conditions
What are the 3 levels of germicide?
High - all microbes plus spores
Intermediate - vegetative pathogens, fungal spores, protozoans
Low - vegetative only
What are the 3 mechanisms by which agents kill or inhibit microbes?
Damage to proteins:
- Proteins regulate everything (metabolism) - denaturing proteins/enzymes disrupts cell function
Damage to nucleic acids:
- Direct protein synthesis and replication (mutate = deadly) - destroy or damage is fatal
Alter the cytoplasmic membrane or cell wall:
- Cell wall counteracts the effect of osmosis (prevent bursting) - disruption makes cells more vulnerable
- Cytoplasmic membrane - controls passage of things in and out (disruption of lipid and proteins = inside leaks out)
How does heat kill microbes?
Denatures DNA in proteins
What is thermal death point (TDP)?
Lowest temperature at which all cells in a culture are killed in 10 minutes
What is thermal death time (TDT)?
Time during which all cells in a culture are killed at a specific temperature
Compare the effectiveness of moist and dry heat
Moist heat penetrates better, so it is more effective
Water conducts heat better than air, so the temperature will be distributed better in moist heat methods, making it a better method
Moist heat denatures proteins while dry heat kills by oxidation effects
What are the 3 methods of moist heat and how do they kill microbes?
Boiling: Protein denaturation
- Does not kill all microbes (spores and viruses are not always killed)
- Kills within 10 minutes, sometimes up to 30 minutes for heartier microbes
- Easily accessible
Autoclaving: Protein denaturation
- Pressurized steam - most dependable
- Sterilizing agent is steam, not pressure
- All vegetative cells and their spores are killed in about 15 minutes
- Limitations: plastics, degrade chemicals, oils don’t mix with water
Pasteurization: Protein denaturation
- Reduces spoilage organisms and pathogens
- Not all are killed - those who withstand heat survive
- Batch method - 63℃ for 30 minutes
- High-temperature short-time - 72℃ for 15 seconds
- Ultra-high-temperature pasteurization - 135℃ for 1 second
- Ultra-high temperature sterilization - 140℃ (kills all)
What are the 3 methods of dry heat and how do they kill microbes?
Direct flaming: Burning contaminants to ashes
- Very effective for sterilization
- Used for inoculating loops
Incineration: Burning to ashes
- Very effect for sterilization
- Used for disposal of contaminated dressings, animal carcasses, and paper
Hot-air sterilization: Oxidation
- Very effective for sterilization
- Requires temperature of 170℃ for 2 hours
- Used for empty glassware that is placed in an oven
How does filtration suppress microbial growth?
Separation of bacteria from suspending liquid
High-efficiency particulate air (HEPA) filters - remove microbes from the air
Membrane filters - remove microbes from liquids
Removes microbes by passage of a liquid or gas through a screenlike material
Most filters in use consist of cellulose acetate or nitrocellulose
Useful for sterilizing liquids that are destroyed by heat (i.e., enzymes, vaccines)
What are the 3 methods of cold temperatures and how do they suppress microbial growth?
Refrigeration: decreased chemical reactions and possible changes in proteins
- Has bacteriostatic effect (slows growth)
Deep-freezing: Decreased chemical reactions and possible changes in proteins
- Effective for preserving microbial cultures, food, and drugs
Lyophilization: Decreased chemical reactions and possible changes in proteins
- Most effective for long-term preservation of microbial cultures, food, and drugs
How does high pressure suppress microbial growth?
Alteration of molecular structure of proteins and carbohydrates
How does desiccation suppress microbial growth?
Disruption of metabolism
Involves removing water from microbes
Primarily bacteriostatic
When water is made available, they can resume growth and division
How does osmotic pressure suppress microbial growth?
Plasmolysis
High concentrations of salts and sugars results in hypertonic environment that causes a loss of water from microbial cells
What are the 3 types of radiation and how do they kill microbes?
Ionizing radiation: Destruction of DNA
- Uses gamma rays, X rays, and high-energy electron beams
- Ionizes water to create reactive hydroxyl radicals
- Damages DNA by causing lethal mutations
- Kills all
- Used for sterilizing pharmaceuticals and medical and dental supplies
Nonizonizing radiation: Damages to DNA
- UV light
- Damages DNA by creating thymine dimers that inhibit correct replication during cell reproduction
- Damages but doesn’t kill spores
- Not very penetrating
Microwaves: Heat
- Not much of a direct effect
- Heat kills most vegetative pathogens
What are vegetative pathogens?
Growing form of bacteria
Not spore form
Is radiation safe to use on food?
Yes - process does not make food radioactive
Radiation passes through the food, killing germs without leaving residual radiation
How does pasteurization differ from other types of moist heat microbial control?
Generally uses mild temperatures compared to autoclaving and boiling (63-72 degrees vs. 121 degrees) - exception is ultra-high temperature pasteurization/sterilization that is 135-140 degrees (not often used)
Primary goal is to kill pathogens and reduce number of spoilage microbes, whereas other methods aim for sterilization and killing all microbes
Commonly used in dairy products and other beverages in order to make them safe for consumption, while other methods are used in medical and laboratory settings to sterilize equipment and materials
What 5 factors can affect the efficacy of a disinfectant?
Concentration of disinfectant
Temperature - activity of disinfectant increases as temperature increases, but too high temperature can inactivate it
Presence of organic materials - can interfere with action of disinfectant
pH of medium - certain disinfectants work optimally at different pHs
Contact with microbes - area may need to be scrubbed and rinsed before disinfectant is applied in order to make effective contact
What are the 3 levels of disinfectants?
Low level: kills some viruses and vegetative bacteria (quaternary ammonium compounds)
Intermediate level: kills mycobacterium, most viruses, and all vegetative bacteria (phenolics, halogens, diluted alcohols)
High level: kills all organisms except high levels of bacterial spores and prions (ethylene gas, formaldehyde, glutaraldehyde, hydrogen peroxide)
What are the 5 methods for evaluating disinfectants and antiseptics?
Phenol coefficient
Use-dilution test
In use test
Disk-diffusion
Minimum inhibitory concentration
What are the steps for a phenol coefficient?
Dilute concentration of phenol and see how long it takes to kill organisms in 10 minutes but not 5 minutes
Also dilute disinfectant concentration to see how long it takes to kill organisms in 10 minutes but not 5 minutes
Then divide disinfectant concentration by phenol concentration to see phenol coefficient
What is the protocol for a use-dilution test?
Current standard in US
Utilize 3 bacteria in lab test
Metal rings dipped in pure culture
Dried at 37 degrees
Rings placed in disinfectant for 10 minutes at 20 degrees
Rings placed on media - count number of colonies grown
What is the protocol for an in use test?
Swab area before and after cleaning
Inoculate broth
See if it grows
What is the protocol for a disk-diffusion test?
Evalutates efficacy of chemical agents
Filter paper disks are soaked in a chemical and placed on a culture
Look for zone of inhibition around disks
What is the protocol for a minimum inhibitory concentration test?
Serial dilutions of an antimicrobial agent of known concentration
Inoculate and incubate the tubes
MIC is lowest concentration of antimicrobial agent needed to inhibit growth
After determining MIC, plate bacteria in tubes where there was no growth and put them on a nutrient agar plate and see if anything grows to determine if bactericidal or bacteriostatic
- MBC - minimum concentration that kills bacteria
What is the recommended optimal concentration of alcohol to kill bacteria?
70% ethanol
What are the 4 targets for a chemical antimicrobial?
Cell walls, cytoplasmic membranes, proteins, or DNA
How are different microbes affected by disinfectants?
Often more effect against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa (endospores harder to kill)
Enveloped viruses have a lipid layer outside – easier to desiccate
Non-enveloped viruses have protein layer outside – harder to kill
Gram+ easier to kills than Gram- because of the presence of an outer membrane in Gram-
What is the order of resistance of microbes to chemical biocides from most resistant to least resistant?
10 total
Prions
Endospores of bacteria
Mycobacteria
Cysts of protozoa
Vegetative protozoa
Gram-negative bacteria
Fungi, including most fungal spores
Viruses without envelopes
Gram-positive bacteria
Viruses with lipid envelopes
What are examples of halogens, its mechanism, and applications?
Examples: Chlorine, iodine
Mechanism: Denatures proteins
Applications: Water treatment, skin antisepsis
What are examples of phenolics, its mechanism, and applications?
Examples: Triclosan, chlorohexidine
Mechanism: Denature proteins, disrupt cell membranes
Applications: Surface disinfection, skin antisepsis
What are examples of alcohols, its mechanism, and applications?
Examples: 70% ethanol, 75% isopropyl
Mechanism: Denatures proteins, dissolves lipids, dehydrating agent
Applications: Instrument disinfection, skin antisepsis
What are examples of quaternary ammonium compounds, its mechanism, and applications?
Examples: Cationic detergents
Mechanism: Disrupt lipids in cell membrane
Applications: Instrument disinfection and skin antisepsis
What are examples of heavy metals, its mechanism, and applications?
Examples: Mercury, copper, silver
Mechanism: Denatures proteins
Applications: Skin antisepsis, disinfectants, algaecide in pool and water supply, antiseptic in eyes
What are examples of peroxides, its mechanism, and applications?
Examples: Benzoyl peroxide, hydrogen peroxide
Mechanism: Denatures proteins, radicals attack DNA, lipids, and proteins
Applications: Skin antisepsis, surface disinfection, contact sterilization
What are examples of aldehydes, its mechanism, and applications?
Examples: Glutaraldehyde, formaldehyde
Mechanism: Denatures proteins, inactivates nucleic acids
Applications: Sterilize surgical equipment, embalming, vaccine production
What are examples of sterilizing gases, its mechanism, and applications?
Example: Ethylene oxide gas
Mechanism: Denatures proteins, inactivates nucleic acids
Applications: Sterilize surgical equipment, heat sensitive objects
How do bisphenols work?
Disrupt plasma membrane
How do biguanides work?
Disrupt plasma membrane
What are the 3 types of surface active agents?
Soaps and detergents
Acid-anionic sanitizers
Quaternary ammonium compounds
How do soaps and detergents work?
Mechanical removal of microbes through scrubbing
How do acid-anionic sanitizers work?
Disrupt plasma membrane
What are the 2 types of food preservatives?
Organic acids
Nitrates/nitrites
How do organic acids work?
Metabolic inhibition
Mostly affecting molds
Action not related to acidity
How do nitrates/nitrites work?
Active ingredient is nitrite, which inhibits certain iron-containing enzymes of anaerobes
What is selective toxicity?
An antimicrobial’s ability to selectively find and destory pathogens without damaging the host
Targets: Cell wall synthesis and ribosomes
What is chemotherapy?
The use of chemicals to treat a disease
What is an antibiotic?
A substance produced by a microbe that, in small amounts, inhibits another microbe
What is an antimicrobial drug?
Synthetic substance that interferes with the growth of microbes
What are broad spectrum antibiotics?
Antibiotics that affect a broad range of Gram-positive and Gram-negative pathogenic bacteria
What are narrow spectrum antibiotics?
Antibiotics that target very specific types of bacteria
What are the 5 modes of action of antibiotics?
Inhibition of cell wall synthesis
Inhibition of protein synthesis
Inhibition of nucleic acid synthesis
Injury to plasma membrane
Inhibition of essential metabolite synthesis
How do antibiotics inhibit cell wall synthesis?
Penicillin prevents synthesis of peptidoglycans (beta-lactams)
Vancomycin binds PG subunits
Cell wall is greatly weakened, and the cell undergoes lysis
How do antibiotics inhibit protein synthesis?
Inhibits translation by targeting bacterial 70S ribosomes (30S or 50S subunit)
How do antibiotics inhibit nucleic acid synthesis?
Interfere with DNA replication and transcription (RNA polymerase)
Rifamycin inhibits RNA synthesis
How do antibiotics injure plasma membranes?
Polypeptide antibiotics change membrane permeability
Antifungal drugs combine with membrane sterols
How do antibiotics inhibit essential metabolite synthesis?
Antimetabolites compete with normal substrates for an enzyme
Often inhibit enzymes
What are the 3 tests for antibiotic effectiveness?
Disk-diffusion method (Kirby-Bauer test)
- Tests effectiveness of chemotherapeutic agents
- Paper disks with agent placed on agar containing organism
- Zone of inhibition around the disk determines sensitivity of organism to antibiotic
E test
- Determines MIC
- Gradients of certain antibiotic concentrations are on a strip so you can see what concentration is effective
Broth dilution tests
- Determine MIC and MBC of antibiotic
- Test organism is placed into wells of a tray containing dilutions of a drug - growth is determined
- Antibiograms - reports that record susceptibility of organisms encountered clinically
What are the 5 clinical considerations in prescribing antibiotics?
Routes of administration
- Topical for external infections
- Oral routes are self-administered and no needles
- Intramuscular delivers drug via needle into muscle
- Intravenous delivers drug directly into bloodstream
Toxicity
- Cause of adverse reactions is poorly understood
- Drugs can be toxic to kidneys, liver, or nerves
- Considerations for pregnant women
- Therapeutic index - ratio of drug dose that can be tolerated to the drug’s effective dose
Allergies
- Allergic reaction are rare but can be life-threatening
- Anaphylactic shock
Disruption of normal microbiota
- May result in secondary infections
- Overgrowth of normal flora causing superinfections
- Greatest concern in hospitalized patients
Overuse and misuse of antibiotics
- Antibiotic resistance
