2. CONTROL OF MICROORGANISMS Flashcards
→ complete removal or destruction of all forms of life, including bacterial spores
→chemical or physical methods
Sterilization
→process that eliminates a defined scope of
microorganisms
→process of killing or removing microorganisms in inanimate surfaces thru the use of chemical agents
Disinfection
→free of life of every kind
Sterile
→destroys pathogenic organisms, but not necessary all microorganisms or spores
→most are chemical substances
Disinfection
→having the property of inhibiting bacterial growth or multiplication
Bacteriostatic
→having the property of killing or destroying bacteria
→precipitates bacterial protein (H2SO4, HCl
Bactericidal
→chemical substance used to kill infection producing microorganisms on surface but too toxic to be applied directly on tissues
Germicide or Disinfectant
→characterized by the presence of pathogenic
microbes in living tissue
Septic
→characterized by the absence of pathogenic
microbes
Aseptic
→chemical substance which opposes sepsis or putrefaction either by killing microorganism or preventing their growth
→applied topically to living tissues
Antiseptic
Examples of Antiseptic
Phisohex, Hexachlorophene, Tincture of Iodine/Povidone Alcohol (Iodophore)
→time in minutes to reduce the bacterial population or spores by 90% at a specified temperature
Decimal Reduction Time (DRT/D/D)
Factors That Influence the Degree of Killing Microorganisms
a. Types of organisms
b. Number of organisms
c. Concentration of disinfecting agent
d. Presence of organic material
e. Nature of surface to be disinfected
f. Contact time
g. Temperature
h. pH
i. Biofilms
j. Compatibility of disinfectants and sterilants
Types of ORGANISMS!
✓Bacterial spores→ resistant
✓Acid-Fast Bacilli
✓Nonenveloped viruses
✓Vegetative bacteria
✓Enveloped viruses
→ most resistant to the actions of heat, chemicals, and radiation
→naked pieces of protein without nucleic acid
Prions
degenerative diseases of the nervous system (transmissible spongiform encephalopathy—mad cow disease)
Creutzfeldt-Jakob disease
blood, mucus, and pus
organic materials
at least 1 to 2 minutes to kill microorganisms
Alcohol and Iodine preparations (Betadine)
Most Resistant → Least Resistant organisms
Prions → Bacterial spores → Mycobacteria → Nonlipid viruses → Fungi → Bacteria → Lipid viruses
PHYSICAL METHODS
Heat
Low/Cold Temperature
Desiccation and Lyophilization
Filtration
Radiation
→most common method used for the elimination of microorganisms
→most reliable and universally applicable method of sterilization
Heat
→kills microorganisms rapidly than dry heat
→Lethal effect is attributed to DENATURATION and COAGULATION of protein and DEGRADATION of nucleic acid
Moist Heat
→fastest and simplest physical method of sterilization
→sterilization method of choice for heat-stable objects
Moist Heat
→ surgical instruments, needles, hypodermic syringes, rubber stoppers
→kills all vegetative organism but not all spores or viruses
Boiling
Boiling (Temperature and Time)
→100°C for 15-30 minutes (20 minutes minimum)
→ Sterilize biohazardous trash and heat-stable objects
→ all microorganisms (except for prions) and their endospores are destroyed within approximately 15 minutes of exposure
Autoclaving
→ chamber which is filled with hot Steam Under Pressure
Autoclave
Two common sterilization temperatures:
121°C (250°F), 15 psi for 15 minutes→ media, liquids, and instruments
132°C (270°F), 15 psi for 30-60 minutes→ infectious medical waste
BIOLOGIC INDICATOR: (Autoclaving)
Geobacillus stearothermophilus (B. stearothermophilus) incubated at 56°C
most commonly used steam sterilizer in the
microbiology laboratory
Gravity Displacement Type
Sterilizing heat-sensitive culture media containing carbohydrates
Flowing Steam
→Destroys vegetative cells and spores after three consecutive days
FRACTIONAL/INTERMITTENT STERILIZATION/ TYNDALLIZATION
Materials are exposed for 30 minutes for 3 successive days -
✓Day 1: Vegetative cells
✓Day 2: Sporulated cells
✓Day 3: Remaining cells
Instrument for flowing steam
Arnold Sterilizer
Flowing Steam (Temperature and Time)
100°C for 30 minutes
Principle: thickening through evaporation
Inspissation
Sterilize high protein containing media that
cannot withstand high temperature
Inspissation
Example of Inspissation:
LJ, Loeffler’s and Dorset egg medium
Inspissation (Temp. and Time)
→70-80°C for 2 hours for 3 consecutive days
→partially sterilizing organic solutions by heat without altering their chemical properties
→used to sterilize milk, dairy products, and alcoholic beverages
Pasteurization/Partial Sterilization
→eliminates food-borne pathogen and organisms responsible for food spoilage
→cannot eliminate bacterial endospores
Pasteurization/Partial Sterilization
3 types of Pasteurization
- Low Temperature Holding (LTH)/Batch Method
- High Temperature Short-Time(HTST)/Flash Pasteurization
- Ultra-high Temperature
→destroys milk-borne pathogens
→63°C for 30 minute
- Low Temperature Holding (LTH)/Batch Method
→72°C for 15 seconds
→quick heating and immediate cooling
- High Temperature Short-Time(HTST)/Flash Pasteurization
→140°C for 3 seconds
→Cooled very quickly in a vacuum chamber
- Ultra-high Temperature
Advantage of Ultra-high temperature Pasteurization
milk can be stored at RT for 2 months without affecting its flavor
→requires longer exposure times (1.5 to 3 hours) and higher temperatures than moist heat (160° to 180°C)
→does not require water
→sterilization of glasswares, oil products or powders
Dry Heat
Lethal Effects of Dry Heat
Protein denaturation, oxidative damage
and toxic effects of elevated levels of
electrolytes
FLAMING OR DIRECT HEATING
Flaming with a Bunsen Burner
Burning with a Bunsen Burner
→flaming mouth of culture tubes or slides
Flaming with a Bunsen Burner
wireloops, forceps and straight wire
Burning with a Bunsen Burner
→most widely used type of dry heat
→used for glasswares, certain metals and oils
→2 hours for 160-180°C kill organisms including all spore formers
Hot Air Oven
Hot Air Oven (QC)
Bacillus subtilis var. niger (Bacillus atrophaeus) at 35-37°C
→Principle: Burning of materials into ashes at 300-400°C
→most common method of treating infectious waste and infected laboratory animals
→destruction of sputum cups, garbage and used dressing
Incineration
considered as HAZARDOUS MATERIAL in Incineration
870-980°C
→burning dead human bodies control the spread of communicable disease
Cremation
Types of Dry Heat
- Flaming or Direct Heating
- Hot Air Oven
- Incineration
- Cremation
→considered BACTERIOSTATIC—reduces the rate of metabolism
Low/Cold Temperature
→exposure to __________ kills the agent of syphilis
2°C for 72 hours
→not reliable method of sterilization
→repeated freezing and thawing are much more destructive than prolonged freezing
→ preservation of bacterial culture via lyophilisation or freeze-drying
Freezing
Lethal effects of Freezing
Protein denaturation, toxic effects of
increased intracellular electrolyte concentration
→disruption of metabolism that involves removing of water from microbes(BACTERIOSTATIC)
Desiccation
→changes in proteins and chemical reactions
Lyophilization
➢Bacteria which remain active in dry environment
✓Neisseria gonorrhoeae →viable for 1 hour
✓MTB →viable for several months
✓Bacillus and Clostridium →viable for ten years
→method of choice for antibiotics solutions, toxic chemicals, radioisotopes, vaccines and carbohydrates, serum, plasma, urea(heat-sensitive solutions)
→may be used with both liquid and air substances
Filtration
Types of Filters
a. Depth Filters
b. Membrane Filter (Circular Filter)
c. Filtration of bacteria, yeasts and molds
d. Critical Sterilizing
→made up of fibrous or granular materials
Depth Filters
(Depth Filters)- Diatomaceous earth
Berkefield Filter
(Depth Filters) - unglazed porcelain
Chamberland Filter
(Depth Filters) - 98% effective
Seitz (Compressed Asbestos)
(Depth Filters) - 100% bacterial sterility
✓Membrane filter(Swinney)Millipore 0.22 um
→porous membranes (almost 0.1um thick)
→composed of cellulose acetate or polycarbonate
→sterilize pharmaceuticals, ophthalmic solutions, culture media, antibiotics and oil products
Membrane Filter (Circular Filter)
→pulling the solution through cellulose acetate or cellulose nitrate membrane with a vacuum
Liquid Filtration
→uses HIGH-EFFICIENCY PARTICULATE AIR filters
→remove microorganisms larger than 0.3 μm from isolation rooms, operating rooms, and biologic safety cabinets (BSCs)
Air Filtration
→uses 0.45 and 0.80 μm pores of membrane filters
→0.2 to 0.45 um in diameter-remove most bacteria as well as fungi but not viruses
→0.01 μm are capable of retaining small viruses
Filtration of bacteria, yeasts and molds
0.22-μm
→ (parenteral solutions and alcohol)
→remove vegetative cells but not viruses
Critical Sterilizing
Principle: when radiation passes through the cells, free hydrogen and hydroxyl radicals and some peroxidase are created which in turn cause different intracellular damage
Radiation
BIOLOGIusC INDICATOR: (Radiation)
Bacillus pumilus
→gamma rays (1500-2500 radiation), electron beams, X-rays
→short wavelength and high energy
Ionizing radiation (Cold Sterilization)
→ used for plastic syringes, catheters, sutures, gloves, hormone solutions and antibiotics
→causes mutation in the DNA and produces peroxidase
→destroys vegetative cells and endospores
Ionizing radiation (Cold Sterilization)
→damage to cellular DNA by producing Thymine dimers
→ultraviolet rays (10 um to 400 um) in which 260 um is the most lethal
→long wavelength(>1um) and low energy
Nonionizing Radiation
→poor penetrability
→used to disinfect exposed surfaces, operating rooms, nursery rooms
→control of airborne infection
Nonionizing Radiation
→no practical value in sterilization and disinfection since there are many survivors after the treatment
Ultrasonic and Sonic Vibrations
used mainly as disinfectants
Chemical Methods
chemical agents used to sterilize
Chemosterilizers
Chemical agents exert their killing effect by the
following mechanisms:
✓Reaction with components of the cytoplasmic
membrane
✓Denaturation of cellular proteins
✓Reaction with the thiol (–SH) groups of enzymes
✓Damage of RNA and DNA
→excellent in vitro bactericidal activity against most gram-positive and gram-negative bacteria
→ also kill Mycobacterium tuberculosis, various fungi, and certain enveloped viruses
→ not sporicidal and have poor activity against certain nonenveloped viruses
Alcohol
Types of Chemical Methods
a. Alcohol
b. ALDEHYDES/COLD/CHEMICAL STERILANTS
c. HALOGENS
d. Quaternary Ammonium Compounds (Quats)/Detergents
e. PHENOLICS
f. Heavy Metals
g. Gas
h. Hydrogen Peroxide and Periacetic Acid
i. Acid and Alkaline Solution
→ inactivated by the presence of organic material
→should be used in concentrations between 60% and 90%
→denaturation of proteins and dissolution of lipid membranes
Alcohol
→used as both antiseptic and disinfectant (bactericidal and fungicidal)
→allowed to evaporate from the surface to achieve complete antisepsis
→Isopropanol and Ethanol
Alcohol
→inactivation of proteins and nucleic acids
→commonly used in sterilizing medical instruments
→8% formaldehyde and 2% gluteraldehyde
Aldehydes/Cold/Chemical Sterilants
→used as formalin, a 37% aqueous solution or
formaldehyde gas
→irritability factor and its potential carcinogenicity
Formaldehyde
often used to disinfectant biosafety
hoods, HEPA filters
Formaldehyde Gas
3%-8% formalin is used with a contact time of at least 30 minutes
Mycobacteria
→saturated five-carbon dialdehyde
→inactivation of DNA and RNA through alkylation of sulfhydryl and amino groups
Glutaraldehyde
→broad-spectrum activity and rapid killing action and remains active in the presence of organic matter
→extremely susceptible to pH changes because it is active only in an alkaline environment
Glutaraldehyde
(Pseudomonocidal, Tuberculoidal, Fungicidal and Virucidal)
Glutaraldehyde
germicidal in approximately 10 minutes and sporicidal in 3 to 10 hours
2% Glutaraldehyde Solution
→does not penetrate organic material well when used as a sterilant
→sterilizer of choice for medical equipment that is not heat-stable and cannot be autoclaved as well as for material that cannot be sterilized with gas
Glutaraldehyde
100% tuberculocidal
2% solution at 25 Celcius to 30 Celcius (Glutaraldehyde)
sporicidal in a minimum of 10 hours’ exposure at room temperature
Cold Sterilants
→destroys through oxidation process
→Chlorine, Iodine, Fluorine, Bromine, Astatine
→tincture of iodine and iodophore—effective antiseptics
Halogens
→effective bleach
→freshly prepared every day with 10-30 minutes of contact time—effective tuberculocide
1:10 NaOCl (Sodium Hypochlorite)
→parasulfone dichloraminobenzoic acid
→contains Cl and is used to disinfect drinking
water
Halozone
→alcohol and iodine solutions
→used mainly as antiseptics
→2%iodine in 70% alcohol
Tincture
→combination of iodine and a neutral polymer(Detergent) carrier that increases the solubility of the agent
→less irritating, nonstaining, and more stable
→used as antiseptics or disinfectants
Iodophor
→slow and continuous release of free iodine
→Free iodine degrades microbial cell walls and cytoplasm, denatures enzymes, and coagulates chromosomal material
→Bactericidal action is due to the oxidative effects of molecular iodine (I2) and hypoiodic acid (HOI)
Iodophor
Contact time: (Iodophor)
30-60 seconds onto the skin prior to blood
collection
→hypochlorite—liquid sodium hypochlorite (household bleach) and solid calcium hypochlorite
→oxidative effects of hypochlorous acid, formed when chloride ions are dissolved in water
→not used as sterilants because of the long exposure time required for sporicidal action
Chlorine and Chlorine Compounds
→inactivated by organic matter
→concentrated bleach solutions should not be used for disinfection—Corossive
→influenced by the pH of the surrounding medium
Chlorine and Chlorine Compounds
→used for disinfection
→stable for no longer than 30 days
0.5% to 1% Sodium Hypochlorite
→ recommended by CDC for cleaning up blood spill
1 : 10 dilution of 5.25% Sodium Hypochlorite
Contact time: (Chlorine and Chlorine Compounds)
3 minutes and longer if organic material is
present and 10-30 minutes for mycobacteria
Disadvantage: (Chlorine and Chlorine Compounds)
ineffective use in the presence of large amount of protein
→derived by substitution of the four-valence ammonium ion with alkyl halides
→cationic, surface-active agents, or surfactants, that work by reducing the surface tension of molecules in a liquid
→effectiveness is reduced by hard water and soap, and they are inactivated by excess organic matter
Quaternary Ammonium Compounds (Quats) / Detergents
→disruption of the cellular membrane, resulting in leakage of cell contents
→not sporicidal or tuberculocidal
→disinfection of noncritical surfaces such as benchtops and floors
Quaternary Ammonium Compounds (Quats) / Detergents
resistant to quaternary ammonium
compounds
Pseudomonas aeruginosa
Example of Detergents
Zephiran (Benzalkonium Choride and Ceepryn) and cetylpyridium chloride
→molecules of phenol (carbolic acid) that have been chemically substituted, typically by halogens, alkyl, phenyl, or benzyl groups
→ortho-phenylphenol and ortho-benzyl-para-chlorophenol
→not sporicidal
Phenolics
→stable, biodegradable, and relatively active in the presence of organic material
→disruption of cell walls, resulting in precipitation of proteins
→able to disrupt enzyme systems—lower concentration
→found in germicidal soaps
Phenolics
→disrupts the microbial cell membrane and precipitates the cell contents
→more effective against gram-positive than gram-negative bacteria and has less activity against fungi and tubercle bacilli (0.5% to 4%)
Chlorhexidine Gluconate
→inactive against bacteria spores except at elevated temperatures
→binds to the skin and remains active for at least 6 hours
→pH-dependent: 5.5 - 7.0
Chlorhexidine Gluconate
Lipid enveloped viruses - rapidly activated
(herpesvirus, HIV, respiratory viruses,
influenza virus, cytomegalovirus)
Nonenveloped viruses - nor inactivated
rotavirus, adenovirus, enterovirus
→effective against gram-positive bacteria
→chlorinated bisphenol
→interrupts bacterial electron transport, inhibits membrane-bound enzymes at low concentrations, and ruptures bacterial membranes at high concentrations
Hexachlorophene
3% hexachlorophene within 15 to 30 seconds
Gram-positive bacteria
3% hexachlorophene for longer time
Gram-negative bacteria
→halogen substituted phenolic compound
→cell wall disruption and enzyme inactivation(0.5% to 4%)
→good activity against gram-positive bacteria, less active against gram-negative bacteria M. tuberculosis, fungi, and viruses
Chloroxylenol (Parachlorometaxylenol [PCMX])
→unaffected by organic materials
→neutralized by nonionic surfactants and polyethylene glycol
→intermediate-acting to slow-acting and has minimal persistent effect of more than a few hours
→low antimicrobial efficacy compared with iodines, iodophors, and CHG in reducing skin flora
Chloroxylenol
→diphenyl ether that disrupts the cell wall
→reaction time is intermediate; persistence is excellent
→good activity against gram-positive bacteria, gram-negative bacteria, and viruses
Triclosan
→fair activity against M. tuberculosis and poor activity against fungi
→not affected by organic matter but affected by pH and the presence of surfactants and emollients
Triclosan
→slowly bactericidal; bacteriostatic
→inactivating and precipitating cell protein
Example: copper, arsenic, mercury, silver and zinc
Heavy metals
→used as a prophylactic treatment to prevent gonococcal (Neisseria gonorrhoeae) conjunctivitis in newborns
1% Silver nitrate (1% eye drop solution)
Factors affecting GAS
temperature, time, and relative humidity are extremely important in determining the effectiveness of gas sterilization
→ most commonly used for sterilization
→explosive in its pure form; mixed with nitrogen or carbon dioxide
→relative humidity of 30% is optimal for
the destruction of spores
→alkylation of nucleic acids in the spore and vegetative cell
Ethylene Oxide
Recommended Concentration of Ethylene Oxide
450 to 700 mg of ethylene oxide/L of chamber space at 55° C to 60° C for 2 hours
→primarily used as a sterilant in the pharmaceutical and medical device
→against all vegetative microorganisms and bacterial and fungal spores
→used in a gaseous form as a sterilant primarily
Hydrogen Peroxide and Periacetic Acid
→hydrolyzes and coagulate proteins
Acid and Alkaline Solutions
→standard reference material for the evaluation of disinfectants
→highest dilution that kills the bacteria after a 10-minute exposure
→potency or bactericidal power of a disinfectant is compared with phenol
Phenol COefficient
Test organisms for PHENOL COEFFICIENT
Staphylococcus aureus and Salmonella
serotype typhi (20°C or 37°C)
lowest concentration that kills the test organisms in 10 minutes at 20°C
End point
