Sterilisation and Disinfection Flashcards
What are the factors determining disinfectant activity?
- Nature of Microorganisms
- Physiologic State of Microorganisms
- Microbial Communities and Biofilms
- Innate Resistance of Microbes
- Concentration of Disinfectant
- Physical and Chemical Factors
- Organic/Inorganic Matter
- Electrolytes
- Additive and Components of Excipients
What are the 4 physiologic states of bacteria?
- Lag Phase – cells preparing for cell replication, not dormant
- Log Phase – cells doubling at constant rate, gradient indicates the speed of the process
- Stationary Phase – growth-limiting factors affect replication, growth rate = death rate
- Death Phase
Why are biofilms more difficult to remove than free microbes?
→ Biofilms are more resistant to drying, UV radiation and microbial agents compared to free microbes
→ Microorganisms may be protected from disinfectants by production of thick masses of cells and extracellular materials or tightly attached biofilms and cannot be easily removed
→ Microbes within biofilms can be resistant to disinfectants by multiple mechanisms – physical layers of biofilms, genotypic variation, neutralising enzymes, physiologic gradients in biofilm etc
→ Tend to have many different strains of bacteria in them – need to be able to cover all
What makes spores resistant to disinfectants?
spore coat and cortex act as barrier
What makes mycobacteria resistant to disinfectants?
waxy cell wall prevents entry of disinfectants
What makes Gram Negative bacteria harder to kill by disinfection?
Gram negative bacteria have outer membrane that acts as barrier to disinfectant uptake
What are the physical and chemical factors that can affect disinfectant activity?
→ Temperature: efficacy generally increases with temperature increase but can lead to degradation
→ pH: increased pH affects differently, by affecting disinfectant molecule or cell surface
Improved antimicrobial activity: glutaraldehyde, QAC
Decreased antimicrobial activity: phenols, hypochlorites, iodine
→ Humidity: affects gaseous disinfectants (most important factor)
→ Water hardness: increasing hardness decreases efficacy as divalent cations react w disinfectant to form insoluble precipitates
What are the factors affecting disinfection efficacy?
- Number of microbes
- Innate resistance of microbes
- Concentration and potency of disinfectant
- Physical and chemical factors
- Organic/Inorganic matter
- Biofilms
- Duration of exposure
What is the mechanism of action of alcohol as a disinfectant?
Leads to denaturation of proteins, mechanism is especially increased by presence of water
What are the uses of alcohol as a disinfectant?
Used for hard-surface disinfection & skin antisepsis, commonly coupled w other excipients/biocides
What is the spectrum of activity of alcohols?
rapid broad spectrum antimicrobial activity against vegetative bacteria (incl. mycobacteria), viruses and fungi, but are not sporicidal
What is ammonia as a disinfectant used for?
Used as general-purpose cleaner for many hard surfaces eg glass, stainless steel
What is the mechanism of action of ammonia as a disinfectant?
Saponifies lipids within envelopes of microorganisms to disrupt lipid layer
What are the dangers of ammonia as a disinfectant?
→ Irritant for eyes and gastric system
→ Cannot be mixed with bleach: may release chloramine gas (toxic)
What is the spectrum of action of aldehydes as disinfectants?
- Bacterial spores: Low concentrations inhibit germination, high concentrations are sporicidal (?Strong interaction with outer cell layers)
- Mycobacteria: Action unknown, but probably involves mycobacterial cell wall
- Other nonsporulating bacteria: Strong association w outer layers of gram-positive and gram-negative bacteria, Cross linking of amino groups in protein, Inhibition of transport processes into cell
- Fungi: Fungal cell wall appears to be a primary target site, with possible interaction with chitin (essential component of cell walls and septa of pathogenic fungi)
- Viruses: Protein-DNA cross links and capsid changes
What is the mechanism of action of aldehydes as disinfectants?
Alkylates sulfhydryl, hydroxyl, carboxyl and amino groups – alter DNA, RNA, protein synthesis
What is paraformaldehyde used to disinfect?
Can be vaporised by heat for gaseous decontamination of laminar flow biologic safety cabinets when maintenance work or filter changes require access to sealed portion of cabinet
What are the benefits of using ortho-phthalaldehyde as a disinfectant?
- OPA is more lipophilic than glutaraldehyde, therefore more likely to be taken up through outer layers of mycobacteria and gram-negative bacteria
- OPA posited to kill spores by blocking spore germination process
- Excellent stability over a wide pH range (pH 3-9), excellent material compatibility
- Not known to irritate eyes & nasal passages, barely any perceptible odour
What are the dangers of using ortho-phthalaldehyde as a disinfectant?
Stains proteins grey (incl unprotected skin)
What is the mechanism of action of chlorhexidine against bacteria?
Damages outer membrane, crosses by passive diffusion, then attacks bacteria cytoplasmic membrane
What is the mechanism of action of chlorhexidine against yeast?
partitions into cell wall, plasma membrane and cytoplasm, then damages yeast plasma membrane
What are the cons of using chlorhexidine as a disinfectant?
dependent on pH, effectiveness greatly reduced in presence of organic matter
How does alexidine compare to chlorhexidine?
Faster onset of bactericidal activity and significantly faster alteration in bactericidal permeability
What are the main mechanisms of action of the chlorine compounds as disinfectants?
Dissociation of hypochlorus acid causes oxidative stress:
- Oxidation of sulfhydryl enzymes, amino acids
- Ring chlorination of amino acids
- Loss of intracellular contents
- Decreased uptake of nutrients
- Inhibition of protein synthesis
- Decreased oxygen uptake
- Oxidation of respiratory components
- Decreased ATP production
- DNA breaks
- Depressed DNA synthesis
What is the spectrum of activity of the chlorine compounds?
Broad spectrum antimicrobial activity, able to remove dried or fixed organisms and biofilms from surfaces
What are the cons/dangers of using the chlorine compounds?
- NaOCl at 5.25-6.15% (household bleach) can produce ocular irritation or oropharyngeal, oesophageal and gastric burns
- Releases toxic chlorine gas when mixed with ammonia or acid
- Corrosive to metals in high concentrations (>500ppm), Discolours or “bleaches” fabrics
- Inactivated by organic matter, overall relatively less stable
- efficacy decreases w increase in pH (encourages conversion of HOCl to OCl- before it can exert disinfecting activity)
What is the mechanism of action of iodine and the iodophors?
Iodine can penetrate cell wall of microorganisms quickly, lethal effects believed to result from disruption of protein and nucleic acid structure and synthesis
What are the disadvantages of using iodine as a disinfectant?
- Skin irritation
- Stain clothes
- inactivated by QACs (widespread in household materials)
- inactivated by organic matter
What is the mechanism of action of hydrogen peroxide as a disinfectant?
Acts as oxidant by producing OH radicals to attack essential cell components, including lipids, proteins and DNA (proposed that exposed sulfhydryl groups and double bonds esp targeted)
What is the spectrum of action of hydrogen peroxide as a disinfectant?
Broad spectrum efficacy against viruses, bacteria, yeast, bacteria spores – Widely used biocide for disinfection, sterilisation, and antisepsis
- Greater activity against gram-positive than gram-negative
- Bacteria can have increased tolerance at lower concentrations if they have catalase
- Higher concs (10-30%) w longer contact times required to be sporicidal, better if gaseous
What are the disadvantages of using hydrogen peroxide as a disinfectant?
- Degrades rapidly
- ocular irritation
- Activity against mycobacteria is limited
- Effectiveness not so much as a liquid, mainly works well as a gas.
What is the mechanism of action of peracetic acid as a disinfectant?
Denatures proteins and enzymes and increases cell wall permeability by disrupting sulfhydryl (-SH) and sulfur (S-S) bonds
What is the spectrum of action of peracetic acid as a disinfectant?
sporicidal, bactericidal, virucidal, fungicidal at low concentrations of ~0.3%
unable to be decomposed by peroxidases and remaining active in the presence of organic loads
What are the uses of peracetic acid as a disinfectant?
Mainly used as a low temperature liquid sterilant for medical devices, flexible scopes, hemodialyzers, also used as an environmental surface sterilant
What are the cons of using chloroxylenol
- Bactericidal – P. aeruginosa & many molds highly resistant
- Generally regarded as relatively nontoxic and nonirritant when used as excipient in topical products, but toxic upon oral consumption or ocular contact
What is the mechanism of action of diamidines as disinfectants?
Inhibit oxygen uptake, cause amino acid leakage
What is the mechanism of action of silver nitrate as a disinfectant?
Interacts with thiol (-SH) groups in enzymes and proteins
What is the mechanism of action of silver sulfadiazine
Produces membrane blebs in bacteria – plasma membrane detached from actin cytoskeleton
What are the dangers of using mercuric chloride as a disinfectant?
Highly toxic, both acutely and as a cumulative poison: Tends to accumulate in the kidneys – severe corrosive damage leading to acute renal failure
What are the mechanisms of QAC as disinfectants?
- Adsorption and penetration into cell wall
- Reaction w cytoplasmic membrane (lipid or protein) followed by membrane disorganisation
- Leakage of intracellular low Mr material
- Degradation of proteins, nucleic acids
- Wall lysis caused by autolytic enzymes
What is the spectrum of action of QAC as disinfectants?
Fungicidal, bactericidal, virucidal (lipophilic viruses only); not effective against TB
What are the advantages of using QAC as disinfectants?
- Low bactericidal concentration of quaternary ammonium salt disinfectants
- colourless
- low odour
- no corrosive bleaching effect
- relatively stable performance, light and heat resistance, easy to store
- low toxicity
- Many MOAs so less likely for resistance since it can work in many ways
What are the disadvantages of using QAC as disinfectants?
- low efficiency
- skin irritation
What are the testing parameters for disinfectants?
→ Exposure time
→ Interfering substance – need to simulate practical conditions
→ Interpretation criteria - Log reduction in a certain amount of time
What is the Sterility Assurance Level?
- The probability of having one viable microorganism in a certain number of drug products
- Defines an acceptable safety level according to pharmacopeial standards
What is the purpose of Aseptic Process Simulation?
→ A method of validating an aseptic process using a microbiological growth medium as a substitute for product and employing method, that closely approximate those used during manufacturing
→ Used to qualify the process at the beginning and to confirm its aseptic capability periodically
→ To check if there are any microbes in the process itself
What are the advantages and disadvantages of moist heat sterilisation?
Adv: Uses biological indicator to verify sufficient heat/pressure
Disadv: Cannot use on heat/moisture sensitive items
What are the advantages and disadvantages of dry heat sterilisation?
Adv: Destroys pyrogens, Easier to operate and maintain
Disadv: Should be used only for materials that might be damaged by moist heat or that are impenetrable to moist heat (eg powders, petroleum products, sharp instruments), Slower rate of heat penetration, Heat may not reach all equipment as effectively as moist heat sterilisation
What are the differences between static air and forced air dry heat sterilisers?
- Forced air sterilizers have a motor-driven fan/blower to circulate heated air throughout the chamber at a high velocity compared to static air steriliser that does not have
- Forced air steriliser is thus faster in heating, takes a shorter time to reach sterilising temperature and is more uniform in temperature control than static air type (but still slow)
What are the advantages and disadvantages of aseptic filtration?
Adv: Can carry out Filter integrity test to verify filter didn’t rupture
Disadv: Filter must be certified for volume and filtrate load
What are the advantages and disadvantages of gamma irradiation?
Adv: Low temperature sterilisation that ensures low penetration and high dose rate, Containers and packaging may remain intact
Disadv: Requires v high radiation to be active, Induces oxidation in polyethylene, not good for some heat-sensitive items
What is the mechanism of action of ethylene oxide as a sterilant?
Alkylating agent – attacks proteins, nucleic acids and other organic compounds
What are the advantages and disadvantages of ethylene oxide as a sterilant?
- Compatible with several materials and is well tolerated; leaves residue but can be avoided with proper aeration
- Flammable and explosive
- Can cause eye pain, sore throat, difficulty breathing, blurred vision, dizziness, nausea, headache, convulsions, blisters, vomiting and coughing; also shown to be carcinogenic
What are the advantages and disadvantages of hydrogen peroxide as a sterilant?
- Inactivates a broad range of microorganisms, including vegetative bacteria (incl mycobacteria), yeasts, fungi, viruses, bacterial spores. (Sporicidal as sterilant, not sporicidal as disinfectant)
- Able to inactivate MRSA, Serratia marcescens, Clostridium botulinum spores and Clostridium difficile from rooms, furniture, surfaces and equipment
- By-products are water and oxygen – nontoxic, don’t need aeration
- Sterilized materials can be handled safely, for immediate use or storage
What are the advantages of peracetic acid as a sterilant?
- Highly biocidal oxidiser, still effective in the presence of organic soil (higher dose 200-500ppm)
What is the spectrum of activity of peracetic acid as a sterilant?
- Inactivates gram-positive and gram-negative bacteria, fungi and yeast in <5mins at <100ppm
- For viruses, higher dose req eg poliovirus inactivated in yeast needs 15mins at 1500-2250ppm
- Bacterial spores inactivated in 15s-30min using 500-10,000 ppm
What are the advantages and disadvantages of lyophilization?
Adv: Ease of processing liquids, High stability of product
Disadv: Expensive, Diluent must be sterile
List the different sterility tests.
- membrane filtration test
- direct inoculation test
- bioburden test
- pyrogen/endotoxin tests (Rabbit, Limulus Amoebocyte, Monocyte, Recombinant Factor C
