Unit 5 Slides (1) PDF - Microbiology

TRADITIONAL CULTURING METHODS • Conditions are usually optimized for rapid growth. • Most studies on bacteria have been carried out on bacteria in this state. • Bacteria grown in a liquid media in a flask are generally fast growing single cells behaving as individuals. One E. coli cell 43 hours o...

TRADITIONAL CULTURING METHODS • Conditions are usually optimized for rapid growth. • Most studies on bacteria have been carried out on bacteria in this state. • Bacteria grown in a liquid media in a flask are generally fast growing single cells behaving as individuals. One E. coli cell 43 hours of exponential growth with doubling every 20 minutes What does the fact that Earth is not a big ball of E. coli tell you about how bacteria live? Bacteria don’t have unlimited resources. They spend most of their life in stationary phase – not multiplying. TRADITIONAL CULTURING METHODS • Conditions are usually optimized for rapid growth. • Most studies on bacteria have been carried out on bacteria in this state. • Bacteria grown in a liquid media in a flask are generally fast growing single cells behaving as individuals. • This style of growth is called “planktonic” growth. Planktonic E. coli All cells are similar and for the most part behave as individuals. May the best germ win. You Your cells have many different specialized roles and cooperate for the greater good – your health. Frequently sacrifice themselves. BIOFILMS – ORGANIZED BACTERIAL COMMUNITIES • Bacteria and fungi frequently live in organized communities stuck on surfaces called “biofilms” Most live in polysaccharide/protein-encased communities attached to surfaces ➢ Cause slipperiness of rocks in stream bed, slimy “gunk” in sink drains, scum in toilet bowls, dental plaque ➢ Cells in a biofilm have physiological properties different than planktonic cells ➢ Grow slowly compared to planktonic ➢ Have water channels ➢ Copyright © The McGraw-Hill Companies, Inc. MEDICAL IMPLICATIONS OF BIOFILMS • Biofilms are hard to kill Far more resistant to antibiotics and disinfectants ➢ Not easy to remove with mechanical force ➢ • Biofilms have important implications Industrial concerns: accumulations in pipes, drains ➢ Dental plaque leads to tooth decay, gum disease ➢ Involved in many tissue infections (e.g., ear infections, cystic fibrosis lung infections, vaginosis, bone infections) ➢ Copyright © The McGraw-Hill Companies, Inc. BIOFILMS ON MEDICAL DEVICES Biofilms have been very problematic for their ability to form on medical devices including contact lenses, artificial bone/ joint replacements, dentures and false teeth, and catheters and intravenous lines. Because they are difficult to kill completely with antibiotics or antifungal medications, biofilms often form the source of recurrent infections like bone and urinary tract infections. Copyright © The McGraw-Hill Companies, Inc. FORMATION OF A BIOFILM Planktonic bacteria move to the surface and adhere. Bacteria multiply and produce extracellular polymeric substances (EPS). Other bacteria may attach to the EPS and grow. Cells communicate and create channels in the EPS that allow nutrients and waste products to pass. Copyright © The McGraw-Hill Companies, Inc. Some cells detach and then move to other surfaces to create additional biofilms. QUORUM SENSING AND CELL-CELL COMUNICATION • Social behaviors (behaviors coordinated between several cells) in bacteria are often controlled by chemicals the bacteria produce called autoinducers. • Bacteria secrete autoinducers as a signal to neighboring cells. The cells producing autoinducers also sense the autoinducers they produce. When the concentrations of these autoinducers reach a threshold concentration the bacteria switch into a different mode of behavior. This mode of regulation is known as “quorum sensing”. • “Signaling molecule” in the figure above is synonymous with autoinducer INTERACTIONS OF MIXED MICROBIAL COMMUNITIES • Prokaryotes regularly grow in close association with other species Interactions can be cooperative ➢ Can foster growth of species otherwise unable to survive ➢ Create microenvironments ➢ Strict anaerobes can grow in mouth if others consume O2 ➢ Metabolic waste of one microbe can serve as nutrient for other ➢ Interactions often competitive ➢ Some synthesize toxic compounds to inhibit competitors ➢ Can use a secretion system to hit competing bacteria with a spike containing an enzyme that destroys peptidoglycan ➢ • Difficult to re-create these conditions in a laboratory setting ➢ Limits our understanding of microbes ENVIRONMENTAL FACTORS INFLUENCING MICROBIAL GROWTH • Prokaryotes inhabit nearly all environments Some live in comfortable habitats favored by humans ➢ Associated with disease & food spoilage ➢ Some live in harsh environments ➢ ➢ Termed extremophiles; most Archaea • Major conditions that influence growth Temperature ➢ Atmosphere ➢ pH ➢ Water availability ➢ http://deepseacreatures.org/amazing/deep-sea-hydrothermal-vents TEMPERATURE REQUIREMENTS • Each species has well-defined temperature range • Optimum growth usually close to upper end of range Psychrophile: –5° to 15°C ➢ Found ➢ in Arctic and Antarctic regions Psychrotroph: 20° to 30°C ➢ Important ➢ Thermophiles: 45° to 70°C ➢ Common ➢ 35° to 40°C Psychrotroph Psychrophile in hot springs Hyperthermophiles: 70° to 110°C ➢ Usually Hyperthermophile Thermophile Mesophile Mesophile: 25° to 45°C ➢ Pathogens ➢ in food spoilage Growth rate ➢ members of Archaea ➢ Found in hydrothermal vents –10 0 10 20 30 40 50 60 70 80 90 100 110 120 Temperature (°C) Copyright © The McGraw-Hill Companies, Inc. TEMPERATURE REQUIREMENTS • Proteins of thermophiles resist denaturing due to amino acid sequence of proteins ➢ Number stability and position of these bonds determines protein structure & • Temperature and food preservation ➢ ➢ Refrigeration (~4°C) slows spoilage by limiting growth of otherwise fastgrowing mesophiles ➢ Psychrophiles, psychrotrophs can still grow but slowly Freezing preserves food; not effective at killing microbes ➢ Recall: Freezing is also a method to preserve stock cultures • Temperature and disease ➢ Microbes cause disease in certain parts of the human body based on temperature differences ➢ E.g., Hansen’s disease (leprosy) involves coolest regions (ears, hands, feet, fingers) due to preference of M. leprae OXYGEN REQUIREMENTS OF MICROBES • Aerobes (obligate aerobes) – Grow only in the presence of oxygen and strictly use oxygen for their respiration • Facultative anaerobes – can get energy via respiration using oxygen and also, when necessary, by fermentation. • Obligate anaerobes – cannot use or tolerate oxygen • Aerotollerant anaerobes – do not use oxygen but can tolerate it • Microaerophiles (microaerobes)– grow in the presence of low concentrations of oxygen. OXYGEN REQUIREMENTS • Growth in a “shake tube” can determine requirement for O2 Boil nutrient agar to drive off O2; cool to just above solidifying temperature; inoculate; gently swirl; allow media to harden ➢ Solidified agar slows gas diffusion - O2 high at top & bottom is anaerobic ➢ ➢ Growth occurs in region of suitable O2 OXYGEN REQUIREMENTS Reactive Oxygen Species • O2 used in aerobic respiration produces harmful reactive oxygen species (ROS) as by-products ➢ Includes superoxide (O2–) and hydrogen peroxide (H2O2) • Damaging to cellular components Targets DNA, RNA, proteins, and lipids ➢ Modifies these components to disrupt cellular processes & structure ➢ • Cells must have mechanisms to protect against ROS ➢ Obligate anaerobes typically do not OXYGEN REQUIREMENTS Reactive Oxygen Species • Almost all organisms growing in presence of oxygen produce enzyme superoxide dismutase ➢ Inactivates superoxide by converting it to O2 and H2O2 • Almost all also produce catalase ➢ Convert H2O2 to O2 and H2O ➢ Exception is aerotolerant anaerobes ➢ Useful test for 2 groups of medically important aerobic bacteria ➢ Staphylococcus species (catalase +ve) & Streptococcus species (catalase –ve) pH • Bacteria survive a range of pH & have an optimum pH Most maintain constant internal pH, typically near neutral (pH 7) ➢ Pump out protons if in acidic environment ➢ Bring in protons if in alkaline environment ➢ Acidophiles grow optimally at pH below 5.5 ➢ ➢ Picrophilus oshimae has optimum pH of less than 1! Alkaliphiles grow optimally at pH above 8.5 ➢ Most microbes are neutrophiles ➢ Range of pH 5 (acidic) to 8 (basic); optimum near pH 7 ➢ Food can be preserved by increasing acidity ➢ H. pylori grows in stomach; produces urease to split urea into CO2 and ammonia to decrease acidity of surroundings ➢ WATER AVAILABILITY • All microorganisms require water for growth • Dissolved salts, sugars interact with water - unavailable to cell • If solute concentration is higher outside of cell, water diffuses out (osmosis) ➢ Causes cytoplasm to dehydrate & shrink from cell wall plasmolysis Cytoplasmic membrane pulls away from the cell wall (plasmolysis). Dissolved substances (solute) Cytoplasmic membrane Cell wall Water flows out of cell Copyright © The McGraw-Hill Companies, Inc. WATER AVAILABILITY • Growth inhibition of high salt & sugar used in food preservation ➢ ➢ High levels of salt in bacon, salt pork, anchovies High sugar in jams, jellies and honey – natural preservative • Some microbes can withstand or even require high salt ➢ ➢ Halotolerant: withstand up to 10% salt ➢ e.g., Staphylococcus – inhabit dry, salty skin environment Halophiles: require high salt concentrations ➢ E.g. Many marine bacteria require ~3% salt ➢ Extreme halophiles (Archea) require ≥ 9% salt (Dead Sea, Utah’s salt flats) SUMMARY PRINCIPLES OF CONTROL • Sterilization: removal or destruction of all microorganisms so they can no longer multiply or revive ! Sterile item is free of microbes including endospores and viruses but does not consider prions (not destroyed by standard sterilization) • Disinfection: elimination of most or all pathogens ! ! ! Some viable microbes may remain Disinfectants used on inanimate objects ! May be called biocides, germicides, bactericides Antiseptics used on living tissues • Pasteurization: brief heating to reduce number of spoilage organisms, destroy pathogens ! Foods, inanimate objects PRINCIPLES OF CONTROL • Decontamination: reduce pathogens to levels considered safe to handle • Sanitization: substantially reduced microbial population that meets accepted health standards ! Not a specific level of control • Preservation: process of delaying spoilage of foods and other perishable products ! ! Adjust conditions to slow microbial growth Add bacteriostatic preservatives (growth-inhibiting but do not kill) SITUATIONAL CONSIDERATIONS H2O treatment Home life • Microbial control methods depend upon situation and level of control required Food industry © Digital Vision Ltd. © Don Tremain/Getty Images © Bob Daemmrich/The Image Works Hospitals Copyright © The McGraw-Hill Companies, Inc.. © Ryan McVay/Getty Images © Royalty Free/Corbis Other industries DAILY LIFE • Washing and scrubbing with soaps and detergents achieves routine control ! ! ! ! Soap aids in mechanical removal of organisms Beneficial skin microbiota reside deeper on underlying layers of skin, hair follicles so they are not NOT adversely affected by regular use Hand washing with soap and water most important step in stopping spread of many infectious diseases Other methods include cooking food, cleaning surfaces, and refrigeration HOSPITALS & OTHER HEALTHCARE FACILITIES • Minimizing microbial population very important because of the dangers of healthcare-associated infections – also called nosocomial infections ! ! ! ! Patients more susceptible to infection because of weakened condition May undergo invasive procedures (e.g., surgery), which cuts intact skin that would otherwise prevent infection Pathogens more likely found in hospital setting because of high concentration of patients with infectious disease ! Feces, urine, respiratory droplets, bodily secretions shed pathogenss Instruments must be sterilized to avoid introducing infection to deep tissues MICROBIOLOGY LABORATORIES • Routinely work with microbial cultures • Use rigorous methods of control microorganism growth • Must eliminate microbial contamination to both experimental samples and environment ! ! Careful treatment both before (sterile media) and after (sterilize cultures, waste) Aseptic techniques used to prevent contamination of samples, self, laboratory • CDC and PHAC guidelines for labs working with microbes ! Biosafety levels range from BSL-1 (microbes not known to cause disease) to BSL-4 (lethal pathogens for which no vaccine or treatment exists) BIOSAFETY LEVELS! (ALSO CALLED “CONTAINMENT LEVELS”) FOOD & FOOD PRODUCTION FACILITIES • Perishables retain quality longer when contaminating microbes destroyed, removed, inhibited ! ! ! ! Heat treatment most common and reliable mechanism ! Can alter flavor, appearance of products Irradiation approved to treat certain foods Chemical additives can prevent spoilage CFIA/FDA regulates use of irradiation & chemical additives because of risk of toxicity • Facilities must keep surfaces clean and relatively free of microbes to avoid contamination of large quantities of product WATER TREATMENT FACILITIES • Ensure drinking water free of pathogens • Chlorine is traditionally used to disinfect water ! ! Saved countless numbers of lives by preventing spread of waterborne illnesses Can react with naturally occurring chemicals to form disinfection byproducts (DBPs), which have been linked to long-term health risks • Some organisms resistant to (traditional) chemical disinfectants parvum (causes diarrhea) ! Regulations require facilities to minimize DBPs and C. parvum in treated water ! Cryptosporidium SELECTION OF AN ANTIMICROBIAL PROCEDURE • Complicated since every method has disadvantages to limit their use ! Ideal, multi-purpose, non-toxic method does not exist • Choice of procedure depends on many factors including ! ! ! ! ! Type of microbe Number of contaminating microbes Environmental condition Risk for infection Composition of the item TYPES OF MICROBES • Products potentially contaminated with highly resistant microbes require more rigorous treatment • Bacterial endospores: ! ! Most resistant form of life encountered. Only extreme heat or chemicals completely destroys E.g. Bacillus, Clostridium sp. • Protozoan cysts and oocysts: ! ! ! Excreted in feces & cause diarrheal disease if ingested Resistant to disinfectants but easily destroyed by boiling E.g. Giardia lambia • Mycobacterium species: ! Waxy cell walls makes resistant to many chemical treatments. Stronger, more toxic chemicals used to disinfect TYPES OF MICROBES • Pseudomonas species: ! ! Common environmental organism that can cause serious heatlhcareassociated infections. Resistant to and can actually grow in some disinfectants. • Naked viruses: ! ! Lack lipid envelope & more resistant to disinfectants and detergents but still susceptible to chlorine ! E.g. Polio virus Enveloped viruses (e.g. HIV) are sensitive to these chemicals NUMBER OF MICROBES • Time for heat or chemicals to kill is affected by population size since only a fraction of population dies during given time interval Large population = more time • Decimal reduction time (D value) gauges commercial effectiveness • Time required to kill 90% of population under specific conditions 108' Log'decrease'of'1' 107' Number'of'surviving'cells'(logarithmic'scale)' ! 106' Logarithmic' killing' 105' 104' 103' Log' decrease' of'1' 102' 101' D ' ' 1 ' 0 D ' 30' 60' 90' Time'(min)' Copyright © The McGraw-Hill Companies, Inc.. 120' 150' ENVIRONMENTAL CONDITIONS • Dirt, grease, body fluids can interfere with heat penetration, action of chemicals • Another reason why is it so important to thoroughly clean items before disinfection or sterilization • pH, temperature can influence effectiveness of microbial death ! ! E.g., sodium hypochlorite (household bleach) solution can kill suspension of M. tuberculosis at 55°C in half the time as at 50°C Even more effective at low pH RISK FOR INFECTION • Medical instruments categorized according to risk for transmitting infectious agents – greater threat, more rigorous procedure • Critical items come in contact with body tissues ! ! Include needles and scalpels Must be sterile • Semicritical instruments contact mucous membranes but do not penetrate body tissues ! ! ! Includes endoscopes and endotracheal tubes Must be free of viruses and vegetative bacteria Few endospores that remain are blocked by mucous membranes & pose little risk • Non-critical instruments contact unbroken skin only ! ! Include countertops, stethoscopes, blood pressure cuffs Low risk of transmission USING HEAT TO DESTROY MICROORGANISMS • Heat treatment useful for microbial control ! ! ! Reliable, safe, relatively fast, inexpensive, non-toxic Can be used to sterilize or disinfect Methods include moist heat, dry heat MOIST HEAT • Destroys microbes by irreversibly denaturing proteins • Boiling (100C, sea level) destroys most microorganisms and viruses but does not sterilize since endospores can survive • Pasteurization destroys pathogens to protect from disease and spoilage organisms to enhance product shelf life ! ! High-temperature–short-time (HTST) ! Currently the most common method used ! Milk: 72°C for 15s; ice cream: 82°C for 20s Ultra-high-temperature (UHT) – also known as ultra-pasteurization ! Shelf-stable boxed juice and milk ! Milk: 140°C for a few seconds, then rapidly cooled THE AUTOCLAVE • Sterilization Using Pressurized Steam ! Heat and moisture-tolerant items (surgical instruments, microbiological media, glassware) are sterilized using an autoclave ! Water in autoclave is heated to form steam, which increases pressure in the chamber ! Increased pressure raises temperature such that endospores are killed – the pressure itself does not play a role in killing ! Sterilization typically occurs at 121°C and 15 psi for 15 min ! Longer for larger volumes because of penetration Exhaust'valve'to' remove'steam' aJer'sterilizaLon' Valve'to' control'steam' to'chamber' Pressure'gauge' Safety' valve' Door' Steam' Air' Jacket' Thermometer' Trap' Copyright © The McGraw-Hill Companies, Inc. Pressure' regulator' Steam'supply' MOIST HEAT • Steam must displace air in the containers of autoclaved items ! Long, thin containers used & they are never closed tightly • Flash sterilization at higher temperature can be used for rapid processing of items that must be readily available • To destroy prions autoclave at 132°C for 1 hour Exhaust'valve'to' remove'steam' aJer'sterilizaLon' Valve'to' control'steam' to'chamber' Pressure'gauge' Safety' valve' Door' Steam' Air' Jacket' Thermometer' Trap' Copyright © The McGraw-Hill Companies, Inc. Pressure' regulator' Steam'supply' MOIST HEAT • Tape with heat-sensitive indicator indicates items heated but may not indicate sterility • Biological indicators ensure autoclave working properly ! G. stearothermophilus – endospores put in a media. If you see a colour change in the media after autoclaving it means the bacterial spores survived and Copyright*©*The*McGraw3Hill*Companies,*Inc.** MOIST HEAT • Commercial Canning Process ! ! ! ! Uses industrial-sized autoclave called retort Designed to destroy Clostridium botulinum endospores but other organisms that grow under normal storage conditions also killed ! Aim to reduce 1012 endospores to only 1 (12 D process) ! Virtually impossible to have so many endospores Critical because endospores can germinate in canned foods; cells grow in low-acid anaerobic conditions and produce botulinum toxin Canned food commercially sterile ! Endospores of some thermophiles may survive but they are usually not a concern as they only grow at temperatures well above normal storage DRY HEAT • Less effective than moist heat and requires longer times, higher temperatures ! ! 200°C for 90 minutes (dry) vs. 121°C for 15 minutes (moist) Hot air ovens oxidize cell components & denature proteins • Incineration a method of dry heat sterilization ! Burns cell components to ashes ! Used to destroy medical waste and animal carcasses FILTRATION • Some material cannot withstand heat treatment • Retains bacteria from heat-sensitive solutions (e.g., beer) ! Membrane filters (microfilters) Thin, small pore size (0.2 µm) ! Pore size can extend below size of smallest virus but flow of liquid slowed ! Vacuum or pressure applied to aid movement of liquid through filter ! ! Depth filters Filter' Flask' Thick porous filtration material Sterilized' fluid' (e.g., cellulose) ! Complex passage – diameter of passage larger than microbe but electrical charges on filter walls trap cells ! Vacuum' pump' Copyright © The McGraw-Hill Companies, Inc. RADIATION • Electromagnetic radiation – e.g. radio waves, microwaves, visible and ultraviolet light, X rays, and gamma rays ! ! High frequency = short wavelength = higher energy Ionizing vs. non-ionizing radiation. 200 Wavelength (nm) 400 500 300 Ultraviolet (UV) light 600 700 Visible light Ionizing radiation Gamma rays 10–5 10–3 X rays UV ' 1 Infrared Microwaves 103 106 Wavelength (nm) 109 Increasing energy Crest Increasing wavelength Radio waves 1012 One wavelength Trough Copyright © The McGraw-Hill Companies, Inc. RADIATION • Ionizing radiation – X rays and gamma rays - enough energy to remove electrons from atoms ! Destroys DNA and damages cytoplasmic membranes but also causes indirect damage by reacting with O2 to produce reactive oxygen species ! Endospores are most radiation-resistant microbial forms while Gram negative species such as Salmonella & Pseudomonas are most susceptible ! Applications of ionization radiation: ! Sterilize heat-sensitive materials & can be used after packing. ! Effectiveness ! Approved in destroying microbes depends on dose applied for use on foods - FDA has approved for fruits, vegetables, and grains (for insect control), pork (for parasite control), and poultry, beef, lamb, and pork (for bacterial control) RADIATION • Ultraviolet radiation ! ! ! ! ! Destroys microbes directly by damaging their DNA Used to destroy microbes in air, water, and on surfaces ! Actively multiplying organisms easily killed whereas endospores are the most UV-resistant Poor penetrating power ! Thin films or coverings (on equipment or microbe) can limit effect, so it cannot be used to kill microbes within solids or turbid liquids ! Most types of glass and plastic block out UV Most effective when used at close range against exposed microorganism Must be carefully used since damaging to skin, eyes and promotes development of skin cancers RADIATION ! Consumer concerns: ! Can cause unwanted changes in taste of certain foods ! Misconception that food becomes radioactive ! Toxins or carcinogens are subsequently present in food - scientific evidence indicates that irradiated food is safe to consume ! People will rely too heavily on irradiation and ignore other foodsafety practices – irradiation intended to complement, not replace, these procedures ULTRA HIGH PRESSURES • High Pressure – also called “pascalization” ! Used in some types of commercial foods (e.g., guacamole) ! Avoids problems with high temperature pasteurization by using high pressure (up to 130,000 psi) ! Destroys microbes by denaturing proteins and altering cell permeability ! Products maintain color, flavor associated with fresh food SUMMARY USING CHEMICALS TO DESTROY MICROORGANISMS & VIRUSES USING CHEMICALS TO DESTROY MICROORGANISMS & VIRUSES • Germicidal chemicals are predominantly used to disinfect but can also sterilize (in some cases). • Typically contain several antimicrobial chemicals & other chemicals (such as buffers) • Generally less effective than heat but useful for treating countertops, surfaces, bathroom fixtures, doorknobs & heatsensitive items. • Typically act by irreversibly binding protein, DNA, cytoplasmic membranes, or viral envelopes but their exact mechanisms of action are often poorly understood POTENCY OF GERMICIDAL CHEMICAL FORMULATIONS METHOD Sterilant DESTROYS CANNOT DESTROY All microbes (incl. virus & endospores All viruses, vegetative High-level disinfectant cells Intermediate-level disinfectant Vegetative bacteria, mycobacteria, fungi, most viruses Low-level disinfectant Destroy fungi, vegetative bacteria, enveloped virus APPLICATIONS Heat-sensitive, critical instruments (scalpels) Endospores Semi-critical instruments (GI endoscope) Certain viruses, endospores Disinfect non-critical instruments (stethoscopes) Mycobacteria, nonenveloped virus, endospores Disinfect furniture, walls, floors in hospitals SELECTING APPROPRIATE GERMICIDAL CHEMICAL • Toxicity: benefits of disinfecting must be weighed against risk of use • Activity in presence of organic material ! Many germicides inactivated • Compatibility with material being treated ! Liquids cannot be used on electrical equipment • Residues: can be toxic or corrosive • Cost and availability • Storage and stability ! Concentrated stock decreases storage space • Environmental risk ! Agent may need to be neutralized before disposal CLASSES OF GERMICIDAL CHEMICALS • Alcohols ! ! ! ! 60–80% aqueous solutions of ethyl or isopropyl alcohol ! Kills most vegetative bacteria and fungi but not reliable against endospores and some naked viruses Denatures essential proteins (e.g., enzymes) & damages lipid membranes ! More effective mixed in water; pure alcohol less effective Commonly used as antiseptic and disinfectant ! Relatively non-toxic & does not leave residue Antimicrobial chemicals such as iodine are sometimes dissolved in alcohol - called tinctures. CLASSES OF GERMICIDAL CHEMICALS • Aldehydes ! ! ! Glutaraldehyde, formaldehyde, and orthophthalaldehyde work by inactivating proteins and nucleic acids. Orthophthalaldehyde is a relatively new and slightly safer alternative to glutaraldehyde. Immersion in 2% alkaline glutaraldehyde for 10–12 hours kills all microbial life. Shorter times for orthophthalaldehyde. Formaldehyde used as gas or as formalin (37% solution) ! Effective germicide that kills most microbes quickly ! Used to kill bacteria, inactivate viruses for vaccines, & preserve specimens ! Limited use – irritant & suspected carcinogen CLASSES OF GERMICIDAL CHEMICALS • Biguanides ! Chlorhexidine most effective & commonly used antiseptics ! Stays on skin, mucous membranes ! Relatively low toxicity ! Destroys vegetative bacteria, fungi, some enveloped viruses ! Applications ! Skin cream, mouthwash ! Chlorhexidine-impregnated catheters and implanted surgical mesh are used in medical procedures. ! Side effects rare but allergic reactions reported CLASSES OF GERMICIDAL CHEMICALS • Ethylene oxide ! ! ! ! ! Useful gaseous sterilant that destroys microbes including endospores and viruses by reacting with proteins Penetrates fabrics, equipment, implantable devices (e.g. pacemakers) Useful in sterilizing heat- or moisture-sensitive items Sterilize disposable laboratory items (e.g. petri dishes, pipettes) Applied in special chamber resembling autoclave when used to sterilize ! Sterilize in 3-12 hours but toxic ethylene oxide removed by heated forced air for 8-12 hours ! Limitations: mutagenic and potentially carcinogenic CLASSES OF GERMICIDAL CHEMICALS • Halogens: oxidize proteins, cellular components • Chlorine: Destroys all microorganisms and viruses ! ! ! ! Used as disinfectant but not an antiseptic since it is caustic to skin and mucous membranes 1:100 dilution of household bleach effective Very low levels disinfect drinking water but not enough to kill Cryptosporidium oocysts or Giardia cysts Presence of organic compounds a problem • Iodine: Kills vegetative cells but unreliable on endospores ! ! ! Some Pseudomonas species can survive in stock solution Commonly used as iodophore (iodine slowly released from carrier) Iodophores not as irritating to skin as tincture nor are they as likely to stain CLASSES OF GERMICIDAL CHEMICALS • Ozone ! ! ! O3 - unstable form of oxygen but powerful oxidizing agent Decomposes quickly, so generated on-site Used as alternative to chlorine to disinfectant drinking & wastewater CLASSES OF GERMICIDAL CHEMICALS • Peroxygens: powerful oxidizers used as sterilants ! Biodegradable, no residue, less toxic than ethylene oxide, glutaraldehyde • Hydrogen peroxide: effectiveness depends on surface ! Aerobic cells produce enzyme catalase which breaks down H2O2 to O2 & H2O ! Doesn’t damage most materials (rubber, plastics, steel, glass) ! Hot solutions of H2O2 used in food industry ! Vaporized H2O2 can be used as sterilant • Peracetic acid: more potent than H2O2 ! Effective on organic material, no residue, useful on variety of material ! Strong, sharp odour – irritates skin & eyes CLASSES OF GERMICIDAL CHEMICALS • Phenolic Compounds (Phenolics) ! ! ! ! ! ! Phenol (a.k.a. carbolic acid – used by Lister) one of earliest disinfectants Phenol has unpleasant odor, irritates skin Phenolics kill most vegetative bacteria, & mycobacterium (at high concentrations – 5-19%) but are not reliable on all virus groups Phenolics destroy cytoplasmic membranes, denature proteins Wide activity range, reasonable cost, remain effective in presence of detergents and organic contaminants and leave antimicrobial residue which can be good or bad depending on the specific purpose Triclosan is a phenolic compound that is sufficiently non-toxic to be used in soaps, lotions, and toothpaste. The most common antimicrobial found in commercial “antibacterial” soaps. Inhibits the bacterial synthesis of fatty acids used to make membranes. CLASSES OF GERMICIDAL CHEMICALS • Quaternary Ammonium Compounds (Quats) ! ! ! ! ! Cationic (positively charged) detergents Most household soaps, detergents are anionic Nontoxic, used to disinfect food preparation surfaces Charged hydrophilic and uncharged hydrophobic regions ! Reduces surface tension of liquids, which facilitates removal of dirt, organic matter, organisms Positive charge of quats attracts them to negative charge of cell surface ! Reacts with membrane ! Destroys vegetative bacteria and enveloped viruses but not effective on endospores, mycobacteria, naked viruses ! Pseudomonas resists, can grow in solutions SUMMARY