PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 9 Controlling Microbial Growth in the Environment Principles of Microbial Control Terminology of Microbial Control Death Rate Action of Anti-microbial agents Selection of Anti-microbial agents Factors affecting efficacy BioSafety Levels Methods of Microbial Control Physical Methods Chemical Methods How to Evaluate AntiMicrobial Agents Bacteristatic vs cidal vs sterilize? Strength and time? In what situation to use them? 1
Operation Room Aseptic (free of pathogens) Disinfection (most) Floor/non-living DeGerming (your hands) mechanical Sterilization(all) instruments Antisepsis (patient) Living tissue Sanitization (most) floor Public place Applies to Pathogens Microbicidal agents do not simultaneously kill all cells. They kill a constant percentage over time. Figure 9.1 A plot of microbial death rate. To evaluate the efficacy of an antimicrobial agent Constant percentage of the extant population is killed each minute Decimal Reduction Time (D) is the time it takes to kill 90% of the population All? Thermal Death Time = time it takes to completely sterilize The Selection of Microbial Control Methods Ideally, agents should be Inexpensive Fast-acting Stable during storage Capable of controlling microbial growth while being harmless to humans, animals, and objects 2
Basic Principles of Microbial Control Action of Antimicrobial Agents Alteration of cell walls and membranes (1/2) Cell wall maintains integrity of cell Cells burst due to osmotic effects when damaged Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell Cellular contents leak out when damaged Proof: Non-enveloped viruses have greater tolerance of harsh conditions Basic Principles of Microbial Control Action of Antimicrobial Agents Damage to proteins and nucleic acids (2/2) Protein function depends on 3-D shape Extreme heat or certain chemicals denature proteins STOP protein synthesis through action on RNA (ribosomes) Eukaryotic 80S ribosomes Prokaryotic 70S ribosomes Mitochondria/Chloroplast also 70S ribosomes Basic Principles of Microbial Control Action of Antimicrobial Agents Damage to proteins and nucleic acids (2/2) Nucleic acids can be altered or destroyed by chemicals, radiation, and heat 3
The Selection of Microbial Control Methods Factors Affecting the Efficacy of Antimicrobial Methods Site to be treated (1/3) Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects Method of microbial control based on site of medical procedure (bed vs scalpel) The Selection of Microbial Control Methods Factors Affecting the Efficacy of Antimicrobial Methods Relative susceptibility of microorganisms (2/3) Endospores protozoan cysts Mycobacterium tuberculosis waxy cell walls Bacteria Vegetative cell endospore boil Waxy lipids Protozoa trophozoite cyst 4
Figure 9.2 Relative susceptibilities of microbes to antimicrobial agents., RNAse enzyme The Selection of Microbial Control Methods Factors Affecting the Efficacy of Antimicrobial Methods Relative susceptibility of microorganisms (2/3) Germicide classification (imagine for medical instruments that cannot be sterilized by heat) High-level germicides (invasive contact) Kill all pathogens, including endospores Intermediate-level germicides (non-invasive but mucous contact) Kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria Low-level germicides (surface contact) Kill vegetative bacteria, fungi, protozoa, and some viruses The Selection of Microbial Control Methods Factors Affecting the Efficacy of Antimicrobial Methods Environmental Conditions (3/3) Temperature (high) ph (low) Good Contact 5
The Selection of Microbial Control Methods Biosafety Levels Four levels of safety in labs dealing with pathogens Biosafety Level 1 (BSL-1) Handling pathogens that do not cause disease in healthy humans Biosafety Level 2 (BSL-2) Handling moderately hazardous agents (hepatitis/influenza virus, MRSA) Biosafety Level 3 (BSL-3) Handling microbes in safety cabinets with HEPA filters (tuberculosis/anthrax bacteria, yellow fever/rocky mountain fever virus) Biosafety Level 4 (BSL-4) Handling microbes that cause severe or fatal disease (smallpox, Lassa fever virus) (E.coli) (Ebola virus) Figure 9.4 A BSL-4 worker carrying Ebola virus cultures. Physical Methods of Microbial Control 5 Physical Methods Heat-Related Methods (extremes of heat) (1.1/5) Heat-Related Methods (extremes of cold) (1.2/5) Dessication and Lyophilization (2/5) Filtration (3/5) Osmotic Pressure (4/5) Radiation (5/5) 6
Physical Methods of Microbial Control Heat-Related Methods (extremes of heat) (1.1/5) Effects of high temperatures Denature proteins Interfere with integrity of cytoplasmic membrane and cell wall Disrupt structure and function of nucleic acids Thermal death point Lowest temperature that kills all cells in broth in 10 min Thermal death time Time to sterilize volume of liquid at set temperature Physical Methods of Microbial Control Heat-Related Methods Moist Heat e.g.? Moist heat Used to disinfect, sanitize, sterilize, and pasteurize More effective than dry heat (water conducts heat better than air) Methods of microbial control using moist heat Boiling Autoclaving Pasteurization Ultrahigh-temperature sterilization Physical Methods of Microbial Control Boiling Kills vegetative/growing cells of bacteria and fungi, protozoan trophozoites, and most viruses Boiling time is critical Endospores (20 hrs), protozoan cysts, and some viruses can survive boiling 7
Physical Methods of Microbial Control Heat-Related Methods Autoclaving Pressure applied to boiling water prevents steam from escaping Boiling temperature increases as pressure increases Autoclave conditions: 121ºC, 15 psi, 15 minutes not for vitamins or some plastics Figure 9.6 The relationship between temperature and pressure. Figure 9.7 An autoclave. How to tell it is working Manual exhaust to atmosphere Pressure gauge Safety valve Valve for steam to chamber Exhaust valve Steam Air Door Steam jacket Material to be sterilized Trap Thermometer Steam supply 8
Figure 9.8 Sterility indicators. Cap that allows steam to penetrate Flexible plastic vial Crushable glass ampule Nutrient medium containing ph color indicator Endospore strip Incubation After autoclaving, flexible vial is squeezed to break ampule and release medium onto spore strip. Yellow medium means spores are viable; autoclaved objects are not sterile. Red medium means spores were killed; autoclaved objects are sterile. B. Stearothermophilus endospores Physical Methods of Microbial Control Pasteurization (kills Brucella melitensis / mycobacterium Bovis / E.coli) Used for milk, ice cream, yogurt, and fruit juices Not sterilization Heat-tolerant (thermoduric) and heat-loving (thermophillic) microbes survive (expiration date on milk) Pasteurization Yeast = Alcohol Bacteria = Acid Beer and wine spoiled due to growth of bacteria Pasteurization = heat it just enough to kill bacteria that generate acid 9
Physical Methods of Microbial Control Pasteurization of milk Batch method Flash pasteurization Ultrahigh-temperature pasteurization/sterilization Dairy Industry Procedures Then cool rapidly Figure 9.6 The relationship between temperature and pressure. Ultra-high temperature pasteurization of milk requires 134 ºC - so what pressure needs to be applied? Physical Methods of Microbial Control Heat-Related Methods Dry Heat e.g.? Dry heat Used for materials that cannot be sterilized with moist heat (powders, oil, metals) Denatures proteins and oxidizes metabolic and structural chemicals Requires higher temperatures for longer time than moist heat 10
Extreme Dry heat = fire Incineration is ultimate means of sterilization Physical Methods of Microbial Control Refrigeration and Freezing (extremes of cold) (1.2/5) Decrease microbial metabolism, growth, and reproduction Chemical reactions occur slower at low temperatures Liquid water not available Refrigeration halts growth of most pathogens Some microbes can multiply in refrigerated foods 11
Physical Methods of Microbial Control dessication e.g.? Desiccation and Lyophilization Desiccation (drying) inhibits growth due to removal of water Lyophilization (freeze-drying) used for long-term preservation of microbial cultures Prevents formation of damaging ice crystals Figure 9.9 The use of desiccation as a means of preserving apricots in Pakistan. Dessication and what? 12
Figure 9.10 Filtration equipment used for microbial control. Nonsterile medium Membrane filter To vacuum pump Sterile medium Nitrocellulose/plastic Pore size important and not thickness Sterilization? Figure 9.11 The roles of high-efficiency particulate air (HEPA) filters in biological safety cabinets. Outside Exhaust HEPA filter Safety glass viewscreen Blower Supply HEPA filter Light High-velocity air barrier 13
Osmotic Pressure Osmotic Pressure To preserve food by inhibiting microbes use Low or High concentrations of salt or sugar in foods Why? 14
Physical Methods of Microbial Control Osmotic Pressure High concentrations of salt or sugar in foods to inhibit growth Why? Cells in hypertonic solution of salt or sugar lose water Fungi have greater ability than bacteria to survive hypertonic environments Physical Methods of Microbial Control Radiation Ionizing radiation e.g? Wavelengths shorter than 1 nm Electron beams, gamma ray, some X rays Why? - Ejects electrons from atoms to create ions Ions disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxyl radicals, denature DNA Gamma rays penetrate well but require hours to kill microbes Figure 9.12 A demonstration of the increased shelf life of food achieved by ionizing radiation. Gamma rays FDA approved 15
Physical Methods of Microbial Control Nonionizing radiation - UV Wavelengths greater than 1 nm Why? Excites electrons, causing them to make new covalent bonds - Affects 3-D structure of proteins and nucleic acids UV light does not penetrate well Suitable for disinfecting air, transparent fluids, and surfaces of objects UV light causes pyrimidine dimers in DNA Figure 7.25 A pyrimidine (in this case, thymine) dimer. Ultraviolet light How to create point mutations Thymine dimer G C T G T T = G G T A C G A C A A C C A T Figure 9.9 The use of desiccation as a means of preserving apricots in Pakistan. Dessication and UV 16
Good Summary Good Summary Chemical Methods of Microbial Control Affect microbes cell walls, cytoplasmic membranes, proteins, or DNA Effect varies with differing environmental conditions Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa Fungal spores, protozoan cysts, bacterial endospores are particularly resistant 17
Chemical Methods of Microbial Control Phenol and Phenolics Denature proteins and disrupt cell membranes Effective in presence of organic matter Remain active for prolonged time Commonly used in health care settings, labs, and homes Have disagreeable odor and possible side effects Figure 9.13 Phenol and phenolics. Phenol Orthocresol Orthophenylphenol Triclosan Hexachlorophene Bisphenolics Phenolics 18
Chemical Methods of Microbial Control Alcohols (rubbing alcohol = isopropanol) Intermediate-level disinfectants Denature proteins and disrupt cytoplasmic membranes 100% not as effective as 70-90% because proteins need water to denature More effective than soap in removing bacteria from hands Swabbing of skin with alcohol prior to injection removes most microbes Fungal spores, bacterial endospores are resistant Chemical Methods of Microbial Control Halogens Why? - Damage enzymes by denaturation Widely used in numerous applications Iodine tablets, iodophores, chlorine treatment, bleach (NaOCl), chloramines (Cl and NH 3 ), and bromine disinfection (evaporates slower than Cl hot tubes), Fluorine 19
Figure 9.14 Degerming in preparation for surgery on a hand. Iodophor (Betadine) iodine containing organic compound that slowly releases iodine Chemical Methods of Microbial Control Oxidizing Agents (release oxygen radicals) Peroxides, ozone, and peracetic acid Kill by oxidation of microbial enzymes Especially effective against anaerobic pathogens High-level disinfectants and antiseptics Hydrogen peroxide can disinfect and sterilize surfaces Not useful for treating open wounds due to catalase activity Peracetic acid is effective sporicide used to sterilize equipment (Not adversely affected by organic contaminants and leaves no residue) 20
Ozone (O 3 ) treatment of drinking water (more effective than Cl) Chemical Methods of Microbial Control Surfactants "Surface active" chemicals Why? - Reduce surface tension of solvents Soaps and detergents Soaps have hydrophilic and hydrophobic ends Good degerming agents but not antimicrobial Detergents are positively charged organic surfactants (More soluble than soaps) Figure 9.15 Quaternary ammonium compounds (quats). Quats colorless/tasteless and harmless Low-level disinfectants Disrupt cellular membranes Ideal for many medical and industrial applications Ammonium ion Cetylpyridinium Quaternary ammonium ions (quats) Benzalkonium Hydrophobic tail 21
mouthwash Chemical Methods of Microbial Control Heavy Metals (As, Zn, Hg, Ag, Cu) Heavy-metal ions denature proteins (by combining with sulfur in cysteine) Low-level bacteriostatic and fungistatic agents 1% silver nitrate to prevent blindness caused by N. gonorrhoeae Thimerosal (Hg) used to preserve vaccines Copper controls algal growth in fish tanks, pools, storage tanks Figure 9.16 The effect of heavy-metal ions on bacterial growth. Dental amalgam 22
Copper kills MRSA Chemical Methods of Microbial Control Aldehydes Compounds containing terminal CHO groups Cross-link functional groups to denature proteins and inactivate nucleic acids 2% Glutaraldehyde disinfects (10 mins) and sterilizes (10 hrs) Formalin (37 % formaldehyde) used in embalming and disinfection of rooms and instruments (carcinogenic) Chemical Methods of Microbial Control Gaseous Agents Microbicidal and sporicidal gases used in closed chambers to sterilize items (4-18 hrs -ethylene oxide, propylene oxide, beta-propiolactone) Cannot expose to heat or water and need to sterilize use ethylene oxide Denature proteins and DNA by cross-linking functional groups Used in hospitals and dental offices Disadvantages Can be hazardous to people, highly explosive, Extremely poisonous, Potentially carcinogenic (beta-propiolactone) 23
Chemical Methods of Microbial Control Enzymes Antimicrobial enzymes act against microorganisms Human tears contain lysozyme Digests peptidoglycan cell wall of bacteria Use enzymes to control microbes in the environment Lysozyme used to reduce the number of bacteria in cheese Prionzyme can remove prions on medical instruments Chemical Methods of Microbial Control Antimicrobials Antibiotics (antimicrobial agents produced naturally by microbes) and semisynthetic and synthetic chemicals Typically used for treatment of disease and not for environmental control (like earlier chemicals) Some used for antimicrobial control outside the body Nisin (kill bacteria in cheese) natamycin (kill fungi in cheese) is Lysozyme an antibiotic? 24
Good Summary Questions from here 4 Methods for Evaluating Disinfectants and Antiseptics 1) Phenol coefficient (1/4) Evaluates efficacy of disinfectants and antiseptics Compares an agent's ability to control microbes to phenol Greater than 1.0 indicates agent is more effective than phenol Phenol coefficient (a) of 100 =?, (b) 0f phenol =? Methods for Evaluating Disinfectants and Antiseptics Methods for Evaluating Disinfectants and Antiseptics 2) Use-dilution Metal cylinders dipped into broth cultures of bacteria, dried Contaminated cylinder immersed into dilution of disinfectant Cylinders removed, washed, and placed into tube of medium Most effective agents entirely prevent growth at highest dilution Current standard test in the U.S. 25
Methods for Evaluating Disinfectants and Antiseptics Kelsey-Sykes capacity test (3/4) Alternative assessment approved by the European Union Bacterial suspensions added to the chemical being tested Samples removed at predetermined times and incubated Lack of bacterial reproduction reveals minimum time required for the disinfectant to be effective Methods for Evaluating Disinfectants and Antiseptics In-use test (4/4) Swabs taken from objects before and after application of disinfectant or antiseptic Swabs inoculated into growth medium and incubated Medium monitored for growth Methods for Evaluating Disinfectants and Antiseptics Development of Resistant Microbes Little evidence that products containing antiseptic and disinfecting chemicals add to human or animal health Use of such products promotes development of resistant microbes 26
FDA bans antibacterial soaps - Sep2016 Rule removes triclosan and triclocarban from overthe-counter antibacterial hand and body washes The agency after some data suggested that longterm exposure to certain active ingredients used in antibacterial products for example, triclosan (liquid soaps) and triclocarban (bar soaps) could pose health risks, such as bacterial resistance or hormonal effects. 27