6-Ch.7 Control of Microbial Growth Physical & Chemical.pdf

Ch.7 – Control of Microbial Growth Norovirus “winter vomiting bug” causes gastroenteritis or “infectious diarrhea” virus infects/replicates in small intestine  Ch.7 – Control of Microbial Growth o microbial death rate o microbial control...

Ch.7 – Control of Microbial Growth Norovirus “winter vomiting bug” causes gastroenteritis or “infectious diarrhea” virus infects/replicates in small intestine  Ch.7 – Control of Microbial Growth o microbial death rate o microbial control - mechanisms of action o physical methods of control o chemical methods of control o evaluating chemical methods of control  Learning Objectives: o Present the modes of action, microbial spectrum, and clinical use of disinfectants and other antimicrobial agents used to treat infections. o Differentiate sterilization, disinfection and antiseptic. o Describe the roles of antagonism and synergism in treating infections.  The Terminology of Microbial Control Sepsis: refers to bacterial contamination Asepsis: is the absence of significant contamination Aseptic surgery techniques prevent the microbial contamination of wounds Sterilization: removing and destroying all microbial life heat most common method Commercial sterilization: killing Clostridium botulinum endospores from canned goods Clostridium botulinum produces deadly neurotoxin – canned goods: enough heat applied to kill endospores other thermophilic endospores survive – cause food spoilage but not human disease Disinfection: destroying harmful microorganisms Antisepsis: destroying harmful microorganisms from living tissue De-germing: the mechanical removal of microbes from a limited area Sanitization: lowering microbial counts on eating utensils to safe levels Biocide (germicide): treatments that kill microbes Bactericidal: kills the bacteria Bacteriostatic: inhibiting, not killing, the bacteria. Keeping them in the stationary phase of growth  The Rate of Microbial Death Effectiveness of treatment depends on: 1. Number of microbes 2. Environment (organic matter, temperature, pH, biofilms) 3. Time of exposure 4. Microbial characteristics Exponential Death Rate: logarithmic death rate: o When heated (or disinfectant): bacteria die at constant rate: o plotting death rate logarithmically if 90% of 1million microbes killed in 1 minute – 100k survivors linearizes curve 90% of 100k in 1min. = 10k survivors etc…  It takes longer to kill ALL MEMBERS of a larger population than a smaller one If rate of killing is the same – will take longer to kill larger population logarithmic plot of death rate: large vs. small population  Ch.7 – Control of Microbial Growth o microbial death rate o microbial control - mechanisms of action o physical methods of control o chemical methods of control o evaluating chemical methods of control  Microbial Control Agents: Mechanisms of Action 1. Alteration of cell wall or plasma membrane permeability damage to sugars, lipids or proteins causes cell contents to leak out Cleenol disinfectant 2. Damage to proteins (enzymes) denaturing proteins – lose “folding” structure, thus function enzymes often targeted 3. Damage to nucleic acids damage – can not replicate or produce proteins usually heat, radiation or chemicals 4. Interfering with protein synthesis ribosomes, DNA or RNA polymerase Heat or Chemicals o most common methods of control  Ch.7 – Control of Microbial Growth o microbial death rate o microbial control - mechanisms of action o physical methods of control o chemical methods of control o evaluating chemical methods of control  Physical Methods of Microbial Control I. Heat - denatures enzymes o Thermal death point (TDP): Lowest temperature at which all cells in a liquid culture are killed in 10 min o Thermal death time (TDT): Minimal time for all bacteria in a liquid culture to be killed at a particular temperature o Decimal reduction time (DRT): Time to kill 90% of a population at a given temperature Ia - Moist Heat Sterilization moist heat denatures proteins boiling free-flowing steam protein denaturing  Autoclave: steam under pressure autoclave 121⁰C at 15 psi for 15 min kills all organisms and endospores steam must contact the item's surface large containers require longer sterilization times test strips are used to indicate sterility Sterilization Indicators 1b - Pasteurization high temp / short time kills pathogenic organisms reduces number of spoilage microbes to safe level (expiration date!) does not denature proteins (taste!) equivalent treatments for treating at 63C for 30 min: o high-temperature short-time (HTST): 72C for 15 sec kills pathogens / lowers bacterial counts – keeps well refrigerated o ultra-high-temperature (UHT): 140C for 4 sec sterilization – milk stored room temp. several months milk pasteurization plant thermoduric organisms: organisms able to survive pasteurization 1c - Dry Heat Sterilization “Bacinerator” o kills by oxidation flaming incineration hot-air sterilization – 170C / 2hrs.  II. Filtration passage of substance through a screen-like material used for heat-sensitive materials high-efficiency particulate air (HEPA) filters remove microbes >0.3 µm membrane filters remove microbes >0.22 µm membrane filtration technique  SEM – wet vs. dry bacterial growth III. Low temperature - has bacteriostatic effect inhibits growth – doesn’t kill Refrigeration – slows or prevents growth Listeria grows at low temp. IV. High pressure - denatures proteins V. Deep-freezing VI. Desiccation - absence of water prevents metabolism can remain viable for years!!! add H2O – will resume growth and division Lyophilization - freeze drying VII. Osmotic pressure – use of salts and sugars to create hypertonic environment causes plasmolysis VIII. Radiation Sterilizing Radiation – radiation that kills microorganisms 1. Ionizing radiation – very short wavelength, very high energy (gamma, xray, electron beams) 2. non-ionizing radiation – longer wavelength, less energy (UV – thymine dimers)  Ionizing radiation - X rays, gamma rays, electron beams o super, super, super high energy o ionizes water to create reactive hydroxyl radicals hydroxyl radical (OH) neutral form of hydroxide ion (OH-) o damages DNA by causing lethal mutations thymine dimer light energy wavelength spectrum Nonionizing radiation - UV, 200-400 nm o damages DNA by creating thymine dimers Microwaves – low energy wavelength o kills by heat; reacts with water to heat o not affective sterilization with solid / thick foods DNA “break” from ionizing o not antimicrobial radiation physical methods of controlling microbial growth: summary tables  Ch.7 – Control of Microbial Growth o microbial death rate o microbial control - mechanisms of action o physical methods of control o chemical methods of control o evaluating chemical methods of control  Chemical Methods of Microbial Control Principles of effective disinfection: o concentration of disinfectant o pH o time – longer contact increases effectiveness table of chemical disinfectants and effect on different microorganism groups  Disinfectant types: 1. Phenol and Phenolics injure lipids of plasma membranes, causing leakage phenolic phenol = carbolic acid o rarely used currently as antiseptic or disinfectant skin irritant and odorous phenolics essential oils: phenol mixture extracted from plants o altered phenol to reduce  centuries old traditional medicine used to preserve food irritating qualities and increase antimicrobial activity 2. Bisphenols o contain two phenol groups connected by a bridge Triclosan – antibacterial soaps, toothpaste, mouthwash inhibits enzyme that synthesizes lipids for membrane o disrupt plasma membranes especially effective against gm+ bacteria o examples: Hexachlorophene and Triclosan Hexachlorophene particularly effective against gm+ Staphylococci and Streptococci 3. Biguanides: o organic compound (formula: HN(C(NH)NH2)2) 4. Halogens – reactive! o water soluble - gives highly basic solution o iodine (I2) – one of oldest/most effective antiseptics o broad spectrum of activity Impairs protein synthesis and alters membranes o primarily targeting bacterial cell membranes o most effective against gm+ o are effective against gm-: except Pseudomonas Chlorhexidine: periodic used in surgical hand scrubs table disrupt plasma membranes Chlorine highly electronegative oxidizing agent! shuts down cellular enzyme systems Bleach: hypochlorous acid (HOCl) Chloramine: chlorine + ammonia  5. Alcohols (-OH) o denature membrane proteins and dissolves membrane lipids o no effect on endospores and non-enveloped viruses o best effect on capsulated/enveloped bacteria and virus o ineffective as antiseptic against wounds – cause coagulation layer of protein – bacteria can grow o ethanol - pure ethanol less effective than diluted requires water to react and denature proteins o isopropanol – “rubbing alcohol” best “alcohol” antiseptic and disinfectant  6. Heavy Metals and Their Compounds oligodynamic action — very small (oligo) amounts of heavy metals exert antimicrobial activity o several metals are bactericidal or antiseptic - Ag, Hg, Cu, Zn denatures protein – metal ions combine with sulfhydryl groups on proteins - denaturing  mercuric chloride prevents mildew in paint  copper sulfate is an algicide (kills algae)  zinc chloride is used in mouthwash  silver nitrate is used to prevent ophthalmia neonatorum Ophthalmia Neonatorum bacterial infection as path through birth canal silver nitrate used as treatment antimicrobial effects of sulfur containing proteins of bacteria metals  7. Surface-Active Agents or Surfactants substance which lowers the surface tension of the medium in which it is dissolved soaps and detergents Soaps: good de-germing method but little value as antiseptic “emulsifies” fats – breaks them up - then wash away Acid-anionic sanitizors: combinations of acid with surface-active agents effective only at low pH phosphoric acid combined with surface agent Quaternary ammonium compounds (quats): strong bases and their salts derived from ammonium modifications of ammonium ion (NH4+) - affect membrane permeability; loss of fluids - plasmolysis strongly bactericidal against gm+; less for gm- surfactant summary table do not kill endospores (breaks down fat) quat example  Chemical Microbial Control effect of different chemical methods on microbial death rate  8. Chemical Food Preservatives o Sulfur dioxide (SO2) long used as disinfectant prevents wine spoilage – used for 2800 years! = = o Organic acids (or salts of organic acids) interfere with bacteria / fungus metabolism or plasma membrane SO2 “bleaching” on grapes non-toxic - body readily metabolizes / “safe” in foods sorbic acid, benzoic acid, and calcium propionate prevent molds in acidic foods nitrites and nitrates prevent endospore germination Common Food Preservatives Sorbic acid bread mold  9. Antibiotics o molecule produced by one species of bacteria that inhibits or kills another species o antibiotics are “broad spectrum” – target wide range of bacteria o used to treat infection mostly o not often used as food preservative Antibiotic sensitivity test – the Kirby Bauer test  10. Aldehydes: functional group with the structure −CHO o inactivate proteins by covalently cross-linking with protein functional groups –NH2, –OH, –COOH, –SH o used for preserving specimens and in medical equipment o formaldehyde – used for years to preserve biological specimens o glutaraldehyde – relative of formaldehyde; less irritating, more effective than formaldehyde 11. Chemical Sterilization: o gaseous sterilants: causes alkylation — replacing hydrogen atoms of a chemical group with a free radical cross-links nucleic acids and proteins gaseous sterilant used for heat-sensitive material cross-linking DNA and protein ethylene oxide Plasma: o fourth state of matter, consisting of electrically excited gas laboratory plasma sterilizer o free radicals destroy microbes, including endospores o used for tubular instruments Peroxygens and other forms of oxygen o oxidizing agents – “steal” electrons o used for contaminated surfaces and food packaging O3, H2O2, and peracetic acid Microbial Characteristics and Microbial Control: Summary Table  Ch.7 – Control of Microbial Growth o microbial death rate o microbial control - mechanisms of action o physical methods of control o chemical methods of control o evaluating chemical methods of control  evaluating disinfectant and antiseptic efficacy (effectiveness) 1. Use-Dilution Tests: metal cylinders are dipped in test bacteria at different dilutions and dried cylinders are placed in disinfectant for 10 min at 20C cylinders transferred to culture media to determine whether the bacteria survived treatment use-dilution test  2. The Disk-Diffusion Method o evaluates efficacy (effectiveness) of chemical agent - including antibiotics o filter paper disks are soaked in a chemical and placed on a culture lawn o special “diffusion” agars can be used o look for “zone of inhibition” around disk disk diffusion method with different disinfectants and antibiotics VIDEO: crash course – controlling microbial growth How pathogenic microorganism are transmitted through feces The removal or destruction of ALL forms of microbial life is called… A. sterilization. B. disinfection. C. pasteurization. D. sanitization. The destruction of vegetative pathogens is called… A. sanitization. B. sterilization. C. disinfection. D. antisepsis. What treatment is intended to lower microbial counts on eating and drinking utensils to safe public health levels? A. sanitization B. sterilization C. pasteurization D. antisepsis The absence of significant contamination is referred to as… A. sepsis. B. bacteriostasis. C. asepsis. D. biocide. Commercial sterilization is a limited heat treatment to destroy… A. Streptococcus pyogenes. B. Clostridium tetani. C. Staphylococcus aureus. D. Clostridium botulinum endospores.  Ch. 7 Learning Objectives Define the following key terms related to microbial control: sterilization, disinfection, antisepsis, degerming, sanitization, biocide, germicide, bacteriostasis, and asepsis Describe the four factors related to effective treatment; exponential and logarithmic death rate Describe the patterns of microbial death caused by treatments with microbial control agents. o How is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria? Describe the mechanisms of action for microbial control agents on cellular structures. o How would a chemical microbial control agent that targets the plasma membranes affect humans? Physical methods of control: Compare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat. o How is microbial growth in canned foods prevented? What specifically do they aim to kill? o Why would a can of pork take longer to sterilize at a given temperature than a can of soup that also contained pieces of pork? Describe how filtration, low temperatures, high pressure, desiccation, and osmotic pressure suppress microbial growth. Describe the difference between ionic and non-ionic radiation and their specific mechanisms of action  Ch. 7 Learning Objectives Describe the three main principles of effective chemical disinfection. Identify the methods of action and preferred uses of the different chemical disinfectant groups o Describe halogen mechanism of action. o Identify the appropriate uses for surface-active agents. Define and interpret the results of use-dilution tests and the disk-diffusion method