CFU What are some ways you could make water safe to drink? Controlling Microbial GrowthPhysical Measures Oliver Yuan BIOL 240 Learning Objectives Be able to order vocabulary in terms of most clean to least Calculate D-value from a microbial death curve Vocabulary Sepsis: microbial contamination Asepsis: the absence of significant contamination Aseptic surgery techniques prevent the microbial contamination of wounds Antisepsis: destroying harmful microorganisms from living tissue Sanitization: lowering microbial counts to safe levels for public health Commercial sterilization: removal of foodborne pathogens while maintaining food quality Killing Clostridium botulinum endospores from canned goods How Clean Does it Need to Be? Item Cleaning Procedure Critical item Sterilization: removing and destroying ALL microbial life, including endospores and viruses Semicritical item Thorough disinfection: destruction of most harmful microorganisms from a fomite Noncritical item Degerming: mechanical removal of microbes from a limited area Example Controlling Bacteria Biocide (germicide): treatments that kill microbes Bactericidal agents: Chemicals result in bacterial death in the culture, no growth Subculture: no bacteria grows Bacteriostatic agents: Chemicals cause temporary inhibition of growth, no growth Subculture: bacteria grow Effectiveness of Treatment Depends on: Number of microbes Environment (organic matter, temperature, biofilms) Time of exposure Microbial characteristics Microbial Death Curve D-value (decimal reduction time): time it takes to reduce microbe population by one magnitude (90%) CFU What is the Dvalue of this agent? Physical Methods of Microbial Control Heat Filtration Low Temperatures High pressure Desiccation Osmotic pressure Heat Thermal death time (TDT): shortest time during which all cells in a culture are killed at a given temperature Thermal death point (TDP): lowest temperature at which all cells in a culture are killed in 10 min Moist Heat Sterilization Moist heat denatures proteins Boiling Autoclave: steam under pressure that kills all organisms and endospores 121°C at 15 psi for 20 min Steam must contact the item's surface Pasteurization Reduces spoilage organisms and pathogens Equivalent treatments 63°C for 30 min High-temperature short-time (HTST): 72°C for 15 sec Ultra-high-temperature (UHT): 138°C for 2-4 sec Dry Heat Sterilization Kills by oxidation Flaming Incineration Hot-air sterilization 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 Physical Methods of Microbial Control Low temperature inhibits microbial growth Refrigeration Deep-freezing Lyophilization: freezing dry High pressure denatures proteins Desiccation prevents metabolism Osmotic pressure causes plasmolysis Radiation damages DNA Controlling Microbial Growth- Cleaning Agents and Antimicrobial Drugs Oliver Yuan BIOL 240 Learning Objectives Compare and contrast different kinds of cleaning agents, their mechanisms, and their evaluation of effectiveness Chemical Methods of Microbial Control Phenol and Phenolics Bisphenols Alcohols Halogens Heavy Metals Surfactants Phenol and Phenolic Compounds Disrupt plasma membranes and denaturing proteins a) Phenol b) o-Phenylphenol c) hexachlorophene (pHisoHex) Triclosan: another bisphenol Inhibits an enzyme needed for the biosynthesis of fatty acids Effective against gram-positive, certain gram-negative and yeasts FDA banned in 2016 from soaps and body washes Alcohols Denature proteins and disrupting membranes No effect on endospores and nonenveloped viruses 70% works better than 100% since the latter coagulates proteins too fast Halogens and Heavy Metals Halogens Iodine: Alters protein synthesis and membranes Chlorine Bleach Oxidizing agents Heavy Metals Ag, Hg, and Cu Bind to and denature proteins Oligodynamic: very effective at very low concentrations For a disk diffusion assay, the zone of inhibition is: ⓘ Start presenting to display the poll results on this slide. CFU For a disk diffusion assay, the zone of inhibition is: A. How far the antimicrobial agent spread into the agar B. The growth around a disk with an antimicrobial agent C. Measured in mm D. The same for all antimicrobial agents for a given species CFU Why is 70% ethanol a more effective antimicrobial agent than 100% ethanol? Surfactants Soap: salts with hydrophobic tails; only degerming Quaternary ammonium salts (quats): Disrupt membrane integrity Effective against fungi, protozoans, and enveloped viruses, but endospores are unaffected Chemical Food Preservatives Sulfur dioxide (SO2) prevents wine spoilage Organic acids Inhibit metabolism Sorbic acid, benzoic acid, and calcium propionate prevent molds in acidic foods Nitrites and nitrates prevent endospore germination in processed meat Creates carcinogenic nitrosamines when heated Evaluating a Disinfectant Use-Dilution Tests Metal cylinders are dipped in test bacteria and dried Cylinders are placed in disinfectant for 10 min at 20°C Cylinders are transferred to fresh culture media without disinfectant to determine whether the bacteria survived treatment Disk Diffusion Method Filter paper disks are soaked in a chemical and placed on a culture Look for zone of inhibition around disks Antimicrobial Drugs Compound produced by microorganisms that inhibit growth of other microorganisms Selective toxicity: specifically inhibits or kills microbial targets without harm to the host Actions of antimicrobial drugs Alteration of membrane permeability Damage to proteins (enzymes) Damage to nucleic acids Damage to cell walls Alteration of metabolic pathways Targets of Antibacterial Drugs Evaluating Antibacterial Drugs Kirby-Bauer Disk Diffusion Test Filter paper disks are soaked in antimicrobial drug and placed on Mueller-Hinton agar Look for zone of inhibition around disks Variability in Disk Diffusion Test Water solubility of agent Ability to diffuse into agar Concentration of agent Concentration of agar Type of bacteria selected Evenness of agar and of bacterial lawn Other Antimicrobial Drugs More difficult to treat, either because they are eukaryotes or because they use much of the host’s own machinery Antifungal Antiprotozoan Quinoline: chloroquine thought to disrupt heme breakdown in malaria and Entamoeba histolytica Antihelminth Imidazole: Disrupts cell membrane by targeting ergosterol in Candida vaginal yeast, athlete’s foot, etc. Praziquantel: thought to cause influx of Ca2+ into tapeworms and flukes (Schistostoma), paralyzing them Antiviral Remdesivir: adenosine analog that binds to RdRP and initiates termination; hepatitis C, previous emergency treatment for COVID-19
