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sample/2021week-6-control-of-microorganismshaov-qw51qzc3.pdf 11 Dr Hao Van thithuhao.van@rmit.edu.au Microbial Growth Control & Antimicrobial Resistance Microbiology 2020 BIOL2158 / BIOL2173 Primary References Brock 15th edition‐ Chapter 5 and 28 Brock 14th edition‐ Chapters 5 and 27 Brock 13th edition‐ Chapters 26 and 36 2 What do the following images have in common? 3 4 © 2019 Pearson Education Ltd. Learning Objectives • To understand the different microbial control processes and how , when and why they are used Physical Microbial Control, Heat, filtration, radiation Chemical Microbial Control in vitro Sterilants, disinfectants, sanitizers, antiseptics, preservatives Antimicrobial agents used in vivo Synthetic antimicrobial drugs Naturally occurring antimicrobial drugs – Antibiotics • To understand how microbial control processes affect the cell © 2019 Pearson Education Ltd. Learning Objectives • Overview of the tests used to measure Antimicrobial activity • Overview of Microbial Resistance How resistance is acquired and spread. Review Antibiotic resistance in some pathogens ( Students are advised to review the structure of the cell and factors controlling microbial growth as these topics are linked) • Humans are constantly exposed to bacteria fungi and viruses from other people, air, water, the environment, products and foods • We employ many different methods to control microbial growth with the aim of: Eliminating or Reducing microbial load Preventing or limiting growth Limiting the effects of microbial growth . 7 Industrial – leather , plastics, wood ,paper, petroleum, textiles, paints, metal working fluids Environment – air, air conditioning units, water potable and swimming pools, surfaces, home, food, hospitals Foods and food production facilities Household cleaners and Personal care products – shampoos, cosmetics, hand washes Medical – in vitro and in vivo o Sterilization of equipment, disinfection of surfaces o Topical and in vivo antibacterial agents 8 The mode of action of all physical and chemical control agents can be divided into 2 basic categories ( the action of antibiotics will be discussed later) 1. Alteration of cell walls or membranes • Cell wall – cell integrity protects against osmosis – damage by physical or chemical agents – water moves in or out of the cell - cell bursts or collapses • Cell membrane controls passage of chemicals in and out of the cell – damage to proteins or phospholipids – leakage or failure of uptake systems - can lead to cell death 9 . 2. Damage to Proteins or Nucleic acids Proteins Regulate cellular metabolism Function as enzymes Structural components of membranes and cytoplasm Function depends on 3D shape – hydrogen, disulphide bonds Heat, chemicals – denature proteins – cellular death Nucleic acids Chemicals , heat, radiation alter or destroy nucleic acids – lead to fatal mutations Damage to enzymatic RNA molecule – interferes with or stops protein synthesis 10 . Sterilization The killing or removal of all viable organisms bacteria and viruses. Inhibition Effectively limiting microbial growth – prevention or slowing Decontamination The treatment of an object to make it safe to handle eg used petri dishes, used surgical equipment Disinfection Directly targets the removal of all pathogens, not necessarily all microorganisms, on non-living objects Antisepsis Inhibition or killing organisms (particularly pathogens) on the skin Sanitization (Sanitizing) reduction of pathogens and non pathogenic bacteria from surfaces and utensils ( ‘sanitizers’ are used in food industry) Pasteurization A heat process used to kill pathogens and control some other microbes that can cause spoilage in food and beverages Temperature Sterilization Pasteurization Radiation Non ionizing - UV Ionizing Filtration HEPA filters Membrane filters 13 Heat sterilization is the most widely used method of controlling microbial growth Sterilization kills all living cells and spores – High temperatures denatures macromolecules – heat killing faster as temperature rises – Moist heat works better than dry heat- has better penetrating power. • Endospores can survive heat that would rapidly kill vegetative cells. • Autoclave at least 121ºC for 15 minutes to kill endospores • Amount of time required to reduce viability of the organism tenfold is called the decimal reduction time • thermal death time: time to kill all cells at a given temperature; affected by population size Figure 26.1 Decimal reduction time (D) Time (min) Su rv iv al fr ac tio n (lo g sc al e) 70ºC 60ºC 50ºC © 2012 Pearson Education, Inc. 1 log redn The autoclave (retort) is a sealed device that uses steam under pressure At 121ºC, the time to achieve sterilization of small amounts of endospore-containing material is about 15 minutes – Not the pressure that kills, but the high temperature – Used to produce canned foods – Sterilize microbiological media – Sterilize dressings, surgical gowns, equipment etc Figure 26.3 Chamber pressure gauge Steam exhaust valve Door Thermometer and valve Steam supply valve Steam enters here Steam exhaust Jacket chamber Air exits through vent Total cycle time (min) Te m pe ra tu re (C ) Autoclave time Stop steam Begin pressure Flowing steam Sterilization time Temperature of object being sterilized Temperature of autoclave © 2012 Pearson Education, Inc. Autoclave Pasteurization • is the process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids – Does not kill all organisms • Designed to kill relevant pathogens and reduce potential spoilage organisms. Is pasteurization a method of sterilization? Pasteurization – To achieve pasteurization, the liquid is passed through a tubular heat exchanger – Times and temperatures vary depending on the organism and matrix • milk - 71˚C for 15 seconds – kills E, coli, Salmonella, Brucella, Mycobacterium (TB) Coxiella burnetti (Q fever), Listeria monocytogenes etc – Does not kill spores of Bacillus or Clostridium – Some non sporing gram positive organisms will also survive eg Lactobacillus • UHT (Ultrahigh-temperature pasteurization) 135ºC 1min kills spores- shelf stable 6-9 months • Bulk pasterurization: 63-66 °C for 30 min: less satisfactory © 2019 Pearson Education Ltd. • Ultraviolet (UV) radiation (between 220 and 300 nm): cause mutations or other serious effects on DNA death of the exposed organism. • UV useful for disinfecting surfaces and air: laboratory laminar flow hoods, air circulating in hospital • Has very poor penetrating power © 2019 Pearson Education Ltd. Figure 5.32 Clean work station - Laminar flow cabinet HEPA Filter UV light used to ‘decontaminate’ a clean surface after use © 2019 Pearson Education Ltd. • Ionizing radiation • electromagnetic radiation that has sufficient energy to produce ions and other reactive molecules upon collision • Generate higher energy electrons, hydroxyl radicals (OH·) and hydride radicals (H·) • Can damage macromolecules and kill irradiated cells Gamma rays • Used for sterilizing disposable hospital equipment syringes, needles, cannulas etc can affect material some plastics change density and become opaque, brittle • Gamma ray continuously emitted from a radioactive isotope Cobalt 60 – rods stored in a water filled pool Electron beams • On/ off operation • Higher dose than gamma (less time) but less penetrating X rays • Electricity based – on/off operation • Used for large pallet loads – uniform dose © 2012 Pearson Education, Inc. © 2019 Pearson Education Ltd. • amount of energy required to reduce viability tenfold (D10) is analogous to D value for heat sterilization Figure 5.33 © 2019 Pearson Education Ltd. • Some microorganisms more resistant to radiation than others (e.g., endospores vs. vegetative cells, viruses vs. bacteria) Table 5.7 © 2019 Pearson Education Ltd. • Radiation is used for sterilization in the medical field and food industry. • plastic labware, drugs, tissue grafts etc. • fresh produce, meat products, spices etc. • In Australia, the doses permitted range from a maximum of 1 kilogray (kGy) for tropical fruits; persimmons, tomatoes and capsicums, and up to 30 kGy for herbs and spices. For sterilization of food products, ionizing radiation is more effective than UV radiation © 2019 Pearson Education Ltd. • Filtration avoids the use of heat on sensitive liquids and gases. • pores of filter are too small for living organisms to pass through but do not trap most viruses • pores allow liquid or gas to pass through Figure 5.35 Types of filters in routine use in microbiology Depth filters – Filtration material arranged randomly – Traps particulate matter, dust, pollens, allergens – HEPA filters high energy particulate air filter - will trap bacteria used in safety cabinets – control airflow in and out, also clean rooms Membrane filters – Functions more like a sieve trapping particles and bacteria on the surface – Most common in laboratory – Made of high grade tensile polymers – Size of pore can be adjusted – Application - syringe, pump, or vacuum • A type of membrane filter is the nucleopore filter – Very uniform holes and an even surface – Used for isolating specimens for electron microscopy © 2019 Pearson Education Ltd. Figure 5.34 SEM showing the structure of (a) a depth filter, (b) a conventional membrane filter and (c) a nucleopore filter (d) bacteria trapped on nucleopore membrane filter (d) Pore Size (micron) Smallest organism trapped 5 Multicellular algae, fungi (hyphae, some spores) 3 Yeasts and larger unicellular algae, some fungal spores 1.2 Protozoa, small unicellular algae, most fungal spores 0.45 Largest bacteria 0.22 Largest viruses and most bacteria 0.025 Larger viruses and pliable bacteria (mycoplasmas, chlamydia some spirochetes) 0.01 Smallest viruses 32 . Chemical Microbial Control Chemical Antimicrobial Agents External Use In Products 33 Chemical Microbial Control An antimicrobial agent is a natural or synthetic chemical that kills or inhibits the growth of micro-organisms Antimicrobial agents can be classified as bacteriostatic, bacteriocidal, and bacteriolytic – Bacteriostatic - inhibit – Bacteriocidal - kills – Bacteriolytic - kills and destroys cells 34 Chemical Microbial Control Effect of Antimicrobial agents on growth Bacteriostatic agents frequently inhibit protein synthesis - eg bind to ribosomes If the concentration drops the agent can be released from the ribosome and growth of the organism resumes Bacterocidal agents bond tightly to cellular targets Are not removed by dilution and eventually kill the cell – however dead cells are not destroyed Bacteriolytic agents kill by cell lysis and release of cytoplasmic contents eg. detergents that rupture cytoplasmic membrane. 35 Figure 26.9 Total cell count Viable cell count Time Lo g ce ll nu m be r Lo g ce ll nu m be r Lo g ce ll nu m be r Bacteriostatic Bacteriocidal Bacteriolytic Total cell count Total cell count Time Time Viable cell count Viable cell count © 2012 Pearson Education, Inc. Viable cells measured by plate counts 36 Total cell count measured microscopically with a counting chamber Measuring Antimicrobial Activity Minimum inhibitory concentration (MIC) is the