1/17/2016 Control of microbial growth began ~100 years ago Microbes following the discovery that microbes caused disease Lister and Semmelweis were the first to develop microbial control practices-aseptic surgery Invisible Invaders Before that time,10% Amazing Allies of surgical deaths and 25%of delivering mothers deaths were due to infections. During the American Chapter 7 Civil War,a surgeon The Control of Microbial Growth may have cleaned his scalpel on his boot sole between incisions. Terminology of Microbial Control Microbial Control Terminology Sterilization-a process that destroys all living microbes, including viruses and endospores:microbicidal Learning Objectives Commercial sterilization:food is heated enough to destroy endospores of Clostridium botulinum-absolute sterility would degrade the food Define the following key terms related to microbial control: Complete sterilization may not be required in many settings.such as a fork or drinking glass in a restaurant sterilization,disinfection,antisepsis, Aim:to prevent the spread of possible pathogens degerming,sanitization,biocide, Disinfection-a process to destroy vegetative pathogens germicide,bacteriostasis,and asepsis (not endospores)on inanimate objects Antisepsis:disinfectant treatment applied to living tissue Antiseptic:disinfectants applied directly to living tissue
1/17/2016 1 Invisible Invaders Amazing Allies Chapter 7 The Control of Microbial Growth Control of microbial growth began ~100 years ago following the discovery that microbes caused disease Lister and Semmelweis were the first to develop microbial control practices - aseptic surgery Before that time, 10% of surgical deaths and 25% of delivering mothers deaths were due to infections. During the American Civil War, a surgeon may have cleaned his scalpel on his boot sole between incisions. Learning Objectives Terminology of Microbial Control g j Define the following key terms related to microbial control: sterilization, disinfection, antisepsis, degerming, sanitization biocide sanitization, biocide, germicide, bacteriostasis, and asepsis Sterilization – a process that destroys all living microbes, including viruses and endospores; microbicidal Commercial sterilization: food is heated enough to destroy Microbial Control Terminology endospores of Clostridium botulinum - absolute sterility would degrade the food Complete sterilization may not be required in many settings, such as a fork or drinking glass in a restaurant Aim: to prevent the spread of possible pathogens Di i f ti Disinfection – a t d t t ti th process to destroy vegetative pathogens (not endospores) on inanimate objects Antisepsis: disinfectant treatment applied to living tissue Antiseptic: disinfectants applied directly to living tissue
1/17/2016 Microbial Control Terminology trol of Mic obinl Growth oe2 w.w..oe before an injection Sanitization-any cleansing technique that mechanically lowers the microbial count to safe public health levels and minimizes the chance of spreading disease Biocide.germocide.or bacteriocide:treatments that kill microbes:fungicide,virucide Bacteriostasis:treatments that inhibit growth of the bacteria Sepsis:indicates bacterial contamination Asepsis:absence of bacterial contamination Rate of Microbial Death treated with ant Learning Objectives die at a constant rate. A Microbial Death Curve plotted Describe the patterns of microbial death caused by treatments with microbial 28hg2osdcosionraeaihroie control agents 2
1/17/2016 2 Degerming – mechanical removal of microbes, which reduces their number, such as swabbing the skin before an injection Microbial Control Terminology Sanitization – any cleansing technique that mechanically lowers the microbial count to safe public health levels and minimizes the chance of spreading disease Biocide, germocide, or bacteriocide: treatments that kill microbes; fungicide, virucide Bacteriostasis: treatments that inhibit growth of the bacteria Sepsis: indicates bacterial contamination Asepsis: absence of bacterial contamination Learning Objectives Rate of Microbial Death g j Describe the patterns of microbial death caused by treatments with microbial control agents When bacterial populations are heated or treated with antimicrobial chemicals, they die at a constant rate. A Microbial Death Curve plotted logarithmically shows a constant death rate as a straight line
1/17/2016 Plotting the typical microbial death curve logarithmically(red line)results in a straight line. Microbial Exponential Death Rate: 1.000.000 TABLE 7.2 An Example 60 Time(min) Deaths per Minute Number of Survivors 0 1,000,000 3.0 600,000 1 900,000 100,000 90,000 10,.000 0,000 100.000 3 9000 1000 7 900 100 Time(min) A microbial death 5 90 10 9 3 mi he cells would 9 ance betwee 100 000 and the baseline. Several factors influence the death rate: Number of microbes:more there are the longer it takes Environmental influences: organic matter inhibits action of chemical antimicrobials biofilms -temperature fats and proteins are protective Time of exposure:longer time for more resistant microbes Time(min) Microbial characteristics:such as (b)Logarithmic plotting(red)reveals that if the rate of killing is the same,it will take longer to kill all members of a endospore or not larger population than a smaller one,whether using heat or chemical treatments
1/17/2016 3 Plotting the typical microbial death curve logarithmically (red line) results in a straight line. r of ser of s One log decrease = 90% of population log10 of numbe r surviving cell s Arithmetic numb e surviving cell skilled Time (min) A microbial death curve plotted arithmetically (blue line) is impractical: at 3 minutes the population of 1000 cells would only be a hundredth of the graphed distance between 100,000 and the baseline. Several factors influence the death rate: • Number of microbes: more there are the longer it takes • Environmental influences: - organi tt i hibit ti f ic matter inhibits action of chemical antimicrobials - biofilms - temperature - fats and proteins are protective • Time of exposure: longer time for more resistant microbes • Microbial characteristics: such as endospore or not r of ls sterile surgical equipment log10 of numbe surviving cel l Time (min) (b) Logarithmic plotting (red) reveals that if the rate of killing is the same, it will take longer to kill all members of a larger population than a smaller one, whether using heat or chemical treatments
1/17/2016 Actions of Microbial Control Agents Alteration of Membrane Permeability Plasma membrane is the target of many Learning Objectives antimicrobial agents. The membrane regulates Describe the effects of microbial passage of nutrients control agents on cellular structures into and wastes out of the cell. Damage to the lipids and content to leak into the surroundina medium and interferes with growth. Damage to Proteins and rily Nucleic Acids Physical Methods of Microbial Control Enzyme activity requires a 3D- structure that is maintained by Learning Objectives chemical bonds connecting portions of the polypeptide. Compare the effectiveness of moist heat (boiling,autoclaving.pasteurization)and akage by h dry heat ing to prote denaturati Describe how filtration,low temperatures. high pressure,desiccation,and osmotic enzyme. pressure suppress microbial growth. Nucleic acids (DNA,RNA)are damoged by heat and chemicals Explain how radiation kills cells which is often lethal for the cell as it can no longer replicate or carry out normal functions
1/17/2016 4 Learning Objectives Actions of Microbial Control Agents g j Describe the effects of microbial control agents on cellular structures Alteration of Membrane Permeability Plasma membrane is the target of many antimicrobial agents. The membrane regulates passage of nutrients into and wastes out of the cell. Damag p e to the lipids and phospholipids causes the cytoplasmic content to leak into the surrounding medium and interferes with growth. Damage to Proteins and Nucleic Acids Enzymes are primarily protein and are vital for cellular activities. Enzyme activity requires a 3Dstructure that is maintained by chemical bonds connecting portions of the polypeptide. The bonds are susceptible to breakage by heat or chemicals leading to protein denaturation, enzyme restructuring, or binding. All of which inactivate the en mzy e. Nucleic acids (DNA, RNA) are damaged by heat and chemicals, which is often lethal for the cell as it can no longer replicate or carry out normal functions. Learning Objectives Physical Methods of Microbial Control • Compare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat • Describe how filtration, low temperatures, high pressure, desiccation, and osmotic pressure suppress microbial growth. • Explain how radiation kills cells
1/17/2016 As early as the stone age,humans have been When selecting the method of microbial control, using some type of physical method of microbial control to preserve food. you must consider what else besides the microbe will the method affect. Drying and Salting were probably the most common early techniques. Heat will inactivate vitamins or antibiotics in a solution Repeated heating damages laboratory and hospital equipment. Economic considerations:using presterilized,disposable plasticware versus washing and resterilization of glassware. Heat:Mode of Action and Relative Thermal Death Measurements Effectiveness Heat resistance varies among different microbes and Moist heat is expressed as TDP. .lower temperatures and shorter Thermal Death Point (TDP)-the lowest temperature at which all microbes in a particular exposure time liquid will be killed in 10 minutes coaqulation and denaturation of proteins Length of time to sterilize is expressed as TDT. Thermal Death Time (TDT)-minimal length of Dry heat time for all bacterial in a particular liguid to be killed at a given temperature .moderate to high temperatures .dehydration Third concept related to heat resistance: Decimal reduction time(DRT)-the time,in .alters protein structure minutes,in which 90%of the population of .incineration bacteria at a given temperature will be killed
1/17/2016 5 As early as the stone age, humans have been using some type of physical method of microbial control to preserve food. Drying and Salting were probably the most common early techniques. When selecting the method of microbial control, you must consider what else besides the microbe will the method affect. • Heat will inactivate vitamins or antibiotics in a solution • Repeated heating damages laboratory and hospital equipment. • Economic considerations: using presterilized, disposable plasticware versus washing and resterilization of glassware. Heat: Mode of Action and Relative Effectiveness Moist heat •lower temperatures and shorter exposure time • coagulation and denaturation of proteins Dry heat • moderate to high temperatures • dehydration • alters protein structure •incineration Thermal Death Measurements Heat resistance varies among different microbes and is expressed as TDP. Thermal Death Point (TDP) – the lowest temperature at which all microbes in a particular liquid will be killed in 10 minutes Length of time to sterilize is expressed as TDT. Thermal Death Time (TDT) – minimal length of time for all bacterial in a particular liquid to be killed at a given temperature Third concept related to heat resistance: Decimal reduction time (DRT) – the time, in minutes, in which 90% of the population of bacteria at a given temperature will be killed
1/17/2016 A comparison of the effectiveness of various Heat Resistance and Thermal Death antiseptics Bacterial endospores are most resistant to heat and 100 require femperatures above boiling fo destroy them. Soap and water 80 TAREE 114 Ihermal Death Iimes of various Endospores 40 Topical antiseptics 12℃ 120C 10填 90 C.NoME 105c 121 13m B.mimberopl l4C Smin t20r℃ 80 t00 120 120min C. TABLE 7.4 Size Moist Heat Methods Steam under pressure-sterilization Contuiner Sire ·Autoclave15 psi at121°Cfor10-40min 18150mm 10 ml Steam must reach surface of item being sterilized yeoak. 95 ml 5 1500ml Item must not be heat or moisture sensitive 0 Mode of action-denaturation of proteins, 58c6oabote 6750ml 70 destruction of membranes and DNA Preferred method of sterilization 6
1/17/2016 6 A comparison of the effectiveness of various antiseptics Topical antiseptics Heat Resistance and Thermal Death Bacterial endospores are most resistant to heat and require temperatures above boiling to destroy them. Moist Heat Methods • Steam under pressure – sterilization • Autoclave 15 psi at 121 • Autoclave 15 psi at 1210C for 10 40 min 0C for 10-40 min • Steam must reach surface of item being sterilized • Item must not be heat or moisture sensitive • Mode of action – denaturation of proteins of proteins, destruction of membranes and DNA • Preferred method of sterilization
1/17/2016 Figure7.2An Autoclave Example of sterilization indicators Boiling Water Pasteurization Pasteurization-heat is applied to kill possible Boiling at 100'C for 30 minutes to microbes that are causative agents of disease destroy non-spore-forming pathogens or food spoilage without destroying the food flavor or value ·Disinfection .63C-66C for 30 minutes (batch method) 720C for 15 seconds (flash method or high- temperature short-time(HTST)pasteurization) Not sterilization-kills non-spore-forming pathogens and lowers overall microbe count Does not kill endospores or many nonpathogenic microbes
1/17/2016 7 Autoclave Example of sterilization indicators Boiling Water • Boiling at 100°C for 30 minutes to destroy non-spore-forming pathogens • Disinfection Pasteurization • Pasteurization – heat is applied to kill possible microbes that are causative agents of disease or food spoilage without destroying the food flavor or value • 63oC – 66oC for 30 minutes (batch method) • 720C for 15 seconds (flash method or hightemperature short-time (HTST) pasteurization) • Not sterilization - kills non-spore-forming pathogens and lowers overall microbe count • Does not kill endospores or many nonpathogenic microbes
1/17/2016 Dry Heat Filtration ·Dry heat uses higher Physical removal of microbes by temperatures than moist heat passing a gas or liquid through filter Mode of action:oxidation Used to sterilize heat sensitive liguids and air in hospital isolation Incineration-flame or units and industrial clean rooms electric heating coil High-efficiency particulate air -ignites and reduces (HEPA)filters remove almost all microbes and other microorganisms larger than about substances to ash 0.3μm Hot-air sterilization or dry Pore size for bacteria:0.2-0.4 jm ovens-150-1800C for 2-4 hrs Pore size for viruses:0.01 um Cold High Pressure .Microbiostatic-slows the growth of microbes High pressure applied to liquid solutions is Refrigeration 0-159C and freezing OC transferred instantly and evenly throughout the sample Used to preserve food,media,and cultures ·If pressure is high enough, molecular Ice crystals that form with freezing are structure of proteins and carbohydrates are damaging to microbes cellular structure. altered inactivating vegetative bacterial cells. Bacterial cell numbers are reduced. Fruit juices have been treated this way and Many eukaryotic parasites are killed after has the advantage that it preserves the flavor,color,and nutrient value of the several days at freezing temperatures. product. 8
1/17/2016 8 Dry Heat • Dry heat uses higher temperatures than moist heat • M d f ti Mode of action: id ti oxidation • Incineration – flame or electric heating coil - ignites and reduces microbes and other substances to ash • Hot-air sterilization or dry ovens – 150-1800C for 2-4 hrs Filtration • Physical removal of microbes by passing a gas or liquid through filter • Us d t st ili h t s siti Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms • High-efficiency particulate air (HEPA) filters remove almost all microorganisms larger than about 0.3 m • Pore size for bacteria: 0.2 – 0.4 m • Pore size for viruses: 0.01 m Cold • Microbiostatic – slows the growth of microbes • Refrigeration Refrigeration 0-150C and freezing < and freezing <00C • Used to preserve food, media, and cultures • Ice crystals that form with freezing are damaging to microbes cellular structure. • Bacterial cell numbers are reduced. • Many eukaryotic parasites are killed after several days at freezing temperatures. High Pressure • High pressure applied to liquid solutions is transferred instantly and evenly throughout the sample • If pressure is high enough, molecular structure of proteins and carbohydrates are altered inactivating vegetative bacterial cells. • Fruit juices have been treated treated this way and has the advantage that it preserves the flavor, color, and nutrient value of the product
1/17/2016 Desiccation Osmotic Pressure Use of high concentrations of sugar or salt to Gradual removal of water from cells leads to preserve food is based on the effects of osmotic metabolic inhibition pressure. Not an effective microbial control as many cells A hypertonic environment retain ability to grow when water is reintroduced is created that causes water Lyophilization-freeze drying method used to preserve bacteria Bacteriostatic for some bacteria as the bacteria Used in curing of meats and to preserve fruits cannot grow or reproduce but many remain viable Viruses and bacterial endospores are resistant to Molds and yeast are able to grow on materials under osmotic pressure.This combined with their desiccation. ability to grow at lower pH is why molds and yeast may be the cause of fruit spoilage. Radiation Ionizing Radiation Radiation has various effects on cells depending on its wavelength,intensity,and duration. Ionizing radiation-short-wave length of radiant Radiation that kills microbes is of two types: energy gives deep penetrating power that has ionizing and nonionizing radiation sufficient energy to cause ionization of water forming highly reactive hydroxyl radicals, which are particularly damaging to DNA Radiant Energy gamma rays,X-rays,cathode rays Spectrum -used to sterilize heat sensitive materials, such as medical supplies(suture material) and food products(vitamins,herbs)
1/17/2016 9 Desiccation • Gradual removal of water from cells leads to metabolic inhibition • Not an effective microbial control as many cells retain ability to grow when water is reintroduced • Lyophilization – freeze drying method used to preserve bacteria • B ct i st tic B acteriost atic f s m b ct i s th b ct i for som e b acteria as th e b acteria cannot grow or reproduce but many remain viable • Viruses and bacterial endospores are resistant to desiccation. Osmotic Pressure • Use of high concentrations of sugar or salt to preserve food is based on the effects of osmotic pressure. • A hypertonic environment is created that causes water to leave the microbial cell inhibiting growth. • Used in curing of meats and to f preserve fruits • Molds and yeast are able to grow on materials under osmotic pressure. This combined with their ability to grow at lower pH is why molds and yeast may be the cause of fruit spoilage. Radiation • Radiation has various effects on cells depending on its wavelength, intensity, and duration. • Radiation that kills microbes is of two types: ionizing and nonionizing radiation Radiant Energy Spectrum Ionizing radiation – short-wave length of radiant energy gives deep penetrating power that has ffi i i i i f Ionizing Radiation sufficient energy to cause ionization of water forming highly reactive hydroxyl radicals, which are particularly damaging to DNA - gamma rays, X-rays, cathode rays - used to sterilize heat sensitive materials used to sterilize heat sensitive materials, such as medical supplies (suture material) and food products (vitamins, herbs)
1/17/2016 wavelength Nonionizing lonizing Radiation epenernhs Radiation Must be directly exposed for Fon ationof thymine dimer sterilization UV light creates thymine dimers that interfere with DNA replication. Actively dividing microbes are more TT sensitive Radiation source Barrier Cell DNA (breakages) Sunlight contains UV radiation but ozone filters out shorter wave. Antimicrobial effect of sunlight is r due to the formation of singlet G92121112T oxygen in the cytoplasm. Nonionizing Radiation:Microwave Chemical Methods of Microbial Control Indirect killing of bacteria through heat Moisture containing foods are heated by Learning Objectives microwave action and the heat kills vegetative pathogens List factors related to effective Solid food heat unevenly due to the uneven disinfection distribution of water in the food. Interpret the results of use-dilution tests and the disk-diffusion method Identify the methods of action and preferred uses of chemical disinfectants Differentiate halogens used as antiseptics from halogens used as disinfectants 10
1/17/2016 10 • Nonionizing radiation – wavelength longer than ionizing radiation so it has little penetrating power • Must be directly exposed for sterilization Nonionizing Radiation • UV light creates thymine dimers that interfere with DNA replication. • Actively dividing microbes are more sensitive • Used to reduce microbial numbers in the air and on surfaces • Sunlight contains UV radiation but ozone filters out shorter wave. • Antimicrobial effect of sunlight is due to the formation of singlet oxygen in the cytoplasm. • Indirect killing of bacteria through heat • Moisture containing foods are heated by Nonionizing Radiation: Microwave g y microwave action and the heat kills vegetative pathogens • Solid food heat unevenly due to the uneven distribution of water in the food. Learning Objectives Chemical Methods of Microbial Control • List factors related to effective disinfection • Interpret the results of use-dilution tests and the disk-diffusion method • Identify the methods of action and preferred uses of chemical disinfectants • Differentiate halogens used as antiseptics from halogens used as disinfectants