Micro Test 3 PDF

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PPT 8 INCfatsiaitnpurpos.es tomakeproteins c structures That Microbial aÉlde qq.irstrate attended thotene initimin Requirements initial.itiinainainiiiiinanie one wayfatto 899991 Translation Mostly concentrated mechanster DNS mechanism © 2013 Pearson Education, Inc.  Chemical Analysis of Bacterial Cells  70% water _________  Proteins  96% of cell is composed of 10 elements = macroelements ____________:  carbon CHO carbohydrates NANUCIGIC and  hydrogen  oxygen Components of CHOs/NA/lipids/proteins  phosphorous Needed in gram quantities  sulfur  Nitrogen  Potassium  calcium  Magnesium Needed in milligram (mg) quantities 1mg=0.001g  Iron © 2013 Pearson Education, Inc. Other Nutrients Important in Microbial Metabolism  Potassium – essential to protein synthesis and membrane function  Calcium – cell wall and endospore stabilizer  Magnesium –membrane and ribosome stabilizer allowingfor betterproteinsynthesis  Iron – part of the ETC=important for ATP ___________ production also some likemetalsbut know these arent apartof © 2013 Pearson Education, Inc. thebelowgroup  Trace elements- 4%  All microorgs require several trace elements - the minerals manganese, zinc, cobalt, molybdenum, nickel and copper  are normally part ofenzymes ___ and ______ cofactors  they aid in the catalysis of reactions make them run go faster  Aid in the maintenance of :____________ proteinstructure enzymes  Needed in Microgram _________ (ug) quantities  (1ug=0.000001g)= usually obtained from air & water sound like metal © 2013 Pearson Education, Inc.  Nutritional Complexity  number of nutrients an org must obtain to live is determined by the kind and number of its _____________. depends enzymes on how much  Why? extra nutrients needed  Enzymes drive metabolic reactions  If enzyme is not present = organism is incapable of using a metabolic reaction to produce a certain substance=> must obtain from environment ______________ If can’t make it Must take it (from the environment) live essential nutrients © 2013 Pearson Education, Inc.  E. coli contains > 5000 different compounds  yet it needs to obtain only a few compounds from the environment to synthesize this great diversity  (glucose, trace elements, H2O, and 6 others)  What does this say about the number of enzymes E.coli has?  E. coli hasatleast __________= Gotenzymes uses just a few compounds to metabolize ~5000 compounds! © 2013 Pearson Education, Inc.  Nutrients  Inorganic nutrients–molecule that contains a combination of atoms other than carbon ______ and ____________ hydrogen inorganic has to lack  Can have neither C or H atlea t of these  Can have C or H- just can’t have both __________ CTH  Examples: metals and their salts (magnesium sulfate, ferric nitrate, sodium phosphate), gases (oxygen (O2), carbon dioxide (CO2) and water (H2O) © 2013 Pearson Education, Inc.  Nutrients  Organic nutrients- containcarbon ___ and ______(must Hydrogen have both C&H) atoms and are usually the products of _____________ livingthings  Can have other elements present  methane (CH4), carbohydrates (CHO), lipids, proteins, and nucleic acids © 2013 Pearson Education, Inc.  All organic compounds have a _______________ carbonbackbone Glucose molecules serving as carbon sources usually also contribute both hydrogen & oxygen © 2013 Pearson Education, Inc.  Environmental Factors That Influence Microbes  Environmental factors fundamentally affect the whateffected function of metabolic enzymes __________. usually Enzymes drive metabolic efflux  Factors include: reactions= no/inactive enzymes→ ________  temperature 819m98wh cell death  pH audits absentorpresent  Gas requirements- Oxygen  Osmotic pressure Understanding microbial ecological niches= ability to  radiation sont other ________________ control microbialgrowth  barometric pressure09th A  Ecological associations maintakeaway © 2013 Pearson Education, Inc. 3 Cardinal Temperatures 370C body temp 250C ambient roomtemp define microorganism growth curve 406 triage 10 C freezer most single cell organisms are poikilothermic =assume ambient To Afreezingtbreakingcwrkillingitor 1) Minimum temperature – lowest temperature that permits a microbe’s growth and metabolism enzymestoo slow to work 2) Maximum temperature – highest temperature that permits a microbe’s growth and metabolism denaturing enzymes 3) Optimum temperature – promotes the fastest rate of growth and metabolism  cardinal temperatures are not “_________”- rigidly fixed because influenced by other ______________________! environmentalfactors © 2013 Pearson Education, Inc. 5 Temperature Adaptation Groups 0oC=freezing, 37oC=body T, 100oC=boiling 1.Psychrophiles (cold loving) = -10C to 20C (opt T is 10 to 13C) Ocean ~5C, can grow in refrigerator, ~not human pathogens lipids in CM=highly unsaturated ________= a lot of double bonds 2.Psychrotolerant= 4 to 35C (opt T 15 to 30C) grow slowly in cold Iii 3.Mesophiles = 10C to 45C (opt T is 20o to 40oC) 2Wks __________and human pathogens _____________, normal flora environment microbes caustertopeon 4.Thermophiles (heat loving)= 45C to 80C (opt 67-72C) Compost pile, hot water heater liketogrowhotter thanmost 5.Extreme Thermophiles= 70C-and greater (opt?, max?) Hot springs, deep ocean vents Archaea- unique __________, enzymes DNA ↑C+G%, no PG, lipids in CM=highly saturated= no double ____________= bonds more resistant to heat saturated 5 mesophilesmostpathogenic c © 2013 Pearson Education, Inc. morestable it  Ecological Groups by Temperature of Adaptation which of these would grow in Frigid hotspringshumanbody © McGraw Hill 13 © 2013 Pearson Education, Inc. Sacrophile Figure 6.2 Food preservation temperatures. Temperatures in this range destroy most microbes, although lower temperatures take more time. Very slow bacterial growth. 37 C Rapid growth of bacteria; some may produce toxins. Danger zone Many bacteria survive; some may grow. Refrigerator temperatures; may allow slow growth of spoilage bacteria, very few pathogens. No significant growth below freezing. © 2013 Pearson Education, Inc.  pH ions Ht hydrogen protons  pH is a measure of the Hydrogen _________ activity of a solution  defined as the degree of acidity or alkalinity of a solution. 0-14 scale  pH of pure water is 7- neutral ___________ intracellular 72 74blood  Each species has a definite pH growth range  The optimum pH range for most orgs is pH ___68 because acid and base can be: denaturing  Damaging toproteins __________- especially enzymes! enzymes  Damaging to ________ CM and other parts of the cell (even the DNA) © 2013 Pearson Education, Inc. © 2013 Pearson Education, Inc.  pH 6 7 14 and neutral base  The effects of pH =  related to the concentration of acid ______ in the medium and  to the protection that bacterial cell walls sometimes provide.  Changes in pH can lead to:  denaturing of enzymes and other proteins and  can interfere with _____________at pumpingions the cell membrane. At ishigh youwannapumpoutwatorktnogeg.ptcal  Many bacteria produce large quantities of acids as they metabolize and grow = leads to high acid concentration= ___________ toxic environment! © 2013 Pearson Education, Inc.  Most acidic = high [ _____ Ht ] acid / blood © 2013 Pearson Education, Inc. basic  Effects of pH  Majority of microorganisms grow at a pH from 6 to 8  Acidophiles – grow at extreme acid pH (pH 0 to 5.5) - archae in hot springs  _____________ Neutrophiles growth between pH 6-8  fungi can grow at lower ______pHs than bacteria!  Alkalophiles-growth between pH __8.5-12____ Natronomonas pharaonis is isolated from soda lakes where it has to cope with two extreme conditions, high salt concentrations and an alkaline pH of 11. It grows optimally in 3.5 M NaCl and at pH 8.5. Returns a lot ofoxygen _____to environment via photosynthesis © 2013 Pearson Education, Inc. www.biochem.mpg.de Effects of pH – stomach cancer /esophageal cancer Helicobacter pylori (gm- spirochete, highmotility __________) rate  found in the stomach ~pH 2.5  causes peptic ulcers, gastric and esophageal cancer  is not and _____________ tolerant itsa neutrophile  Produces toxins that cause inflammation & damage  Disease symptoms made worse by ↑stress & envir factors & diet  protects itself from the stomach by growing in protective _____________of mucouslayer the stomach.  Breaks down urea in the stomach=pdces NH4+ (ammonia) basic= neutralize the microenvironment  Treat: _____________ antibiotics © 2013 Pearson Education, Inc. Helicobacter pylori Gas Requirements Oxygen- important respiratory gas, highly reactive  As oxygen is utilized it is transformed into several toxic products: -  singlet oxygen (O2), superoxide ion (O2 ), - hydrogen peroxide (H2O2), and hydroxyl radicals (OH )  Most cells have developed (protective) enzymes_ that neutralize these toxic O2 products:  Ie. catalase __________, superoxide dismutase oxidase  If a microbe is not capable of dealing with toxic oxygen= lacksprotective ____________ enzymes -so it is forced to live in oxygen free habitats= _________ anaerobe © 2013 Pearson Education, Inc. 86 Categories of Oxygen Requirement Aerobe – utilizes oxygen and can detoxify it  Has protective enzs 1. Obligate aerobe - cannot grow without oxygen  Must have O2 to growlung infections blood 2. __________anaerobe Falculative – utilizes oxygen but can also grow in its absence aerobe butcanalso  Prefers O2 but can survive without it dofermentation 3. Microaerophilic – requires only a small amount of oxygen – grows best at ___% 5 O2 (normal atmosphere is~20% O2) © 2013 Pearson Education, Inc. Categories of Oxygen Requirement Anaerobe – does not utilize oxygen  Lacks protective ___________ enzymes 1. Obligate anaerobe - lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment 2. Aerotolerant ___________ anaerobes –  Grows in the presence of O2 but does not use it  Has different protective mechanisms- use ___________ metalions © 2013 Pearson Education, Inc. Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria onewillbeonexam take youcan anunknowfmphGrowth patternsto figure outthe organism © 2013 Pearson Education, Inc.  Osmotic Pressure  Availability of water in an environment varies and depends 1. on the actual amount of water, 2. but also on the [____] solute in the water.  Most microbes exist under hypotonic (or isotonic) conditions _____ – require a high concentration of salt  Halophiles  Osmotolerant – do not require high concentration of solutes but can tolerate them when it occurs organismsfoundonskin © 2013 Pearson Education, Inc.  Osmotic Pressure _____(A  Water activity w) is a measure of the water that is available for use by an organism.  Aw is lowered by adding ______solutes____ to a solution thus   the solute concentration of the solution and  thus  osmotic pressure & ↓Aw (______________) inversely related  Enzymes require an aqueous environment- in order to be __________ functional  ↓Aw →↓enz function→↓metab death of cell © 2013 Pearson Education, Inc.  Osmotic Pressure  Aw and osmotic pressure are inversely related- with an  osmotic pressure then you have a decrease in Aw.  The aw of pure water is ~_________ 100  Most organisms require an Aw of.90-1.00 for metabolic activity and growth. Examples  Staphlyococcus aureus → can grow at ~0.85 Aw= grows on our skin which is fairly salty (can cause wound infections ie. MRSA)  Fungi →can grow at ~0.70Aw ifit itbecomesMRSA becomes antibioticresistant bichardonitsmoreresistant Salts/sugars= ↓Aw= used as food _______________ preservatives Jamstrellies © 2013 Pearson Education, Inc.  Radiation  Ultraviolet light can cause mutations in DNA (_________= T Tdimers toxic) and even kill organisms.  Some orgs have enzyme ______systems that can repair certain kinds of DNA damage.  Ionizing radiation can be used to sterilize items  _____________can Endospores survive large doses of ionizing radiation __________________________________________  Bacillus stearothermophilus endospores=  Purple sugar broth in ampule & spores on a piece of filter paper  Autoclave and then crush ampule to release broth to coat filter paper with spores  If turns yellow, spores germinated and grew & produced acids= so spores not killed= testing ___________ sterilization did not take place! autoclaves © 2013 Pearson Education, Inc. _____ now wavelengths = high E = Used for sterilization o dont have →generates ions that destroy own ___ to memoriz © 2013 Pearson Education, Inc. © 2013 Pearson Education, Inc.  Pressure  Many orgs spend their lives on land or on the surface of water so are subjected to a pressure of 1 atm so are never affected significantly by pressure.  __________pressure Hydrostatic is the pressure exerted by a water column as a result of the weight of the column with each 10 m of water depth equivalent to ____ I atm © 2013 Pearson Education, Inc.  Pressure  Hydrostatic pressures of > 200 atm generally inactivate enzymes ______ and disrupts cell _________and membrane transport systems______________  Hydrostatic pressure in the deep ocean can reach > 1000 atm (with a temp of 2-3C). Despite these extremes bacteria survive and adapt.  These Barophiles _______ will actually rupture when exposed to normal atmospheric pressure  Barophile enzymes need pressure to maintain their 3-D shape.  No 3-D shape= enzyme isnon _____________! functional © 2013 Pearson Education, Inc. Applications of Microbiology 6.1 A white microbial biofilm is visible on this deep-sea hydrothermal vent. Water is being emitted through the ocean floor at temperatures above 100°C. © 2013 Pearson Education, Inc. Ecological Associations Among Microorganisms  Symbiosis  Organisms living in a close relationship  at least one member is dependent on (requires) the relationship 1. mutualism – obligatory, dependent;both ________benefit members 2. commensalism – commensal member is dependent and benefits, other member not harmed 3. parasitism – parasite is dependent and benefits; _______ most is harmed parasitic © 2013 Pearson Education, Inc. Mutualism  Example: Lichens Association between fungus = an association between fungus and a & photosynthetic microbe = photosynthetic microbe ie. either an unique structure _______ or _____cyanobacteria_ Given a Genus & species algae name based on the  The fungus gets its Organic _________from carbonompa the fungus photosynthetic organism and in return:  protects it from excessive light intensities  and supplies water and minerals  while providing a support structure Lichens are resistant to To ___extremes and ________ dessication But sensitive to _________ airpollution © 2013 Pearson Education, Inc.  fruticose foliose crustose © 2013 Pearson Education, Inc. Symbiosis→the 3 divisions are not always clear cut Human Gut Microbe Using host Antibiotics wipe out Using host much of normal flora metabolic metabolic Sporeformers byproducts & byproducts to germinate and actively their benefit produce producing vitamins toxins=tissue damage ________ mutualism commens Parasite C diff © 2013 Pearson Education, Inc. Biosafety Levels- for handling microbes -each level builds on the previous level  BSL-1: no special precautions  Microbes not known to ___________ causedisease  BSL-2: lab coat, gloves, eye protection (206 lab)  Microbes can be associated with human disease especially those who are immunocompromised  BSL-3: biosafety cabinets to prevent __________ airborne transmission  Microbes that do cause human disease when encountered  Treatable &/or have vaccines  BSL-4: sealed, negative pressure  Exhaust air is filtered twice  Deadly organisms for which we have no treatment or vaccine © 2013 Pearson Education, Inc. Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory. Room negative air pressure spins Waitretiina Biosafety suit positive air pressure © 2013 Pearson Education, Inc.  The Metabolism of Microbes metabolism – all chemical reactions and physical workings of a cell Two types of chemical reactions: anabolism – building process 2 small substrates → 1 large molecule= makes ________ bonds Eo ______ used catabolism – degradative process 1 large molecule → 2 small substrates= bonds _______ broken Eo ________ released Rxns are cyclic and self-regulating © 2013 Pearson Education, Inc.  Enzymes  are biological catalysts  That Increase _____ the rate of a chemical reaction  by lowering the energy _____of activation.  The enzyme is _____ not permanently altered in the reaction. © 2013 Pearson Education, Inc.  reactants products Enzymes substrate enzyme  ___________a physically reaction promote = serve as a physical site upon which the reactants (or substrates) can be positioned for various rxns.  Since enzymes are not a part of the products, it is not used up by the rxn Enzymes  can be used over and over  Are not changed _____  Eventually wear __________ out © 2013 Pearson Education, Inc.  /Reactant Active site 2 small products produced= Catabolic __________ reaction © 2013 Pearson Education, Inc.  Lock and Key Model of Enzyme Function One large product= _______ reaction anabolic Bond made © 2013 Pearson Education, Inc.  Enzyme Structure  Enzymes can be simple or conjugated.  Simple enzymes – _________ proteinonly  Conjugated enzymes– protein + nonprotein molecule  non protein molecules are called __________ cofactors Cofactors are : either organic molecules which can be called ________oftenvitamins coenzymes or inorg elements such as _________ metations (cofactor)_. treatments © 2013 Pearson Education, Inc. Enzyme-Substrate interactions (when cofactor is inorganic) For a reaction to take place → a temporary union between enzyme and substrate ________ occurs Once the enzyme-substrate complex has formed – appropriate reactions occur on the substrate, – often with the aid of a _________, cofactor – a product(s) is/are formed and released. Function: “_________” better fit between enzyme & substrate © 2013 Pearson Education, Inc. Enzyme-substrate interactions (coenzyme)  The general function of a coenzyme is to remove a functional group (amino groups, hydrogen atoms, etc) from substrate 1 and add the functional group to substrate 2  coenzyme function:  Serves as a transient carrier of the functional group between substrate 1 and substrate 2  Increases rate of catalysis faster 1000x © 2013 Pearson Education, Inc. © 2013 Pearson Education, Inc. Enzymes sensitivity  When enzymes are subjected to changes in environmental conditions = ie. To or pH extremes= Unfolded/degraded/___________ denature  Low temps inhibit catalysis lowerskineticenemy  High temperatures, certain chemicals and low and high pH= denature enzymes. contramnational Active (functional) protein Denatured protein  nonfunctional enzymes block metabolic rxns = cell death ___________ © 2013 Pearson Education, Inc. © 2013 Pearson Education, Inc. © 2013 Pearson Education, Inc.  The Study of Microbial Growth ▪ Microbial growth occurs at two levels: ▪ growth at a cellular level with increase in ________ cell size ▪ increase in ___________ cell population ▪ Division of bacterial cells occurs mainly through ______________ fission Binary ▪ parent cell enlarges haploid ▪ duplicates its chromosome (__________) copies DNA ▪ and forms a central transverse septum (crosswall) dividing the cell into: _______________ 2 daughter cells (new/ identical) 1 © 2013 Pearson Education, Inc. © 2013 Pearson Education, Inc. © 2013 Pearson Education, Inc.  ▪ DNA replication in E. coli takes ____minutes 40 to completely copy the bacterial chromosome ▪ However E. coli can reproduce every __ 20 minutes. How does this work!? ▪ New DNA synthesis is initiated before a previous round of DNA synthesis is completed. Initiation site 4 © 2013 Pearson Education, Inc. ▪ Therefore, when a bacterial cell divides into two cells: ▪ each cell receives a complete _______________ chromosome ▪ and an additional portion of the genome whose synthesis was initiated part way through the fission cycle. 5 © 2013 Pearson Education, Inc. during the cell cycle: ▪ the cell must partition the DNA and cytoplasmic components by synthesizing a ____________. septum or crosswall ▪ the septum is initiated by invaginations of the cell envelope layers. (CM &CW) Initiation signal?? ▪ At least 12 ______ proteins in E. coli are responsible for the occurrence of septation ▪ It is believed that the chromosome attaches to the cytoplasmic membrane and is separated as the cell plasmamembrane all thesame membrane _cell wall & cell membrane continue to enlarge and separate cell components_. (_______) cellenvelopegrows The cytoskeleton also helps in the process of separating components into 2 new daughter cells © 2013 Pearson Education, Inc. 6 Figure 6.12a Binary fission in bacteria. Cell wall Cell elongates and DNA is replicated. Plasma membrane Cell wall and plasma DNA membrane begin to constrict. (nucleoid) (cell envelope invaginates) Cross-wall forms, completely separating the two DNA copies. Cells separate. 2 new identical daughter cells produced clones of eachother © 2013 Pearson Education, Inc. (a) A diagram of the sequence of cell division Figure 6.12b Binary fission in bacteria. Partially formed cross-wall DNA (nucleoid) Cell wall (b) A thin section of a cell of Bacillus licheniformis starting to divide © 2013 Pearson Education, Inc.  Rate of Population Growth ▪ Time required for a complete fission cycle is called the generation _______time (gt) ▪ Each new fission cycle increases the population by a factor of 2 =__________________ population doubles each time ▪ Bacteria average generation time is _~_____ 20 60 minutes under optimum conditions. ▪ Leaving food at room temp even for a short period = means that afew ________can bacteria grow to large #s in a short time ▪ E. coli gt= 20min, Vibrio sp. gt =10 min ▪ Mycobacterium leprae (leprosy)- has a very waxy cell wall due to presence of mycolic acid gt= is 14 days 9 © 2013 Pearson Education, Inc. Fig. 7.15.a Exponential growth= populationdoubles ____ with each division 10 © 2013 Pearson Education, Inc.  ▪ Predicting the number of cells that arise during a period of time: ▪ Nf=(Ni)2n the 2 is constant – population always doubles _________ ▪ Nf = final no. of cells, ▪ Ni= initial no. of cells, ▪ n= # of generations 30min ▪ How many bacteria in broth if it is inoculated with 1 bacterium for 1 hour? hour 2 ▪ Gt=30 min patherations ▪ n=# of generations= Nf 1 Shour Ni 2n 3 2 2 4 a (Convert all times to minutes) final initial 2 2 2 8 Nf 1 12 11 © 2013 Pearson Education, Inc. 1114  I example ▪ How many S. aureus will be present in an egg salad sandwich after it sits on the picnic table for 4 hrs? ▪ Ni=10 bacteria and the generation time is 20 minutes. So: ▪ 4 hr =240 minutes divided by 20 is 12 generations 4 1 104 ▪ Nf=(10)212=(10)(4096)= 40960 or __________bacteria 12 © 2013 Pearson Education, Inc. ▪ Bacteria do not maintain their potential growth rate because in most systems → influenced by environmental factors-especially ____________because inthelab bacteria in flasks or test tubes with just some media=limiting conditions ▪ If environmental factors are not optimum→ ▪ Decrease in nutrients ____________&_____________ 2 ▪ increase in acids & waste = decrease ______ growth rate ▪ Instead a population typically displays a predictable pattern or ___________over growthcurve time. aperformgrowthcurve iiI iii iii iiIiiii Iiii go.ie 13 © 2013 Pearson Education, Inc. Figure 6.15 Understanding the Bacterial Growth Curve. In a closed system where ___________are nutrients finite (limited) and the growth period over a few hours to few days produces a curve consisting of a: Lag Phase Log Phase Death Phase Intense activity Logarithmic, or Population Is Stationary Phase preparing for exponential, decreasing at a Period of equilibrium; population growth, increase in logarithmic rate. microbial deaths but no increase in population. balance production of population. new cells. The logarithmic growth in the log phase is due to reproduction by binary 88 fission (bacteria) or mitosis (yeast). Staphylococcus spp. 0088800000 88808898 Why knowing about the growth curve is important © 2013 Pearson Education, Inc.  Lag Phase ▪ When microbes are introduced into fresh medium→usually no immediate increase in cell numbers. Why? ▪ Cells may be old and depleted: of enzymes ______, ribosomes _____, and/or nutrients (any and/or all of these factors)= needed to metabolize & grow ▪ The medium may be different from the previous one ▪ Different sugar→different metabolic pathway→different _______(s) ie. bacteria in glucose broth transferred to lactose enzymes broth ▪ Most of the time the bacteria : are ____________________ gettingreadyfordivision ▪ Cells are making necessary components ▪ Cells are increasing in size 15 © 2013 Pearson Education, Inc.  Log Phase (Exponential Phase) ▪ Bacteria are growing and dividing at the _______________/optimum Maximum rate_____ possible. ▪ The pop is most ___________ uniform in terms of chemical and physiological properties ▪ Actively growing cells are more vulnerable __________ to conditions that disrupt _________and binaryfission __________________ie. cell metabolism drugs/antibiotics/chemicals/ envir factors/ immune system ▪ the organisms divide at their most rapid rate= a regular, genetically determined interval called the _________________ generationtime (gt= always determined during log phase) ▪ Industry/Medicine/Research= use bacteria in log phase when most uniform!= reliable/comparable results 16 © 2013 Pearson Education, Inc. Log Phase Exponential growth phase – a period of maximum growth continues as long as cells have : nutrients __________ are present ______________ environmentalconditionsare good 17 © 2013 Pearson Education, Inc.  Stationary Phase ▪ Eventually growth ceases and the curve becomes horizontal (usually around _109 bacteria/ml_). ▪ the total number of Viable _______________remains microoras constant: 1. This may result from a balance between cell division and cell death _______________ 2. or the population may simply cease to divide though remaining metabolically active – but at a much _____________ reducedrate ▪ Or a combo of #1 & #2 18 © 2013 Pearson Education, Inc.  Stationary phase – rate of cell growth equals rate of cell death caused by: ▪ ↓ nutrients 02 is a nutrient ▪ ↓space & ↓pH (toxic accumulation of ____&_______) adds wastes 19 © 2013 Pearson Education, Inc. Death Phase (exponential) ▪ detrimental (toxic/deadly) environmental changes = decline in the number of viable cells characteristic of the death phase. Death phase –limiting factors intensify = loss of nutrients (nutrients used up) and ________________is low PH now toxic (toxic waste & acids) ▪ Die off exponentially/logarithmically 20 © 2013 Pearson Education, Inc. ▪ Growth patterns in microorgs also account for stages in an infection: ▪ Microorgs in the log phase of growth are more: ____/______/metabolically infectious virulent active/ rapidly growing (max rate) /robust compared those in the death phase. ▪ So you are usually more infectious during the log phase= so usually more infectious at ___________of thebeginning the infection ▪ Later in the infection phase the immune response kills the invader (death phase) ▪ The shorter the generation time the more easily a pathogen can____________quickly= reachhigh s thus the pathogens large numbers initially overwhelm the immune system 21 © 2013 Pearson Education, Inc. Controlling Microbial Growth in the Environment Genetics 1  Terminology and Methods of Control sterilization ________ – a process that destroys all viable microbes, including viruses and endospores; kills everything/ can only be used on inanimate objects____autoclave example _________ Disinfection – a physical/chemical process to destroy vegetative pathogens, not endospores;not______(only sterility use on inanimate objects)_____ – Ie. steam/UV light destroysDN Antiseptic (prevent infection) – disinfectants applied directly to exposed _____ body surfaces Sepsis/Septic is defined as the growth of microorgs in blood or other tissues.verysick woman'damage Therefore in lab aseptic techniques refers to : used to prevent entry of infectious agents into tissues (thus prevent infection) 2  Terminology and Methods of Control Degerming: removing microbes from a limited area ( types of antisepsis)term oftenused onwounds Sanitization: lowering microbial counts on eating utensils→it’s any cleansing method that ____________microbes & debris mechanicallyremoves *safe but not necessarily microbe free -Cide/cidal vs –static/stasis killing inhibitsgrowth Biocide/germicide: _______ Killing microbes Bacteriostasis/static: inhibiting, not killing, bacteria 3 Factors that influence the effectiveness of antimicrobial agents 4 Factors That Affect Death Rate- of antimicrobials 1)Number of microbes the larger the population the longer time is required to kill off the population Remember death is also exponential _________ 16 bact 10 min 8 bact 10 min 4 bact 10 min 2 bact 10 min 1 bact 10 min 0 bact Factors That Affect Death Rate- of antimicrobials 2)Microbial characteristics -________in Nature the population: Prok vs Euk Gram + vs Gram negative Enveloped vs Naked Viruses all cleaned different rates 6 Factors That Affect Death Rate- of antimicrobials 3)Temperature and other environmental factors an increase in temp often enhances activity of chemicals – – Local Environment- environmental factors may offer protection for or aid in microbial destruction. For example:  Slightly acidic pH  Small change in To  Increase in water= Aw water activity 7 Factors That Affect Death Rate- of antimicrobials 4)Concentration or dosage of agent usually the more concentrated the more rapidly orgs are destroyed. Exceptions: betterthan purebleach  Bleach →10% working solution use10 bcbleachisoiln torah andcantgetintocellmembrane hinta soyouneedtodilutethebleach  70% alcohol vs 95% alcohol (disinfectant) (used in staining) Some H2O= cross orinteractwith cellwall cell membrane system 8 Factors That Affect Death Rate- of antimicrobials 5)Duration of action of the agent the longer pop is exposed to agent the better for killing  70% alcohol = is bacteriocidal  disadvantage→ ____________! Only a short evaporation time to exert its killing ability! 9 Factors That Affect Death Rate- of antimicrobials 6) Presence of solvents, organic matter, or inhibitors  Dirt/blood/other organics→  Causes physical blockage  Blood contains many ________ - enzymes may enzymes be able todegrade _____/__________ breakdown the agent cleaning  Blood contains catalase- breaks down H2O2 10 Figure 7.11 Decreasing order of resistance of microorganisms to chemical biocides. Proteinhardet hardtokill use ←protective waxy CW knowledge biggercells b c not Eukaryotessharder memorize ←protective outer membrane moreresistant nooutermembrane ←destroyed by adverse environmental factors Hair Table 7.7 The Effectiveness of Chemical Antimicrobials against Endospores and Mycobacteria Mercury based skin antiseptics -stings -poisonous -color blocked ability to see Infection -inhibited skin healing  Antimicrobial Drugs __________: Chemotherapy the use of drugs to treat a disease Antimicrobial drugs: interfere with the growth of microbes within a host Antibiotic: a natural metabolic product produced by one microbe to inhibit/kill another microbe bacteria Type chemotherapies Selective toxicity: killing harmful microbes without damaging ____________ thehost 14  Antibiotics do not work against they viruses!! Egret Karethealotolerant organisms Gm+ bacteria 15  16 Very difficult to get a drug that meets all these characteristics  The Spectrum of Antimicrobic Drugs Spectrum – range of activity of a drug – narrow-spectrum – effective on a small range of microbes target a specific cell component that is found only in certain microbes ie. isoniazid is only against Mycobacterium ( mycolic acid in the CW) – broad-spectrum – greatest _______________ range ofactivity target →highlyconserved ___________ie. structure Peptidoglycan 1 1he have whodont yet teeth permanent child Ie. tetracycline blocks protein synthesis: by binding to the ribosome= blocking translation= no ____________ proteinsmade 17 Antibiotics are not effective against viruses! youdonthaveto memorizebuthelpspiecethingsto gsmatesbectrum tmall.tn broader mm a'TokaYMYYanwaktfntoaytynqa.pe testingsomeone youthinkhasstrep donoudoabrador Start w handatiahaiseantibiotic narrowspectrum w anarrowspectrum ifpatientisntrllysick keeps healthybacteriaand broad spectrumkillsalot inoutthat Of begoodforboaul bacteria avoids antibioticresistance 18 can soitcanbetherewhenpeathtic.is  a 1 1a Transcription DNA-transcription to RNA- translation to protein Cells must constantly synthesize proteins to survive Transcription is the production of various kinds of RNAs from __DNA_. Three kinds of RNA:  messenger ________mRNA,  transfer ______ tRNA,  ribosomal ________ rRNA involved in protein synthesis All types of RNA are formed through transcription of the DNA, but only mRNA is further __translated to protein _________ 19  RNAsina.fiEn m RNA:  bears the message for __protein synthesis_.  Info coded in mRNA acts during translation to dictate the sequence ofamino _______(aa)__for acids the new protein AUG start codon  In PROK: Each molecule of mRNA starts with the codon for the aa _______________ methionine and ends with a ___________codon. stop  Codon= triplet of nucleotides that code for a certain amino acid 20  DNA structure DNA is read 3’ to 5’ And RNA is read 5’ to 3’ 21 OSC_Microbio_10_02_DoubHelix.jpg  DNA-G,C,T,A RNA-G,C,U,A TAC Triplets/codons→ carry AUG the message for which amino acid to add when making a protein methionine Codon chart: All possible triplets/codons found on mRNA Gives the aa for each triplet 22  Codon triplets on mRNA RNA: G,C,A,U= 4 nucleotides that are used in mRNA 43=__- possible 64 triplets 61 triplets code for aa 3 triplets are stop codons fMet= always first aa added in PROK (AUG anywhere else along mRNA= regular Met added) For each aa there is at least one codon to code for it (some have more than one =Redundancy _____= helps allow protection against mutations/variations= same aa= same protein (message doesn’t always change with a mutation) wobble effect 23  win rel in 1 PROK: all components coming together for translation ______of mRNA→_________ proteins String of amino acids= growing polypeptide P A mRNA 5’ 3’ 24  Translation Translation converts the nucleotide _________of the mRNA sequence into the sequence of amino _______comprising acids a protein. protein In translation all of the elements needed to synthesize a protein are brought together on the _________ ribosome Protein synthesis uses 80-90% of a bacterial cell’s energy. 25 1st tRNA carrying All other tRNAs enter at A site fMet enters atPsite ___ methioninestarts atpshepf.ua ___ bond forms peptide between 1st 2 aa 3’ AUG=Startcodon ______= fMet added If AUG appears elsewhere on the mRNA a regular Met is added 26 Process of Translation occurs in 3 steps: Ribosome reads mRNA 5’→3’ 1) Initiation All steps before 1st _______ bond peptide 50S subunit moves 1st followed by 30S→moves 2) Elongation 1 codon at a time Ist peptide bond to last peptide bond (tRNAcan _______ with aa) get recharged 3)Termination All reactions afterlast ________ peptidebond stopcodon at the Asite Stop codon= ______added no aa Release factors cause Peptide, ribosome subunits, tRNA release 27 ______- multiple ribosomes on 1 strand of mRNA polysome (found in euk & prok) Are all of the ribosomes making the same protein? __ yes In PROK mRNA starts degrading within 5min of ~400 aa protein ____________ transcription Uses 1200 ATP ~30 sec to make protein 1 KittenEthan nhypen 28 In Prokaryotes: (occurs simultaneously on the same mRNA) Read 3’ to 5’ (transcription) mRNA degrades within 5min of transcription (translation) In Eukaroyotes: Transcription & translation physically separated so cannot occur e Nucleus RNA is used by simultaneously on the same mRNA DNA→RNA 1 ribosomes for translation by the nucleus 29  Differences in transcription/translation Prokaryotes Eukaryotes ½ life of mRNA ~ 5 minutes > 4 hours # of ribosomes on > 20 double the ~10 ofr ibosomes mRNA AA added per second able 15 aa/second 119 ~7 aa/ second Rate of translation ~45 ~21 my name nucleotides/second nucleotides/second percodon Presence of introns no yes 30  Figure 10.2 Mechanisms of action of antibiotics Inhibition of cell know wall synthesis Penicillins Carbapenems Cephalosporins Vancomycin Bacitracin Isoniazid Ethambutol Echinocandins Inhibition of pathogen’s (antifungal) attachment or entry into host cell Arildone Inhibition of mudits's Pleconaril protein synthesis Enfuvirtide Aminoglycosides Tetracyclines Chloramphenicol Human Macrolides cell membrane Antisense nucleic acids onemore maybe Inhibition of DNA or RNA synthesis Actinomycin Disruption of Nucleotide cytoplasmic membrane analogs Polymyxins Quinolones Polyenes (antifungal) Rifampin If itson a slideafter Inhibition of general metabolic pathway thisone its fair game Sulfonamides toknowmode of Trimethoprim 5949 keen Dapsone arvas © 2015 Pearson Education, Inc. 1. Antimicrobials That Affect Bacterial Cell Walls Examples: β-lactams (Penicillins and cephalosporins) Free block synthesis of peptidoglycan= by cleaving _________ Lysis towardsendofinfection Most effective against young, growing cells (log phase) Penicillins do not penetrate the outer membrane =less effective against Gram negative ______ bacteria. 2 3 2. Antimicrobial Drugs That Disrupt Cell Membrane Function A cell with a damaged cell membrane dies from ____ differences in _____ in their cell membranes. lipids 1s have specificity for a particular microbial group, based on Example _______________ affect s interact with phospholipids helm gam polymyxinsCpolypeptideclass =leakage, particularly in Gram negative bacteria. Bind to ______ (disrupts outer membrane)→ goes to the cell membrane and disrupts Very toxic-effects kson renal fxn & can cause respiratory paralysis Usually only topical but use to combat antibiotic resistant strains 4 3. Drugs That Inhibit Nucleic Acid Synthesis May block synthesis of nucleotides or stop transcription DNA → DNA → RNA → Proteins replication __________ translation __________ transcription 3’ _______________________5’ RNA DNA polymerase → X RNA 5’ Example: Rifampin attacks _______ polymerase during transcription enzyme that makes a DNA RNA 5  Polymer 4. Drugs That inhibit Protein Synthesis block translation Antibiotics usually have a selective action against prokaryotes but can also damage eukaryotes because _ mitochondria have 705____ ribosomes__ I Streptomycin & gentamycin insert on sites on the L 30S subunit and cause _incorrect reading of the pii _____ 100k below code MRNA Tetracyclines block attachment of tRNA on the A acceptor site and stop further synthesis- binds _____subunit 305 blocks tRNA docking E Am 8 16 2254 3 2 2532 6 Target.fi 5 25 atkp.ttn Figure 20.4 The inhibition of protein synthesis by antibiotics. Protein Growing synthesis site Ontestpolypeptide Tunnel Growing polypeptide 50S 5′ Chloramphenicol Binds to 50S portion and inhibits formation of proteinsynthesis peptide bond 30S mRNA 3′ 50S portion Jun Protein synthesis site Erythromycin tRNA Binds to 50S and blocks movement of the Messenger ribosome RNA 30S portion Direction of ribosome movement 3codding Gentamycin & Streptomycin EEW.ae Tetracyclines 70S prokaryotic Changes shape of 30S portion, causing code on mRNA to be ribosome 5Esb Interfere with attachment of tRNA to mRNA–ribosome read incorrectly Translation complex Diagram indicating the different points at which chloramphenicol, the tetracyclines, and streptomycin exert their activities  5. Drugs that Affect Metabolic PathwaysAhhhh Sulfonamides block enzymes required for folic ______synthesis adds needed for DNA and RNA synthesis. fine iii ______ inhibition=competes for active site on enz competitive Humans acquire folic acid from __________= eating we lack this Kompetitive metab pathway inhibitor 8 Figure 20.2 Major Action Modes of Antimicrobial Drugs. 1. Inhibition of cell wall synthesis: penicillins, 2. Inhibition of protein synthesis: chloramphenicol, cephalosporins, bacitracin, vancomycin erythryomycin, tetracyclines, streptomycin gentamycin QhEmEÑn DNA mRNA Protein Transcription Translation Replication Enzyme 4. Injury to plasma membrane: polymyxin B ribosome 5. Inhibition of essential metabolite synthesis: sulfanimide, trimethoprim 3. Inhibition of nucleic acid replication sulfadrugs and transcription: quinolones, rifampin  Figure 20.17 The disk-diffusion method for determining the activity of antimicrobials. on amera The Acquisition of Drug Resistance Adaptive response in which microorganisms begin to tolerate an amount of drug that would ordinarily be inhibitory= due to genetic versatility or variation Acquired resistance: – spontaneous mutations in critical chromosomal genes – acquisition of new bact genes : 1)conjugation- transfer of DNA from 1 bact to another via pili 2)_______- acquiring DNA via bacteriophage transfer 3)__________- taking up free DNA from the environment 11 Mechanisms of Drug Resistance- due to new gene expression=new proteins with new _________ 1) Decreased permeability to drug = blocked entry Change _________or block receptor 2)Drug inactivation by acquired enzymatic activity penicillinase- cleaves ______________ 3) Change in drug receptors blocks drug binding site on ________ 4)increased elimination of drug from cell protein pumps drug out 5)Change in metabolic pathway New enzyme bypasses blocked enzyme 12 sulfonamides Figure 20.20 Bacterial Resistance to Antibiotics. 1. Blocking entry Antibiotic 2. Inactivation by enzymes Antibiotic Antibiotic Altered target 5. Enzyme catalyzes new metabolic molecule Enzymatic action pathway Inactivated 3. Alteration of target molecule antibiotic 4. Efflux of antibiotic Figure 20.9 The nuclear structures of a cephalosporin and penicillin compared. Bacteria produce β lactamase Insert Fig 20.9 Cephalosporin nucleus –lactam ring cuts Blactamring Penicillin nucleus  Antibiotic Resistance Misuse of antibiotics selects for resistance mutants Misuse includes: – Using outdated or weakened antibiotics – Using antibiotics for the common cold (____) virus and other inappropriate conditions – Using antibiotics in animal feed – Failing to complete the prescribed regimen – Using someone else’s leftover prescription MDD  Why such widespread drug resistance? In hospitals despite efforts to maintain sanitary conditions a hospital provides: perfectconditions forspread of drug resistant bacteria Dealt A lot of sick people in close proximity= ↑________ patients are usually more ill and have lowered resistance to infection use a variety of antibiotics: ↑riskentinffersrista of conjugation+others fbatnitio hfqF system at supressimmune Mmo 16  Prevention of Antibiotic Resistant Strains Limit antibiotic use to situations in which _____________without antibiotic treatment. recovery Sensitivity tests should be done Kirby Bauer test Complete antibiotic regime! Patients with an infectious disease should be isolated from other patients. Nosocomial HAI hospitalaquired infection Treat severe, resistant pathogens with _________ tests 17  Worldwide dilemma Antimicrobial resistance is a major public health Soooooo concern -threatening the practice of modern medicine, food security, and animal health. Roughly 200 million prescriptions for antibiotics are written in the US every year. 50 Estimated that ~1/2 of these prescriptions are inappropriate because the ___________________ infectionagentisnotdiagnosed drugnotneeded Many drugs are also misprescribed as to:t.ua Wrong drug/wrong dosage/wrong iamSeffl ___________ tmofnansman biotics target virus Prokamotic bacteria vs structures 18  N Wantible Low Income/Inadequate Healthcare Countries Inappropriate use of antibiotics __ & poor infection prevention/ asstrategies contribute to antimicrobial resistance control Use of expired drugs Self medication Poor populations During India’s first surge of COVID-19, antibiotic sales soared because the drugs were used to treat cases of COVID-19. Such use is inappropriate because antibiotics are not effective against viral infections and overuse Iffferattoos increases the risk for drug-resistant infections. 19  20 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459633/  There is a tendency of using shotgun antimicrobial therapy for minor infections which involves : use of a ________antibiotic- kill off the original infection but 111now you fnok have:___________ fi This practice can lead to antibiotic _________ as well resistance as toxic reactions. – Are more difficult to treat- more powerful Sholto antibiotic or multidrug regime needed 21 normen I Clostridium difficile C diff 22 Superinfection: pseudomembranous enterocolitis caused by Clostridium difficile (bacterial endospore former) __________ induced disease- occurs because of antibiotic use normal accum of mucus & debris &inflam pus/necrosis-deadly 23  o Medical induced disease oooo How do you re-est NF? ________ Get sick so Why a relapse? 1)NF has not be re- ETIteo_ established 2) ________________ Stop antibiotic orangeadside Different antibiotic to I Do treat C. diff 24  NF Ni 2ⁿ 4 Nt 5 2 2 2 2 2 4 2 8 2 16 16 0