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CHAPTER 6 Bacterial Growth, Nutrition, and Differentiation move work Copyright © 2021 W. W. Norton & Company, Inc. Bacterial Growth, Nutrition, and Differentiation Chapter Objectives ▪ Explain how nutrition and the environment can impact microbial growth and differ...

CHAPTER 6 Bacterial Growth, Nutrition, and Differentiation move work Copyright © 2021 W. W. Norton & Company, Inc. Bacterial Growth, Nutrition, and Differentiation Chapter Objectives ▪ Explain how nutrition and the environment can impact microbial growth and differentiation. ▪ Discuss microbial classifications based on nutritional needs and environmental limits. ▪ Describe how understanding microbial growth helps identify disease-causing pathogens. ▪ Discuss biofilms and their importance to infectious diseases. 2 6.1 Culturing and Counting Bacteria Section Objectives ▪ Explain how pure cultures are obtained and why they are important in medicine. ▪ Distinguish among synthetic, complex, selective, and differential media and their use in clinical microbiology. ▪ Describe the ways bacterial growth is measured, and explain the advantages and disadvantages of each method. 3  Bacterialculturemediumcaneitherbe liquidorsolid Growing Bacteria in Culture pure culture genetically homogenousstrainofasinglespecies ▪ For detailed laboratory studies microbes must be grown separately in pure culture—that is, as a single species. ▪ Bacterial culture media can be liquid or solid. Liquid media allows bacteria to move freely. Solid media is useful to separate mixtures of different organisms. Liquidmediumalso convenient for examining growth rates and changes in microbialbiochemistry Solid medium usufullwhentrying toseparatemixtures ofdifferent organisms environment or in clinical specimens found in the natural 4 If the medium contains thepropernutrients and growthFactors a singlecellwillmultiply Obtaining Pure Cultures – 1 ▪ Solid media Visiblemound cells Bacterial ofcells form colonies on solid media with agar added to make a firm surface. Isolation streaking allows for separation of colonies into pure cultures. Towardstheendofthestreak Fewbacteriasremain on the loop also knownas dilutionstreak a PURECULTURE of thespeciescan beobtained touching a singlecolony by into with asterileinoculatingloopand inserting thatloop freshliquid 5  A viable organism is one that successfully replicate Obtaining Pure Cultures – 2 toform a colony ▪ Pure colonies can also be isolated using the spread plate technique. transfering 30 1 ml of broth containingbacteria - 300 intstanttiff sterile broth andrepeating the dilution into subsequenttubes a glass rod will beused to spread a small amount of the diluted sample to theagarplate Confluent continuous the earlydilutions containg most bacteria that coversthe entire agarsurface ater dilutions containing fewer organisms yieldindividualcolonies Eachcolony on an agarplate represents one viable 6  g True colony if theindividual colony present in the agar platedoes notmove If thecolonymove it isnot considered as a truebacteria some more some does not !  minimalmedium is a synthetic medium in which the components Selective and Differential Media arelimitedtoonlythosenutrientsthe organismneeds inordertogrow ▪ Selective media complex medium richmedium conta Compounds in the media prevent some types of one ormorepoorlydefinedingredients bacteria from growing, favoring the growth of enrichedmedium particular addition addednutritionsneededbythebacter one specific type. Inclinicaldiagnosis its easierto examinepathogens insterileliquids ▪ Differential media two species that well growequally such as CSF butharderforliquids Species grow equally well but compounds in thatcontainsnormalmicrobiota the media are metabolized differently, often distinguished by a color indicator. ▪ Example: MacConkey medium bothselectiveanddifferential Selective agents are bile salts and crystal violet, which prevent growth of bacteria other than distinctive gramnegativebacteriahasthe outermembrane that keep Gram-negative enteric bacteria. intestineBacteria thesedetergentaway thusthey can Differential agentThey can permen is lactose, which some grow bacteria ferment (pink) and others do not. S entericadoes notFermentlactose 8  The mediumcontains lactose as a carbonsource neutralREDdye and amixofpeptidesas analternativesourceofcarbon LactoseFermentors suchas Ecoli secretsacidicproducts that lowerpHaroundthecolonyallowingreddyetoenter 6.2 The Growth Cycle – 1 Section Objectives ▪ Understand the phases of a typical bacterial growth curve. ▪ Explain how bacterial growth correlates to disease. ▪ Describe the purpose of continuous culture and how it correlates to the human digestive tract. 9 Distinguishing the Livingfromthe Dead Detroff Haussercountingchamber hemocytometer microorganism can becounteddirectlyby useof a microscope Con seeing an organism under amicroscopedoesntmeantheyare alive FluorescenceMicroscopy distinguish livingfromdeadcells Forexample propidium iodideinserts betweenDNAbases butcannotfreely penetratethe energized membranesof livingcells DEADcellsappearRED Syto 9 entersbothlivinganddeadcells stainingthembothgreen combination ofbothSyto 9 andpropidiumiodide will distinguish livingfromdeadcells livingcellswillappearGREENand dead cells will appearRED orYellow ORANGE Flowcytometer or Flouvescence activated cellsorter FACS Directcountingofcells withoutmicroscopy FACS bothcounts and separates bacterial cells thathave different properties Count and separate cells that synthesize a flourescentproteinfromcellsthat donot Mixture of cellflowsingle filepast a laserwhichcounts the cell and intensity offlourescence a allowsthemto be separated and collected Proofoflife ViableCounts Viablecells are those that can replicate and form colonies on an for example 100 colonies from 100 agar plate ml of 10 3dilution 1000 cells ml optical density muchcloudierliquidcultures have more calls measured by spectrophotometer  Typicalbacteriagrow by increasing length and mass 6.2 The Growth Cycle – 2 Facilities expansion ofBacterium's nucleoid as its DNA replicates ▪ Bacterial growth is measured at the population level. ▪ Most bacteria reproduce by binary fission (one parent cell divides and forms two offspring cells). Binary fission may be symmetrical or asymmetrical. 2 Hypno microbium ▪ Eukaryotic microbes divide by AS DNA replication nears completion the cell responseby synthesizing an equitoral mitosis. septic thatultimately separates the two daughter cells 10 law as the cell number doubles whatever parameter youuse also doubles Exponential Growth ▪ Growth in which population size doubles at a fixed rate (say, every 20 minutes) is called exponential growth. For example two cells divide to become four cells, then four cells divide to become eight. ▪ In an environment with few bacteria but plenty of resources, bacteria will divide at a constant interval called the generation time (also called doubling time). varies depending on species pH temperature medium ▪ Starting with any number of organisms at time zero (N0), the number of organisms after n generations will be N0 × 2n. 0 because the population of cells doubles over one generation ex E coli in a complexmedium will divide every 20 minutes After I hour 3gen 1 cell 8cal 11 Phases of Growth ▪ Bacterial growth curve, showing the change in growth rate over time 12 Ernentialgoth does not last doubles at a constant rate consumption nutrients and accumulation of toxic by productseventually slow the generation oftime until growthhalts all together Batch Culture simplest to culturbacteria in or test tubes using way flasks liquid medium as conditions in the medium deteriorate cells will change their membrane composition cell size and metabolism The goal is to slow downgrowthbefore they lose viability Lagphase Cellstransferfrom spentculture to newculture needtime to detect new environment express newgenes and synthesizenew components for growth cells DONOT divide From spentto fresh medium short lagphase From richcomplexmedium to poor long lagphase Theyneedto activatenew systems to synthesize new metabolites originally suppliedbythecomplexmedium Logphase starts afterthecells have retooled their physiology to accommodate the new environment theybegin to growexponentially Balancedgrowth cellcomponents are synthesized at constantrates in relation to each other grows at maximum rate possible largest at thisstage late logphase rate of doublingeventually slows atthispointsomebacteria can detect presence ofothermicroorganisms quorumsensing StationaryPhase viablecellsnumber stoprising due to lackofkeynutrients or buildupofwaste bacteriagrownin complexmedium celldensity rises above 10 cells ml growthofindividualcellsslows of cellsdividing cellsdying Theydeveloperesistance toantibiotics and alsohostdefense reactive productssuch as superoxide oxygenradicals test nÉsp by may kill them sothey develope st es somedecreasesize smaller cells needslessnutrients develope new stressresistant enzymes cells at stationaryphasebecomes moreresistant to changes in environment DeathPhase without reprieve in the way of new nutrients cells in stationary phase will eventually succumb to toxicchemicals present in environment death rate exponential as well extremely prolonge only survive formonths years or evendecades Continuous culture classic growth curve grows in a closed system The exponential phase spans only a few generations Opensystem Fresh medium is constantly added to a culture and an equal amount of culture is constantly siphoned off Taken away continuous culture keeps bacterialpopulation in exponentialphase at a constant cell mass for long periods of time All cells are at a steadystate whichpermitsdetailed analysisofmicrobial physiology at differentgrowthrates Chemostat continuousculturesystem inwhichdilutingmedium contains a growth limitingamount of an essential nutrient P Flow rate increases amountofnutrients P nutrients fastercellgrowth The more nutrient a culture receives as a result of increasingflowrate thefastercells can replicate shortergeneration time exampleofchemostat is ourintestine  6.3 Microbial Nutrition Section Objectives ▪ Describe the importance of the nitrogen and carbon cycles and the role of microbes in their maintenance. ▪ Discuss biofilms and their relevance to infectious diseases. GrowthFactor nutrients that is essential For an organisms growth They can either synthesize it or permanently get it from its environment knowing growthfactor arerequired bycertainpathogenic bacteria can helpclinical laboratory technicians the an 13  y ge ng Nutrients and Environmental Niches – 1 ▪ Essential nutrients are ▪ Elements those compounds a Carbon (C) microbe cannot make Nitrogen (N) itself but must gather Phosphorus (P) from its immediate environment if the cell is 7 Hydrogen (H) macronutrients Oxygen (O) to grow and divide. Sulfur (S) Chemicallydefinedminimalmedium Magnesium (Mg2+) some organisms can be grown inmedium Iron (Fe2+) enzymescofactors that contains onlythosecompounds needed Potassium (K+)balanceosmolarity For the organism topropagate Ex ecoli onlyneeds glucose to make all Trace elements such as cobalt, theirproteins nucleicacids and all wall copper, and zinc 14  Obtainingcarbon Autotrophy and Heterotrophy Heterotrophs like E coli relyon otherorganisms to make the organic compounds such as glucose theyuse as carbon sources all bacterial pathogens are heterotrophs organic carbon sources are dissembled togenerate energy and reassemblethem to make other cellcomponents process converts largeamounts of carbon sources to CO2 For life to be continued CO2must be recycled or else heterotrophswilldeplete the world of carbon sources Autotrophs uses the CO2discarded byheterotrophs to make organic compounds such as carbohydrates and aminoacids afterdeath heterotrophs can use all organiccompounds synthesized by autotrophs as carbonsources The Carbon Cycle PhYnaDf pyfgtightabs.ph Polysatharides Glucose GC mineraloxid Lithotrophy Lignin y ate c AcetylCoA Fermentation products ffH ffff Acetate 2C Oxidativerespiration go.fi xqyderived orenergy A Heterotrophs degrade organic fromoxidation compounds to gainenergy of minerals tocaptureCO2andconvert it tocomplexorganisms EnergySource Photo light absorption capturesenergy Chemo preformed organic or inorganic electrondonors are oxidized oxidge Inflation Electron Source Litho Inorganic m écu dilateÉearons Tomes in 2forms Organo Organicmolecules donateelectron oxidizesorganicmolecules  Nitrogen required inlargeamounts by livingorganisms Obtaining Nitrogen – 1 ▪ Nitrogen is needed by cells to make proteins and nucleic acids. The nitrogen cycle converts nitrogen to various forms. ▪ (The following examples are in bacteria outside of the body). Some bacteria perform nitrogen fixation to convert nitrogen gas (N2)to ammonium ions (NH4+), which is a form that can be used for biosynthesis. Other bacteria transform ammonia to nitrate NO3 (nitrification) and then convert nitrate to N2 (denitrification). 15  leukocytes we find in the test are we sure its urine infection if wedon'tfind are we surethat it isnot NO ▪ In the context of UTIs, nitrate reduction is a relevant concept: ▪ Nitrate Reduction: Some bacteria, particularly Escherichia coli (E. coli), can reduce nitrates (NO₃⁻) to nitrites (NO₂⁻) in the urine.  NITROGEN FIXATION N2 makes up 79 of Earth's atmosphere Na unavailable to use because it is a triplebond and requires lots ofenergy tobreak it need to first be FIXED N2 must be converted to NH4t ammonium ions NHyt to nitrate NO and back to Nz Nitrogen is reduced Form nitrogenis removed jiffy Na md converted p Fixers Imamm as a alternative electronacceptor in Nitrogen place of 02 NO NAT Nitrifiers oxidizes NHut to N 5 and generate energy Nitrogen Fixingbacteria may be free living in soil or water or form symbiotic association with plants or otherorganisms symbiont is an organism that lives in intimate association with a secondorganism Nitrogen importantfordiagnosis do we have ammonium in ourbody when we breakingdownprotein do we make the same thing aminoacid breakdown througheating Bacteria in our urine Context of UTI foundnitrate nitratereduction There is an enzymethatworkswithnitrate nitrate from our diet NO absorbed nitratefrom oururinecomingfrom ourdiet came in contact with ei E coli Ñ 3 Moveto kidney andurinate Ecoliwilltake NO andreduce it to nitrites NO If wefindnitritein urine are we 100 sure its UTI  Nitrite test look for the metabolites of nitrite reductase an enzyme generated by a variety of microorganisms this test has sensitivity and specificity of 25 and 94 100 respectively sensitivepeople catcheverything findthedisease truepositive specificpeople are exclusive confirmonlythehealthy or truenegative TRUE t FALSE t 94 100 NEG NEG 0 94 100 0 Positive results usually indicate that we have bacteriapresent because at normal conditions there is nitrite NOI Leukocytes UTI no ET probably yeah Nitrites confirmusingculture UrineTract InfectionDiagnosis urine culture we needcertainamount of CFU bladder and kidney can be infected we haveinfection inflammation and therefore whenwefind inflammation we need SandS Do wegivetreatment Yes because it can get worse and go to the kidneys asymptomatic bacteriarehea bacterias in urine but no symptoms Treatment depends sometimes we don'tneedtreatment because we mightbuild resistant up If female is pregnantyougivetreatment pathogens can acquirethedisease Wewant toavoid infection that can lead tosepsis associated with pretermbirthand maternalsepsis Immunocompromised pt not snow Sands but we need treatment ▪ In the gut, dietary proteins are broken down into amino acids, which are further deaminated to release ammonia. ammonia willtraveltoliver ▪ This ammonia can be absorbed into the willhappeninthe bloodstream → the liver → liver Urea → excreted by the bythe excreted kidneys. kidneys ▪ Microbial Role: Some gut what can wefind if liver is not bacteria may influence working ammonia levels.  The gut microbiota and hepatic encephalopathy ▪ Hepatic encephalopathy: a brain Ifutfeeali.hris notworkingit cantconvertammonia disorder caused by toxin buildup in the brain that occurs in individuals with severe liver disease (cirrhosis) ▪ The gut microbiota in cirrhosis is characterized by overgrowth of potential pathogenic bacteria (Dysbiosis) Theyarechangingtheirmicrobiota Fromthegut ▪ Increased pathogenic bacteria → rise of bacterial metabolites → decreased liver function → the ammonia canenterthebrain and wecan bacterial products enter the find encephalopathy inthe brain brain thus causing disease (ex. Ammonia) 18 6.4 Environmental Limits on Microbial Growth – 1 Section Objectives ▪ List different classes of microbes based on their preferred environmental niches (pH, temperature, and salt). 19  extreme any ecologicalniche outside thenormal growthconditions Extremophiles are bacteria archea and someeukaryoticmicrobes that can grow in extreme environments earliest microbes probably grewin extremeenvironments Hyperthermophile Thermophile Mesophile Psychrophile Temperature above 80 C 176 F between 50 C 122F bet 15 C 597 Below 15 C and 80C 176 F and 45C 1335 595 pH Alkaliphile abovepH9 Neutralophile Acidophile betweenpHsand belowpH3 PH8 Osmolarity Halophile highsalt greater than 2M Nacl Oxygen Strict aerobe Facultativemicrobe Microacrophile Strict anaerobe Conlyoxygen with orwithoutoxygen onlysmallamounts onlywithout ofoxygen oxygen Pressure Barophile Barotolerant at highpressure between 10 and greaterthan 380 atm 500 atm Thermophiles adapted to growth at high temperature 55 C and higher Hyperthermophiles grow at temperature as high as 121 C 250 F which occur under extreme pressure Oceanfloor thermalvents They fold more tightly than those in mesophiles so they do not unfold or denature at high temperature Pyschrophiles microbes that grow at temperatures as low as 10 C 14 F optimum growth temperature is usually around 15 C 59 F prominent Flora beneath icebergs in the arctic and antarctic They cansurvive in cold because their proteins are more flexible than those of mesophiles and require lessenergyto function Their membranes are more fluid than mesophiles at low temperature too flexible and fail to maintain cell integrity Mesophiles F coli and Bascillus subtilislab rats Theirgrowth optima range between 20 C 68 F and 40 C 104 F with a minimum of 15 C 59 F and a maximum of 45 C 113 F human pathogens are also mesophiles  6.4 Environmental Limits on Microbial Growth – 2 ▪ In addition to food, bacterial growth considerations include: Temperature and pressure Osmotic balance pH level Focuson I 20 1 am Normophiles (Mesophiles): – Thrive in moderate environments: as humansdo – Temperature: Around 37°C (human body temperature). pH: Neutral to slightly alkaline (around pH 7.4). – – I Oxygen: Aerobic, anaerobic, or facultative anaerobic. important o Pathogenic Examples: far o Escherichia coli (E. coli) o Staphylococcus aureus o Salmonella species o Streptococcus species  They are rarelypathogenic justfocuson the concepts ▪ Extremophiles: Thrive in extreme environments: Thermophiles: High temperatures (e.g., hot springs). Halophiles: High salt concentrations. Acidophiles: Very acidic conditions. Rarely pathogenic: Human body doesn’t provide extreme conditions needed for survival. 6.4 Environmental Limits on Microbial Growth – 3 23 Variations in Temperature – 1 ▪ Temperature 24 6.5 Living with Oxygen Section Objectives ▪ Differentiate anaerobes from aerobes and describe how each are cultured. ▪ Explain how both aerobes and anaerobes can cause disease. ▪ Discuss the basic differences between respiration and fermentation and how this impacts where an organism grows. 25 Aerobes versus Anaerobes – 1 ▪ Strict aerobes require oxygen as terminal electron acceptor aerobicrespiration Require oxygen for energy metabolism Successfully detoxify reactive oxygen species (ROS) Survive only in environments with oxygen ▪ Strict anaerobes Do not require oxygen for energy metabolism Generally unable to detoxify ROS, making oxygen toxic byproducts Survive only in environments without oxygen 26  STANDINGTESTUBE containg growth culture Egyptians Aerobes versus Anaerobes – 2 27 Aerobes versus Anaerobes – 3 ▪ Microaerophiles Aerobic, but ROS can be toxic Survive in environments with lower oxygen concentration ▪ Aerotolerant anaerobes Anaerobic but less susceptible to ROS, and usually lack catalase Prefer anaerobic conditions Cannot use oxygen but tolerates it's presence ▪ Facultative anaerobes Aerobic AND anaerobic Can use oxygen if it is present but can grow without it 28  Onestin examCatalase ▪ Catalase is an enzyme produced by bacteria that catalyzes the decomposition of hydrogen peroxide into oxygen and water. o This prevents the accumulation of hydrogen peroxide which can cause potential damage to cellular organelles or tissues o Essentially, the catalase enzyme is an antioxidant Catalase test ▪ In a clinical setting, the catalase test can be used to distinguish between different bacteria, as not all bacteria express catalase Ex. Catalase positive Micrococcaceae from catalase- negative Streptococcaceae ▪ In the catalase test, bacteria are exposed to hydrogen peroxide o Bacteria capable of synthesizing catalase will contain bubbles as a result of the reaction 6.6 Microbial Communities and Cell Differentiation Section Objectives ▪ Discuss how biofilms develop and the role of quorum sensing in the process. ▪ Explain the importance of biofilms to infection. ▪ Describe the process of sporulation, and explain how spores impact certain infections. 32 Biofilms: Multicellular Microbes? – 1 ▪ A biofilm is a mass of bacteria that stick to and multiply on a solid surface. Can include a single species or multiple collaborating species Can grow on organic or inorganic surfaces 33  Biofilms: Multicellular Microbes? – 2 ▪ Cells communicate and coordinate actions through quorum sensing. (Individual signaling → Autoinducer → Cells can ‘sense’. ▪ Bacteria in biofilms are very resistant to destruction. doesE.colinonepiliorplgell.at 34 Case History: Death by Biofilm ▪ Cystic fibrosis (CF) is an inherited disease in which chloride ion (Cl–) transport is compromised in many organ systems. The lungs are especially affected by the production of thick, sticky mucous buildup. 35 Case History: Death by Biofilm ▪ In 1951, two women with CF developed acute respiratory distress. From their chest X-rays, they were diagnosed as having an infectious bronchopneumonia. Treatment at the time consisted of intramuscular administration of the antibiotics streptomycin and penicillin. Case History: Death by Biofilm ▪ Repeated bronchial lavage (washing) was attempted in a futile bid to clear airway secretions. Tragically, the two women did not respond to therapy and died within hours. Pseudomonas aeruginosa, a Gram-negative bacillus common in soil and water, was subsequently isolated from both victims. This was the first report of Pseudomonas infection in CF patients. Endospores: Time Capsules for Bacteria – ▪ Endospores Certain Gram-positive bacteria produce destruction-resistant endospores. Bacteria in endospore form are dormant and require no nutrition or energy. – Bacillus, such as B. anthracis – Clostridium, such as C. botulinum and C. tetani Endospore state can last for decades or centuries before reactivation may occur. 38 Clicker Question 1 You are interested in determining the total number of viable cells in a bacterial culture. Which method of counting would you choose for the most accurate results? a. spread plate dilutions b. direct cell counts using a microscope c. flow cytometry d. turbidity with a spectrophotometer 40 Clicker Question 2 A culture of bacteria is growing exponentially under ideal conditions. Its generation time is 20 minutes. If the culture starts with two bacteria, how many bacteria will you find after 1 hour of growth? a. 2 20 mins 60 mins 4 b. 4 2 Bacteria c. 8 243 2 16 d. 16 e. 32 41 Clicker Question 3 How would you classify a bacterial species that grows only in the highlighted area of the culture tube? a. aerotolerant b. strict anaerobe c. strict aerobe d. facultative anaerobe e. microaerophile 42