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Updated: Sept. 8, 2010 Prokaryotic Growth Supplemental instruction Fall 2010 For Dr. Wright’s Bio 7/27 Class Picture from http://www.compostinfo.com/tutorial/microbes.htm Designed by Pyeongsug Kim ©2010 [email protected] Pure culture techniques Pure culture :Individual organisms are isolated and grown from one single cell. •_______(free of microbe) Sterile media, instruments, flaming loop or needle • ________________ Aseptic technique During handling microbes •________________ Culture medium Nutrient for microbes; usually solid Cell are grow in/on; Colonies from one single cell Agar _____ Polysaccharide from algae. •________________ Media Nutrient for microbes Cells are grow in/on “agar plate” = petri dish with agar medium Designed by Pyeongsug Kim, ©2010 www.science-i.com Streak-method - Pure culture - isolated and grown from one single cell. - Using Aseptic technique - Sterile (flaming loop) every streak -Single colony is obtained in 4th . After streak-method, what? Stock culture and incubation. Designed by Pyeongsug Kim, ©2010 www.science-i.com Bacteria growth number of cells. : increase in ______ -How do bacteria grow (reproduce or multiply)? binary fission ________________________ Generation time,” or Doubling time - Time it takes for a population to double in number. *can be 20minutes or 2hours. Nt = N0 X 2n Initial number: 10 cells Doubling time: 20 minutes After 4 hours, how many of bacteria? http://www.bacferm.com.au/silac/micro/files /page4_3.png 40,960 Designed by Pyeongsug Kim, ©2010 www.science-i.com Environmental factor: Temperature, pH, O2, H2O Microbes live EVERYWHERE!!! Where they live? Why? How prevent them? Why do we put food in refrigerator(4oC)? Why do we cook food? Why do we have a fever when infected? Can microbes live in hot spring, artic or North pole? Enzymes for growth!! http://a.abcnews.com/images/Technology/abc_artic5_080509_ssh http://mayhem-chaos.net/photoblog/images/boiling_hot_spring.jpg .jpg Designed by Pyeongsug Kim, ©2010 www.science-i.com http://www.subzerorepairlosan geles.com/images/ge-cafe-eside-by-side-refrigerator-2.jpg Environmental factor: Temperature Psycrophiles __________ : -5 ~ 15 oC - in Artic, Antarctic, & lake with glaciers __________ : -2 ~ 35 oC Psycrotrophs - Grow well in low temperature - Food spoilage __________ : 15 ~ 45 oC E.Coli Mesophiles - Most Human pathogens - live in Hartnell college soil __________ : 42 ~ 80 oC Thermophiles - hot springs, compot heaps, water heaters, nuclear power plant cooling towers. ________________ : 68 ~ 108 oC Hyperthermophiles -hydrothermal vent deep in the ocean - Archea http://www.windenergyplanning.com/wpcontent/uploads/2008/12/didcot_power_station_cooling_tower_zootal ures.jpg Designed by Pyeongsug Kim, ©2010 www.science-i.com Why can some prokaryotes withstand such high temperatures but most cannot? Most prokaryotes have enzymes that are denatured in high temperature. With denatured enzymes they can’t grow. Thermophiles & Hyperthermophiles -have heat stable enzymes Many covalent bonds and hydrogen bonds in these enzymes prevents denaturation of proteins (enzymes). Designed by Pyeongsug Kim, ©2010 www.science-i.com Not all microbes cause disease! Not all microbes spoil food! ____________ Mesophiles Most human pathogens Think the temperature of human body! Spoilage Microbes Fungi(yeast, mold), Bacteria :________________ _______________________ Mesophiles, Psychotrophs in non-refrigerated foods. Psycrotrophs, Psycrophiles in refrigerated foods ______________________ Picture from http://leavingbio.net/FUNGUS/Fungi2.htm Designed by Pyeongsug Kim, ©2010 www.science-i.com Archea -Domain Archea; kingdom Archea -Heat-stable enzymes -Most of hyperthermophiles. can live in hydrothermal vent. -Live ______________ EVERYWHERE!! *Sometimes called extremophiles! http://cvitale.net/ecosystems/pages/ecosystems.html Designed by Pyeongsug Kim, ©2010 www.science-i.com http://www.scientificamerican.com/med ia/inline/what-makes-us-human_1.jpg We, human, are __________. Mesophiles Pathogens for human will be Mesophiles __________. Microbes inside human body are Mesophiles __________. Microbes on the skin are Mesophiles, Psychrotrophs ____________ Fever – makes inactive. inhibit growth. Refrigerator (4 oC) -Mesophiles -limits fast-growing. Psycrotrophs, Psycrophiles -______________________ :can live or multiply. :spoilage occur but slowly. http://www.subzerorepairlosan geles.com/images/ge-cafe-eside-by-side-refrigerator-2.jpg Designed by Pyeongsug Kim, ©2010 www.science-i.com Environmental factor: Oxygen (O2) •In the air •In or beneath the soil of Hartnell College •In aquatic habitats •On the skin •In the human internal organ -heart, lungs, throat, intestine, stomach… http://www.geo.arizona.edu/palynology/geos462/pu http://gloucestercitylibrary.files.wordpress.com/2007/11/aquaticlife.jpg erco1972.jpg Designed by Pyeongsug Kim, ©2010 www.science-i.com Environmental factor: Oxygen (O2) What do you think about Oxygen, O2? ____________ Obligate aerobes “I love O2. I can’t grow without O2 just like you.” http://microbemagic.ucc.ie/ab out_microbes/good_bad_ugly .html -die or not grow in absence of O2 -human=obligate aerobes. -Aerobic respiration to generate ATP. ____________ Facultative aerobes “I like O2, but I can grow without O2.” -can grow in absence of O2 fermentation or anaerobic respiration. -grow better in presence of O2 aerobic respiration. -E.coli, yeast Designed by Pyeongsug Kim, ©2010 www.science-i.com ____________ Obligate anaerobes “I hate O2! I cannot grow with O2.” -cannot grow in presence of O2 only fermentation or anaerobic respiration. ____________ Microaerophiles “I like O2 a little but I’m scared.” “not too much, not too little.” -Require a small of O2 ; die in high concentration fermentation or anaerobic respiration. ____________ Aerotolerants “I don’t know what that is. I don’t care.” -Don’t use O2 ; grow with or without O2. Neither aerobic or anaerobic respiration. -strictly fermentative. Designed by Pyeongsug Kim, ©2010 www.science-i.com _____________________________ require O2 to grow. Obligate aerobes, Microaerophiles _____________________________ Facultative anaerobes, Obligate anaerobes,live in the large intestine or stomach. aerotolerants _____________________________ on the skin. Obligate aerobes, Facultative anaerobes, aerotolerants Microaerophiles, Facultative anaerobes, _____________________________ beneath soil. aerotolerants ALL _____________________________ live in oceans(aquatic habits). _____________________________ common in aquatic habits Microaerophiles Designed by Pyeongsug Kim, ©2010 www.science-i.com Environmental factor: pH •Living in our digestive system - _________ in mouth pH =7 Neutral - _________ stomach low pH Acidic Alkalic - _________ small intestine high pH •Acidify food (lower pH) - inhibit microbial grow - Pickling food http://www.paranormalknowledge.com/article s/wp-content/uploads/2009/01/pickle.jpg All cells(prokaryotes) maintain internal (neutral/acidic/alkalic) pH. How? -raise environment pH eg) H. pylori(neutrophils) in the stomach. proton(H+) pump. eg) acidophiles, alkaliphiles -using _________ Designed by Pyeongsug Kim, ©2010 www.science-i.com http://health.battlecreekenquirer.com/Test Factsheet.aspx?id=29 Environmental factor: pH neutrophils ; pH 5- pH 8 ___________ -favor in neutral -to prevent grow acidify food -H. pylori can grow in stomach produce urease; generates ammonia to raises pH of immediate environment cause peptic ulcer ___________ acidophiles ; below pH 5.5 -to maintain internal neutral pH proton ( in / out ) -Volcanic fissure http://media.photobucket.com/image/Japan%20volcani c%20fissure/milljonas/volcanos.jpg ___________ alkalophiles ; above pH 8.5 -to maintain internal neutral pH Na+/H+ pump proton ( in / out ) -Alkaline lakes and soils http://www.indiana.edu/~sierra2/news_04/day1 2/renee_chemistry.jpg Designed by Pyeongsug Kim, ©2010 www.science-i.com Environmental factor: H2O - interaction b/w H2O and NaCl or Sugars. - Osmosis; dehydration - _____________ Plasmolysis : shrink from the cell wall. *This is not prokaryotic cells! How can bacteria survive high salt environments? * making itself salty! Draw water by increasing internal solute concentration -solute pump -synthesize small organic compound such as amino acid. Designed by Pyeongsug Kim, ©2010 www.science-i.com http://farm4.static.flickr.com/3089/2655340763_87ebe676 8b.jpg http://strawberrygal.wordpress.com/tag/fish/ Environmental factor: H2O ___________ Osmotolerant - can tolerate up to10% NaCl - can grow in dry environments (like skin) ___________ Halophiles -require high level of NaCl to grow. -Many marine bacteria (3%NaCl) http://www.niwa.co.nz/__data/assets/imag e/0009/49239/bacteria3_large.jpg ___________ Extreme halophiles -require 9% level of NaCl or more to grow. -Archaea -salt flats of Utah, Dead sea http://ianaloni.wordpress.com/2009/03/ Designed by Pyeongsug Kim, ©2010 www.science-i.com http://www.bluebird-electric.net/save_the_salt.htm Food preservation using high solutes concentration inhibit growth of bacteria. *shrinking bacterial cell. Designed by Pyeongsug Kim, ©2010 www.science-i.com Fastidious organisms -require many many many growth factors. Designed by Pyeongsug Kim, ©2010 www.science-i.com CO2 -autotroph : Carbon source from ______________ Organic compounds -heterotroph : Carbon source from ______________ Photo: Energy sourc from Sunlight ______________ Chemolitho- : Energy sourc from Inorganic ______________ chemicals(H2, NH3…) Chemoorgano- : Energy sourc from ______________ Organic chemicals (sugars, amino acids…) Heterotroph Phototroph Designed by Pyeongsug Kim, ©2010 www.science-i.com ___________ Photoautotrophs -Energy - sunlight; Cabon – CO2 -make ________ organic compounds -(primary producers/ consumers/ decomposers) -Cyanobacteria, Algae http://www.water.ky.gov/dw/profi/tips/alga e.htm ___________ Chemolithoautotrophs (Chemotrophs or Chemolithotrophs) -Energy – inorganic compounds; Cabon – CO2 (H2, NH3, NO2-….) -Hot springs, hydrothermal vents -(primary producers/ consumers/ decomposers) -Archaea Primary producers: Organisms that posses the capacity to produce organic compounds from inorganic material, with the aid of light or a chemically derived energy source. Primary producers compose the bottom of various food chains. Designed by Pyeongsug Kim, ©2010 www.science-i.com http://cvitale.net/ecosystems/pages/ecos ystems.html ___________ Photoheterotrophs -Energy – sunlight; Cabon – organic compounds; -purple nonsulfur bacteria ___________ Chemoheterotrophs -Energy – organic compounds; Cabon – organic compounds; -(primary producers/ consumers/ decomposers) We, humans, are _________________ Chemoheterotrophs . http://www.leanandmighty.com/Shockme.html Designed by Pyeongsug Kim, ©2010 www.science-i.com Lithotrophs, Autotrophs, Chemotrophs, Heterotrophs, Phtotrophs Autotrophs ____________ carbon is obtained from carbon dioxide (CO2). Heterotrophs carbon is obtained from organic compounds. ____________ ____________ an organism whose energy source is inorganicLithotrophs chemical reactions Chemotrophs energy is obtained from external chemical ____________ compounds (either inorganic or organic). ____________ energy is obtained from light. Phtotrophs Designed by Pyeongsug Kim, ©2010 www.science-i.com Lithotrophs, Autotrophs, Chemotrophs, Heterotrophs, Phtotrophs Hydrothermal vent Designed by Pyeongsug Kim, ©2010 www.science-i.com Media •_______________ Complex medium -most routine laboratory -nutrient for bacteria *bacteria food -nutrient broth or agar, TSA, blood agar, chocolate agar(for fastidious bacteria). -peptone(protein) -used for culture including pure culture. •____________ Defined medium - amounts of pure chemicals - specific research experiments. -To study nutritional requirements of bacteria. -Glucose-salts or depending on needs for study. -may add buffer to maintain neutral pH. *because some bacteria produce acids. Designed by Pyeongsug Kim, ©2010 www.science-i.com http://scienceprofonline.googlepages.com/mic robiologyselectiveanddifferentialmedi Some media are used to detect or to isolate an organism in mixed bacterial population. selective and differential media Both media are ( complex / defined ) media. •________ Selective media - allow “the organism” to grow. inhibit the growth of the other organisms. - MacConkey agar to isolate Gram-negative bacteria. *inhibit gram+ •_________ Differential media - to detect “the organism”. - allow the growth of all organisms. - Blood agar hemolysin – lyses RBC. -MacConkey agar Both differential and selective Fermentation sugar(lactose) Designed by Pyeongsug Kim, ©2010 www.science-i.com When you have very few bacteria you are interested in…….. An organism we want is present at 1 cell/ml but, 10,000 fold of other organism. difficult in pure culture even if selective media are used. Enrichment cultures •___________ -helpful in isolating when the organism is present in very few relatively. -Broth (liquid) favors the growth of the target organism. increase the target population. then, isolating by pure culture. Designed by Pyeongsug Kim, ©2010 www.science-i.com Measurement of bacterial growth: Direct counting * just count! _____________ -Rapid; Don’t have to wait -for organisms that can’t grow in medium. - (Can/Can’t) distinguish live from dead cells. Hard to count motile organisms. - Microscopic Counting chamber with liquid. To estimate effectively total cells at least 107 cells/ml must be present. -Coulter counter (no light) : Count total cells -Flow cytometer (light) :scattering of light by cells :can count total cells :can count the target cells after fluorescent dye or tag Designed by Pyeongsug Kim, ©2010 www.science-i.com Designed by Pyeongsug Kim, ©2010 www.science-i.com _____________ Viable Cell counts - living (and growing) bacteria. - for low number of bacteria. - need special media (selective & differential) - Dilute sample Plate counts Membrane filtration MPN (Most Probable Number) Designed by Pyeongsug Kim, ©2010 www.science-i.com 1)____________ Plate counts - only used if more than 100 organisms/ml - dilute sample with distilled water or 0.85 % NaCl to prevent lysis . - colony from one single cell. - First, dilute sample pour plate or spread plate incubation count colony as a single cell (=colony-forming unit) Calculation Designed by Pyeongsug Kim, ©2010 www.science-i.com 2)____________ Membrane filtration - Relatively low # of organisms in a sample; dilute env. eg) natural water -sterile membrane filter plate incubation count colony as a single cell (=colony-forming unit) Designed by Pyeongsug Kim, ©2010 www.science-i.com 3)____________ Most Probable Number (MPN) - Statiscal estimation theory of probability - used commonly in coliforms. - Dilution 10-1, 10-2, 10-3 incubation note gas production compare MPN table. estimate cell concentration. Coliforms – lactose-fermenting, Gram-negative rods reside in the intestine. Designed by Pyeongsug Kim, ©2010 www.science-i.com http://inst.bact.wisc.edu/inst/images/book_3/chapter_15/15-4.jpg Designed by Pyeongsug Kim, ©2010 www.science-i.com Turbidity (cloudiness) _______ - spectrophotometer - can measure (#of cells / cell mass) *commonly cell mass can estimate # of cells after done to determine correlation b/w cell concentration and tubidity. -relatively assay - Cells grown in liquid medium. Turbid culture indicate bacteria are present. HOWEVER, a clear solution = bacteria absence????? NO! Designed by Pyeongsug Kim, ©2010 www.science-i.com Growth can be measured either by _______ _______ turbidity or by counting to determine growth curve. C D E B A Designed by Pyeongsug Kim, ©2010 www.science-i.com A: Lag phase B: Log phase C:Stationary D:Death phase E: Prolonged decline ___________ Growth curve -As a population in a closed system grow, a pattern of stage. in closed system. -Grow pattern in broth culture. http://ultrapixelshots.blogspot.c om/2009_04_01_archive.html 1) Lag phase - “ready to multiply” - rich nutrient - synthesize macromolecules. enzymes, ribosomes, nucleic acids - generate ATP. Designed by Pyeongsug Kim, ©2010 www.science-i.com 2) Log phase “Happy bacteria” - exponential Phase - Active multiplication - Binary fission - Susceptible to antibiotics - Important medically metabolites - Primary ___________________ required for growth Commercial flavors by gene manipulation Late log phage (resistant) “Ready to be transformer” - transition to stationary phase - depletion of nutrients; build up of waste - initiate the process of sporulation(to be endospores) - Change cell walls, cytoplasmic membranes -______________________ Secondary metabolites Synthesizing different enzymes & proteins Antibiotics!!!! Designed by Pyeongsug Kim, ©2010 www.science-i.com 3) Stationary Phase (more resistant)“Bacteria gangs” - Dying release nutrient for growth of other cells. Multiplying - secondary metabolites - Growth rate = the death rate 4) Death phase - “Bacteria death” - Exponential death; faster death; slower multiply - Growth rate << the death rate 5) Prolonged Decline “What a bacteria!” - Dynamic changes - Use nutrient source from dead cells. - “Survival of the fittest” - Growth rate < the death rate Designed by Pyeongsug Kim, ©2010 www.science-i.com Growth pattern in a solid medium -Colony growth -The center of colony High density of cells rapidly deplete O2 and nutrients. -The edge of colony grow rapidly little competition and can use more O2 and nutrients Log phase Stationary phase Death phase Picture from http://scienceblogs.com/digitalbio/2009/01/how_well_do_you_know_your_bact.php Designed by Pyeongsug Kim, ©2010 www.science-i.com Protective structures glycocalyx endospores biofilm Biofilm -polysaccharides-encased community -attach to surface.; protect communities of microbes -Interactions each other dental plaque, scum in toilet, coats kitchen drains -Resistant to antibiotics 65% of human bacterial infections Designed by Pyeongsug Kim, ©2010 www.science-i.com Biofilms -a layer of slim with open channel for nutrient and waste. - protects communities of microbes in nature (not in the lab.) -Unrelated cells can attach and grow. -Communication with chemicals -So Bad… Resistant to antibiotics, disinfectant Tooth decay (dental plaque) in Pipes, drains, and cooling water towers; damage equipment. -May be good… Bioremediation- degrade harmful chemical. Wastewater treatment Designed by Pyeongsug Kim, ©2010 www.science-i.com http://www.cs.montana.edu/ross/personal/intro-biofilmss1_files/r2003_PSTO_BFIN3STEPS.jpg Biofilm (cont’d) -But, beneficial to human ______________ Bioremediation :natural process of cleaning up organic contaminants through the use of microbes. degrade harmful chemicals Waterwater treatment Biofilm Youtube.com Designed by Pyeongsug Kim, ©2010 www.science-i.com Mixed Microbial Communities - Interaction, cooperation - The activities of one species benefit another 1) Creating anaerobic condition Aerobes consume O2 anaerobic allows the growth of other organism that cannot multiply in the presence of O2 2) Wastes of one species can be nutrient for others Designed by Pyeongsug Kim, ©2010 www.science-i.com