Chapter 5: Microbial Growth and Nutrition

orey f JPL-Caltech/MSSS/NASA of Courtesy © Jones & Bartlett Learning. NOT FOR SALE OR DISTRIBUTION.

5 © NIAID Bacteria: CHAPTER PREVIEWNOT FOR SALE OR Microbial DISTRIBUTION Growth andNOT FOR Nutrition SALE OR DISTRIBUTION 5 . 1 Microbial Reproduction Is Part of the© Jones Cell Cycle & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONooks have been writtenNOT about FOR it; SALEmovies ORhave DISTRIBUTION been made; even a 5 . 2 Microbial Growth Progresses radio play in 1938 about it frightened thousands of Americans. Through Distinct Phases What is it? Martian life. In 1877 the Italian astronomer, Giovanni Clinical Case 5: An Outbreak BSchiaparelli, saw lines on Mars, which he and others assumed were of Food Poisoning Caused by canals built by intelligent beings. It wasn’t until well into the twentieth © Jones & Bartlett Campylobacter Learning, jejuni LLC century that© thisJones notion & Bartlettwas disproved. Learning, Still, when LLC we gaze at the red NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 5 . 3 Culture Media Are Used to planet, we wonder: Did life ever exist there? Grow Microbes and Measure We are not the only ones wondering. Astronomers, geologists, Their Growth and many other scientists have asked the same question. Today MicroInquiry 5: Identification microbiologists have joined their scienti c colleagues, wondering if of a Bacterial Species© Jones & Bartlettmicrobial Learning, life once LLC existed on the Red Planet© or, Jones for that &matter, Bartlett elsewhere Learning, LLC Investigating theNOT Microbial FOR SALE ORin our DISTRIBUTION solar system. NOT FOR SALE OR DISTRIBUTION World 5: The Great Plate Count So, could microbes, as we know them here on Earth, survive on Anomaly Mars, where temperatures are far below 0°C, the atmosphere contains little oxygen gas, and ultraviolet (UV) radiation bombards the planet © Jones & Bartlett Learning, LLCsurface? Researchers, using© Jonesa device & Bartlettto simulate Learning, the Martian LLC soil environment, placed in the soil microbes known to survive extremely NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION cold environments here on Earth. eir results suggested that members of the Archaea, speci cally the methanogens, could grow in the cold, low-oxygen atmosphere, especially if protected from UV light by being buried just under the soil surface. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR140 SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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Hottest environment (Juan de Fuca ridge)—121°C: Strain 121 (Archaea) Coldest environment (Antarctica)— –15°C: Planococcus cryohalophilus (Bacteria) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Highest radiation survival—5NOT MRad, FOR or 5000 SALE× what kills OR humans: DISTRIBUTION Deinococcus radiodurans (Bacteria) NOT FOR SALE OR DISTRIBUTION Deepest—3.2 km underground: Many bacterial and archaeal species Most acid environment (Iron Mountain, CA)—pH 0.0: Ferroplasma acidarmanus (Archaea)

Most alkaline© Jones environment & Bartlett (Lake Calumet, Learning, IL)—pH 12.8: LLC Proteobacteria (Bacteria) © Jones & Bartlett Learning, LLC LongestNOT in space FOR (NASA SALE satellite)—6 OR years: DISTRIBUTION Bacillus subtilis (Bacteria) NOT FOR SALE OR DISTRIBUTION High-pressure environment (Mariana Trench)—1200 times atmospheric pressure: Moritella, Shewanella , and others (Bacteria) Saltiest environment (Eastern Mediterranean basin)—47% salt: Several bacterial and archaeal species © Jones Loneliest& Bartlett (South Learning, Africa)—3.3 kilometers LLC below Earth’s surface: Desulforudis © Jones audaxviator & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Source: http://www.astrobio.net/news/

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC So, microbiologistsNOT have FOR joined SALE the search OR DISTRIBUTIONIn 2004, NASA sent two spacecraNOT to MarsFOR SALE OR DISTRIBUTION for extraterrestrial life. is seems a valid to look for indirect signs of past life. Scientists Extremophile: A microorganism that extremophiles pursuit because the found here here on Earth monitored instruments on the lives in climatic on Earth survive, and even require, living in Mars rovers, Spirit and Opportunity, designed or environmental extreme environments ( TABLE 5.1 )—some to search for signs suggesting water once extremes, such as not so© Jonesdi erent & from Bartlett Mars Learning, ( FIGURE 5.1 LLC ). existed on the planet.© Jones Some & ndings Bartlett suggest Learning, extremely LLC high/low If life (asNOT we FORknow it)SALE did or OR does DISTRIBUTION exist on Mars, there are areas whereNOT salty FOR seas SALE once washedOR DISTRIBUTION temperatures, high it almost certainly was or is bacterial/archaeal. over the plains of Mars, creating a life-friendly acidity, or high salt environment. environments. Opportunity found evidence for ancient © Jones & Bartlett Learning, LLC shores© Joneson what & once Bartlett was believed Learning, to be LLC a sea. NOT FOR SALE OR DISTRIBUTION ScientistsNOT reportedFOR SALE in 2008 OR that DISTRIBUTION a more recent spacecra , the Phoenix Mars Lander, detected water ice near the Martian soil surface. Since 2012, another exploration of the Red Planet has been underway, as the Mars rover © Jones & Bartlett Learning,Curiosity LLC is searching to see if there© Jonesare any & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONchemical signs suggesting life may haveNOT existed FOR SALE OR DISTRIBUTION on the Red planet. Whether microorganisms are here on Earth in moderate or extreme environments or on © Jones & Bartlett Learning, LLC Mars, there are certain© Jones physical & Bartlettand chemical Learning, LLC requirements they must possess to survive, FIGURENOT 5.1 FOR The Martian SALE Surface? OR DISTRIBUTION This barren- NOT FOR SALE OR DISTRIBUTION looking landscape is not Mars but the Atacama Desert in grow, and reproduce. In this chapter, we explore Chile. It looks similar to photos taken by the Mars rovers the process of microbial cell reproduction, Spirit and Opportunity. ❯ ❯ Does this area look like a examine the physical and chemical conditions habitable place for life, even microbial life? required for growth, and discover the ways that © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC © Photodisc/age fotostock. microbial growth can be measured. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE Binary OR Fission DISTRIBUTION Is Part of the CHAPTER CHALLENGE Cell Cycle e series of events involving growth, DNA replication, and cell division is called the cell The United States has sent several spacecra © Jones & Bartlett Learning, LLC cycle. For a bacterial© cell Jones like E. & coli Bartlett, the cycle Learning, can LLC toNOT Mars FOR since theSALE first Viking OR landersDISTRIBUTION in 1976. NOT FOR SALE OR DISTRIBUTION Recently, an international team of scientists be divided into three periods ( FIGURE 5.2A ). carried out studies suggesting terrestrial B Period. Before a bacterial cell actually microbes could hitch a ride to Mars on such a divides into two cells, it goes through a phase cra , and they could even survive the journey. The of metabolic “growth,” called the B period , © Jones & Bartlettteam believes Learning, most spacecra LLC that have touched where© Jones the cell & increases Bartlett in Learning, cell mass andLLC cell down on Mars were not thoroughly sterilized, so NOT FOR SALEthey couldOR haveDISTRIBUTION carried living microbes from size.NOT During FOR this SALE period, OR the DISTRIBUTIONcell is preparing for Earth. NASA scientists have assumed Mars’ thin chromosome replication, so the chromosome atmosphere, which allows intense UV radiation remains a single, circular DNA molecule. to reach the planet’s surface—triple Earth’s C Period. Once the cell has increased in intensity—would kill any life inadvertently © Jones & Bartlett Learning, LLC © Jones & size,Bartlett DNA Learning, replication LLCstarts. During this C carried on the spacecra . However, in laboratory period , DNA synthesis occurs and the bacterial NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION tests, here on Earth the scientists came across chromosome is copied, which will ensure each some spore-forming bacterial species that could survive UV bombardment at a level equivalent daughter cell has one complete set of genetic to that on Mars—if the spores were buried just a information when cell division is completed. few millimeters in the soil. Could such an earthly Chromosome segregation involves the cell’s extremophile© Jones survive& Bartlett on Mars, Learning, or could there LLC cytoskeleton but, unlike© Jones eukaryotic & Bartlett cells, lacksLearning, LLC actuallyNOT FOR be Martian SALE microbes OR DISTRIBUTION on the planet? the mitotic spindleNOT to FORsegregate SALE replicated OR DISTRIBUTION Are there conditions here on Earth that might chromosomes. DNA replication will be give us an idea? Let’s examine what it takes for a bacterial species to survive both “normal” and examined in a later chapter. “extreme” earthly environments. D Period. As DNA replication ends, the © Jones & Bartlett Learning, LLC cell© undergoesJones & binaryBartlett ssion Learning,. During LLC this D NOT FOR SALE OR DISTRIBUTION periodNOT , aFOR partition SALE or septumOR DISTRIBUTION forms at midcell ( FIGURE 5.2B ). e splitting of the cell in half ( cytokinesis ) is coordinated by cytoskeletal KEY CONCEPT 5.1 Bacteria: © NIAID proteins organized into a “ ssion ring apparatus,” which ensures two nearly identical © Jones & Bartlett Learning, Microbial LLC Reproduction Is Part of© the Jones & Bartlett Learning, LLC Cell Cycle daughter cells are formed. Depending on the NOT FOR SALE OR DISTRIBUTION NOT FOR SALEgrowth ORconditions, DISTRIBUTION the septum material may dissolve at a slow rate allowing pairs, chains, Growth in the microbial world usually refers to or clusters of cells to form, representing the an increase in the numbers of individuals; that characteristic arrangements for many bacterial is, an© increase Jones in & the Bartlett population Learning, size with LLC each species. With other© species, Jones like & E.Bartlett coli, the Learning,cells LLC cell carryingNOT FOR the SALEidentical OR genetic DISTRIBUTION instructions completely separateNOT from FOR one another SALE aOR er DISTRIBUTIONthe of the parent cell. Asexual reproduction is the septum is complete. Each of the attached or free process to maintain this genetic constancy. In cells then enters another B period. eukaryotic microbes, an elaborate interaction of microtubules and proteins with pairs of © Jones & Bartlett Learning, LLC Bacterial © Jones and & Bartlett Archaeal Learning, Cells Can LLC chromosomes in the cell nucleus allows for NOT FORthe SALE precise OR events DISTRIBUTION of mitosis and cytokinesis. GrowNOT Exponentially FOR SALE OR DISTRIBUTION Bacterial and archaeal cells divide without e interval of time between successive binary this elaborate division apparatus but still ssions of a cell or population of cells is known as possess similar protein laments to use in the the generation time (or doubling time). Under © Jones & Bartlett Learning,reproduction LLC process. © Jones & optimalBartlett conditions, Learning, some LLC species, especially NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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Cell wall Cell membrane © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Binary fission separates Cells are growing each chromosome into as they prepare a separate cell. for the C period. B period (Cell “growth”) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

FissionNOT ring FOR SALE OR DISTRIBUTION Replicated NOT FOR SALE OR DISTRIBUTION apparatus DNA molecules DNA synthesis and replication generate two daughter chromosomes. (A) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC (B) NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 5.2 The Bacterial Cell Cycle. (A) The bacterial cell cycle can be separated into three stages or periods involved with cell growth (B period), chromosome replication (C period), and binary fission (D period). (B) A false-color transmission electron micrograph of a cell of Bacillus licheniformis undergoing binary fission. The inward growth of the cell envelope is evident at midcell between the segregated chromosomes. (Bar = 0.25 µm.) ❯ ❯ How would binary © Jonesfission & Bartlett di er for aLearning, prokaryotic organism LLC having cells arranged in© chains Jones and another& Bartlett forming Learning, single cells? LLC NOT FOR © Lee Simon/ScienceSALE Source. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONmany pathogens, have a very fast generationNOT FOR SALEfor 36 ORhours, DISTRIBUTION the bacterial cells would cover time; for others, including many environmental the surface of the Earth! ankfully, this will species, it is much slower ( TABLE 5.2 ). In fact, not occur because of the limitation of nutrients for some, it is extremely slow, as MicroFocus and the loss of ideal physical factors required 5.1 reports.© Jones & Bartlett Learning, LLC for growth. e majority© Jones of the& Bartlett bacterial Learning,cells LLC One NOT enterprising FOR SALE mathematician OR DISTRIBUTION calculated would starve to deathNOT or dieFOR in theirSALE own OR waste. DISTRIBUTION that if E. coli binary ssions were to continue e generation time is useful in determining at their optimal generation time (15 minutes) how much time passes before disease symptoms

MICROFOCUS 5.1: Evolution/Environmental Microbiology © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR A Microbe’s SALE ORLife—Zombie DISTRIBUTION Style NOT FOR SALE OR DISTRIBUTION

What do World War Z , Zombieland , Dawn of the Dead and somerock-dwelling microbial zombies could be beneficially a® ecting extreme microbial species have in common? All are examples of the chemistry in the rocks, the deep Earth, and the planet itself. a zombie state, which for the© above-mentioned Jones & Bartlett big screen Learning, flicks LLC Scientifically, these bacterial© Jones communities & Bartlett o® er Learning, an LLC identifies “creatures” that haveNOT died FOR and SALE been reanimated OR DISTRIBUTION as opportunity to learn how cells canNOT survive FOR on SALE so few ORnutrients DISTRIBUTION unconscious, mindless monsters, o en apparently arising from and minimal energy sources. Their very existence helps scientists deep below the Earth. Well, in the microbial world there also are to understand the line between life and death, and to estimate some microbes that exist in a zombie-like state deep below the what the bare minimum is for an organism to stay alive. Earth. As you© Jones already know,& Bartlett microbes Learning, can be found LLC just about © Jones & Bartlett Learning, LLC anywhere NOT on and FOR in planet SALE Earth OR where DISTRIBUTION an energy source and NOT FOR SALE OR DISTRIBUTION nutrients are found. This includes deep below the Earth’s surface. In 2013, scientists with the Integrated Ocean Drilling Program, an international marine research program, announced they had discovered bacterial organisms in rocks 2.5 kilometers below the © Jonesocean & floor. Bartlett More impressive, Learning, these LLC organisms are up to 100 million© Jones & Bartlett Learning, LLC NOT FORyears old,SALE and the OR researchers DISTRIBUTION believe the bacterial cells divide onlyNOT FOR SALE OR DISTRIBUTION once every 10,000 years! The growth is so slow, scientists originally wondered if these cells were alive—or just undead. However, give them a luxury microbial meal and the absorption of nutrients by the cells can be detected. Thus, the apparent snail’s pace for binary fission and growth results from© theJones infinitesimally & Bartlett small amountLearning, of LLC © Jones & Bartlett Learning, LLC energy and nutrients availableNOT in their FOR rocky SALE homes. AtOR these DISTRIBUTION rates, NOT FOR SALE OR DISTRIBUTION it takes thousands of years for the single-celled intraterrestrials (living within the Earth) to produce enough energy to support binary fission. Dr. Beth Orcutt from the Bigelow Laboratory for Ocean Sciences in Maine stated, “Is it really true to call [these © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC microbes] alive when [they’re] doubling every thousands of years? Dr. Beth Orcutt (front row, second from le ) and researchers It’s almostNOT like a zombieFOR SALEstate.” OR DISTRIBUTION are tryingNOT to understand FOR SALE how life belowOR DISTRIBUTIONthe sea floor can Unlike the “human zombies” who attempt to disturb human survive and thrive. society (at least in the movies), the ocean scientists believe the Photo by Jen Magnusson.

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR Examples DISTRIBUTION of Generation 1,000,000 Times—Common Bacterial TABLE 5.2 Species Growing Under Time Number of of day cells Optimal Conditions 1 900,000 8:00 PM 100 Growth© Jones Generation & Bartlett Time Learning, LLC 8:30 200© Jones & Bartlett Learning, LLC Species MediumNOT FOR(minutes) SALE OR DISTRIBUTION 9:00 400NOT FOR SALE OR DISTRIBUTION 800,000 9:30 800 Escherichia coli Synthetic 17 10:00 1,600 Bacillus Synthetic 25 10:30 3,200 11:00 6,400 megaterium 700,000 11:30 12,800 Staphylococcus© Jones Complex & Bartlett Learning, 27–30 LLC 12:00© JonesAM 25,600& Bartlett Learning, LLC aureusNOT FOR SALE OR DISTRIBUTION 12:30NOT FOR51,200 SALE OR DISTRIBUTION 600,000 1:00 102,400 Streptococcus Milk 26 1:30 204,800 lactis 2:00 409,600 2:30 819,200 500,000 Streptococcus Complex 48 3:00 AM 1,638,400 © Jones lactis& Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORLactobacillus SALE OR DISTRIBUTION Milk 66–87 NOT FOR SALE OR DISTRIBUTION

Actual number of bacterial cells 400,000 acidophilus Mycobacterium Synthetic 792–932 tuberculosis (= 12–15.5 hours) 300,000 Treponema Rabbit testes© Jones 1,980 & (=Bartlett 33 hours) Learning, LLC © Jones & Bartlett Learning, LLC pallidum NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 200,000 1 Table modified from microblog.me.uk/138 Adapted from MicrobLog, Doubling Time

100,000 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 0 8 9 10 11 12 1 2 3 appear in an infected individual; faster division Incubation PM Time (hours) AM period: The time times o en mean a shorter incubation period from entry of a for a disease. For example, suppose you eat an FIGURE 5.3 . © Jones & Bartlett Learning, LLC © Jones A &Skyrocketing Bartlett BacterialLearning, Population LLC pathogen into the improperly refrigerated chocolate éclair that The number of Staphylococcus aureus cells progresses body until the first NOT FORwas SALE contaminated OR DISTRIBUTION with Staphylococcus aureus from NOT100 cells FOR to almost SALE 2 million OR cells DISTRIBUTION in a mere 7 hours. symptoms appear. The J-shaped growth curve gets steeper and steeper as the ( FIGURE 5.3 ). If you ingested 100 cells at 8:00 hours pass. Only a depletion of food, buildup of waste, or PM this evening, 200 would be present by 8:30, some other limitation will halt the progress of the curve. 400 by 9:00, and 800 by 9:30 PM . You would ❯ ❯ What is the generation time for S. aureus in this have more than 25,000© Jonesby midnight. & Bartlett By 3:00 Learning, figure? LLC © Jones & Bartlett Learning, LLC AM , the exponential growthNOT willFOR have SALE produced OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION more than 1.6 million cells. Depending on the Bacteria: © NIAID response of the immune system, it is quite likely CONCEPT AND REASONING CHECKS 1 that sometime during the night you would a. Propose an explanation as to how know ©you Jones have food & Bartlett poisoning. Learning, LLC a bacterial cell© Jones “knows” & when Bartlett to Learning, LLC divide. BacterialNOT FOR and archaeal SALE organismsOR DISTRIBUTION are subject NOT FOR SALE OR DISTRIBUTION to the same controls on growth as all other b. If it takes an E. coli cell 7 hours of organisms on Earth. Let’s examine the most growth to reach some 2 million cells, how long would it take a T. important growth factors conferring optimal pallidum cell to reach the same generation times. © Jones & Bartlett Learning, LLC © Jonesnumber & under Bartlett optimal Learning, conditions? LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE A Bacterial OR DISTRIBUTION Growth Curve Illustrates the Dynamics of Growth CHAPTER CHALLENGE A A typical bacterial growth curve for a population illustrates the events occurring over We© Jonesalready know & Bartlett some bacterial Learning, and archaeal LLC © Jones & Bartlett Learning, LLC time ( FIGURE 5.4 ). If a sample of bacterial organismsNOT FOR can surviveSALE in OR hostile DISTRIBUTION environments. NOT FOR SALE OR DISTRIBUTION In fact, these extremophiles not only survive in cells is transferred to a tube of fresh broth, four these environments, they actually can thrive distinct phases of growth occur: the lag phase; there. This means they need to go through binary the logarithmic phase; the stationary phase; and fission as part of their cell cycle to maintain their the decline phase. © Jones & Bartlettpopulation Learning, numbers. LLC ©The Jones Lag & Phase. Bartlett e Learning, rst portion LLC of the NOT FOR SALE Question OR A:DISTRIBUTION growthNOT curve, FOR duringSALE which OR DISTRIBUTION time the bacterial Propose how these extremophiles can carry cells are adapting to their new environment out DNA replication and reproduce in an and compensating for changes in nutritional environment that would otherwise kill a conditions, is called the lag phase . In the broth, temperate microbe? What are the chances some cells may actually die from the shock of © Jones & Bartlett Learning,an Earthly LLC extremophile would have© beenJones & Bartlett Learning, LLC transfer or the inability to adapt to the new NOT FOR SALE OR DISTRIBUTIONon a spacecra to Mars? And if it did,NOT would FOR SALE OR DISTRIBUTION it survive? environment. e actual length of the lag phase Answers can be found in Appendix F . ( B period of the cell cycle) depends on the metabolic activity of the microbial population. ey must grow in size, take up nutrients, © Jones & Bartlett Learning, LLC and replicate their© DNAJones ( C & period Bartlett )—all Learning, in LLC NOT FOR SALE OR DISTRIBUTION preparation for binaryNOT ssion FOR ( DSALE period OR ). DISTRIBUTION KEY CONCEPT 5.2 Bacteria: © NIAID The Log Phase. e population now enters Microbial Growth Progresses Through an active, exponential stage of growth called Distinct Phases the logarithmic (log) phase . In the log phase, © Jones & Bartlett Learning, LLC all ©cells Jones are undergoing & Bartlett binary Learning, ssion LLCand the NOT FORIn SALE the previous OR DISTRIBUTION section, we discovered how generationNOT FOR time SALE is dependent OR DISTRIBUTION on the species and fast some bacterial cells can grow under ideal environmental conditions present. e cells circumstances. Let’s look at the dynamics of exhibit balanced growth because all aspects of bacterial growth in a little more detail. metabolism and physiology remain constant.

© Jones & Bartlett10 Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR9 DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 8 (C) Stationary phase (D) Decline 7 phase 6 5 (B) Log phase ) of viable cells ) of viable 4 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC n NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 3

Logarithm (10 Logarithm 2 (A) Lag © Jones &phase Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 0 NOT FOR01 SALE OR DISTRIBUTION2345 NOT FOR67 SALE OR DISTRIBUTION8 Time (hours) Total cells in population:

Few cells Viable cells Nonviable/dead cells © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC FIGURE 5.4 The Growth Curve for a Bacterial Population. A typical bacterial population goes sequentially through four NOT FOR SALEgrowth OR DISTRIBUTION phases. ❯ ❯ Why would antibiotics work bestNOT to kill orFOR inhibit SALE cells in theOR log DISTRIBUTION phase?

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORAs SALEeach generation OR DISTRIBUTION time passes, the number of logarithmically.NOT FOR For SALE many OR species, DISTRIBUTION the history of cells doubles and the graph rises accordingly on the population ends with the death of the last cell. a logarithmic scale. On the other hand, for some bacterial In a broth tube, the medium becomes species, a few members of the population can cloudy (turbid) due to© increasing Jones & cell Bartlett numbers. Learning, escape death LLC by entering a state of dormancy.© Jones & Bartlett Learning, LLC If plated on solid growthNOT FORmedium, SALE bacterial OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION growth will be so vigorous that visible colonies Some Bacterial Cells Can Exist In a appear within 24 to 48 hours and each colony Metabolically Inactive State may consist of millions of cells ( FIGURE 5.5 ). Vulnerability to antibiotics is also highest at this Environmental conditions vary tremendously active ©stage Jones of growth & Bartlett because Learning, many antibiotics LLC and o en they might© not Jones be favorable & Bartlett to active Learning, LLC a ect metabolicNOT FOR processes SALE likeOR protein DISTRIBUTION synthesis population growth. SuchNOT unfavorable FOR SALE conditions OR DISTRIBUTION in dividing cells. include the presence of toxic chemicals, such as The Stationary Phase. Because a broth antibiotics, and nutrient limitation (potential tube represents a “closed system,” nutrients starvation). erefore, throughout the cell cycle © Jonesare & Bartlettnot available Learning, inde nitely. LLC erefore, a er microbes© Jones must & constantlyBartlett Learning,monitor or LLCsense NOT FORsome SALE hours OR in aDISTRIBUTION broth tube, available nutrients theirNOT surroundings FOR SALE to ensure OR theDISTRIBUTION conditions will become scarce and waste products accumulate. support continued growth. For some bacterial is limitation of nutrients and buildup of species, if the environment is not favorable, waste materials leads to a decline in the growth the cells respond by entering a dormant rate. e vigor of the population changes and (nondividing) state. When environmental the cells enter a plateau,© Jones called the& Bartlett stationary Learning, conditions LLC improve, the cells “revive”© Jones and & Bartlett Learning, LLC phase. Now the numberNOT of FORviable SALE cells equals OR DISTRIBUTIONonce again start dividing. Two suchNOT dormancy FOR SALE OR DISTRIBUTION the number of nonviable or dead cells. strategies are described here. The Decline Phase. If nutrients in the Persister Cell Formation. During the log external environment remain limited or phase of growth, some bacterial populations the quantities© Jones become & Bartlett exceedingly Learning, low, LLCthe produce a subset of© cellsJones that & normally Bartlett stopLearning, LLC populationNOT entersFOR SALEa decline OR phase DISTRIBUTION (or death dividing and remainNOT in theFOR B SALEperiod ORof the DISTRIBUTION phase). Here the number of viable cells declines cell cycle. However, these so-called persister

(A) NOT FOR SALE OR DISTRIBUTION (B) NOT FOR SALE OR DISTRIBUTION

FIGURE 5.5 Two Views of Bacterial Colonies. Bacterial (A) colonies cultured on blood agar in a culture dish. Blood agar is a mixture of nutrient agar and blood cells. It is o en used for growing bacterial colonies. (B) Close-up of typhoid bacterial ( Salmonella enterica serotype Typhi) colonies being cultured on a growth medium. ❯ ❯ How did each colony in © Jones(A) & or Bartlett (B) start? Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR (A) Courtesy SALE of Dr. J. J. Farmer/CDC. OR (B) CourtesyDISTRIBUTION of CDC. NOT FOR SALE OR DISTRIBUTION

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONcells maintain a very low rate of metabolism.NOT FOR SALEdormant OR DISTRIBUTIONstructures called endospores Should the environment change for the worse, ( FIGURE 5.7 ). the persister cells in the population survive Unlike persister cells, most endospores are the even though the rest of the population may result of a focused morphological di erentiation die. For© Jones example, & such Bartlett “altruistic-like” Learning, behavior LLC process speci cally© tied Jones to nutrient & Bartlett limitation Learning, LLC bene NOT ts pathogens FOR SALE like OR M. DISTRIBUTIONtuberculosis. If (starvation) in the NOTenvironment FOR SALE and is initiatedOR DISTRIBUTION antibiotics are used to treat the infection, any by quorum sensing within the “starving” persister cells will survive because they are not population. a ected by the antibiotics, which target only Spore formation requires a complex actively dividing, log phase cells ( FIGURE 5.6 ). program of gene expression that involves © Jones &Should Bartlett the antibioticsLearning, be LLC withdrawn at some hundreds© Jones of &genes. Bartlett e processLearning, involves LLC the NOT FORlater SALE date, OR the persisterDISTRIBUTION cells become active again, replicationNOT FOR of theSALE bacterial OR DISTRIBUTION chromosome and forming a new actively growing population binary ssion that produces an asymmetric cell with some persister cells present. division ( FIGURE 5.8 ). e smaller cell, called Endospore Formation. A few medically- the prespore, will become the mature endospore, © Jones & Bartlett Learning,signi cant LLC gram-positive genera, ©especially Jones & whileBartlett the larger Learning, mother LLCcell will commit itself to NOT FOR SALE OR DISTRIBUTION Bacillus and Clostridium, undergo aNOT di erent FOR SALEmaturation OR DISTRIBUTIONof the endospore before rupturing. dormancy scenario when the cells experience Depending on the exact asymmetry of cell nutrient depletion. Species, such as Bacillus division, the single endospore may develop anthracis (causative agent of anthrax) and at the end of the mother cell, near the end, or Clostridium botulinum (the causative agent of at the center of the cell (the position is useful botulism)© Jones enter &the Bartlett stationary Learning, phase and LLCbegin for species identi ©cation Jones purposes). & Bartlett e entire Learning, LLC sporeNOT formation FOR orSALE sporulation OR DISTRIBUTION that produces process takes aboutNOT 6 to 8FOR hours. SALE OR DISTRIBUTION

Persister cell © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONAntibiotic sensitive cell NOT FOR SALE OR DISTRIBUTION

C B © Jones & Bartlett Learning, LLC Sensitive cells © Jones & Bartlett Learning, LLC Binary fission killed; persister NOT FOR SALE OR DISTRIBUTIONof persister cells NOT FOR SALE OR DISTRIBUTION cells survive. resumes.

Killed cell Antibiotic treatment © Jones & Bartlett Learning, LLC stopped © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning,FIGURE 5.6 LLC Persister Cells. The presence© ofJones persister &cells Bartlett within a bacterial Learning, cell population LLC allows the species to survive environmental changes, in this case the presence of an antibiotic. ❯ ❯ What other way could a susceptible NOT FOR SALE OR DISTRIBUTIONbacterial cell population survive in the presenceNOT of FOR an antibiotic? SALE OR DISTRIBUTION

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(A) (B)

© Jones & Bartlett Learning, LLC FIGURE© Jones 5.7 Three & Bartlett Di erent Learning,Views of Bacterial LLC Endospores. A( lightA ) NOT FOR SALE OR DISTRIBUTION microscopeNOT imageFOR of SALE Clostridium OR cells DISTRIBUTION showing terminal spore formation. Note the characteristic drumstick appearance of the spores. (Bar = 5.0 µm.) ( B )A false-color transmission electron micrograph of Bacillus anthracis spores. The visible spore structures include the core, cortex, and coat layers. (Bar = 0.5 µm.) ( C )A scanning electron microscope view of a germinating spore (arrow). Note that the spore © Jones & Bartlett Learning,coat divides LLC equatorially along the long axis, ©and Jones as it separates, & Bartlett the vegetative Learning, LLC NOT FOR SALE OR DISTRIBUTIONcell emerges. (Bar = 2.0 µm.) ❯ ❯ If an endosporeNOT is resistantFOR SALE to so many OR DISTRIBUTION environmental conditions, how does a spore “know” conditions are favorable for germination? (C) (A) Courtesy of CDC. (B) © Scott Camazine/Alamy. (C) Courtesy of Janice Carr/CDC.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION e highly dehydrated free spore contains rems of radiation, but endospores can survive Rem (Roentgen cytoplasm, highly compacted DNA, and a large one million rems. In this dormant condition, equivalent in amount of dipicolinic acid , a unique chemical endospores can “survive” for long periods of man): A measure of radiation dose compound that helps stabilize the proteins and time, perhaps millennia, as MicroFocus 5.2 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC related to biological DNA. ick layers of peptidoglycan form the reveals. No wonder some scientists believe they e® ect. NOT FORcortex SALE and someOR DISTRIBUTION 70 proteins form the layers of the couldNOT survive FOR a trip SALE to Mars! OR DISTRIBUTION spore coat to protect the cytoplasmic contents. It When the environment is favorable for cell is important to emphasize that sporulation is not growth, the spore coat and cortex break down a reproductive process. Rather, the endospore over a 90-minute period and each endospore represents a “resting stage”© Jones produced & Bartlettin response Learning, rapidly germinates LLC and grows out as a© vegetative Jones & Bartlett Learning, LLC to environmental “hardNOT times.” FOR SALE OR DISTRIBUTIONcell (see Figure 5.7 C). NOT FOR SALE OR DISTRIBUTION Endospores are probably the most resistant Killing endospores can be a tough task. living structures known. Desiccation has Heating them for many hours under high little e ect on the spore. By containing little pressure will do the trick. If they contaminate water,© endospores Jones & alsoBartlett are heat Learning, resistant LLC and machinery, such as ©the Jones mail-sorting & Bartlett equipment Learning, LLC undergoNOT very FOR few SALE chemical OR reactions.DISTRIBUTION ese contaminated in theNOT 2001 FOR anthrax SALE attacks,OR DISTRIBUTION properties make them di cult to eliminate there are potent but highly dangerous chemical from contaminated medical materials and methods to kill the spores. Such methods are food products. For example, endospores can discussed in another chapter. remain viable in boiling water (100°C) for Medical Significance. A few serious © Jones2 &hours. Bartlett ey Learning,can survive inLLC 70% ethyl alcohol diseases© Jones in humans & Bartlett are caused Learning, by spore formers. LLC NOT FORfor SALE 20 years. OR Humans DISTRIBUTION can barely withstand 500 e NOTmost FORnewsworthy SALE hasOR beenDISTRIBUTION B. anthracis,

978128057782_CH05_140_171.indd 149 10/10/14 2:26 PM © Jones & Bartlett Learning. NOT FOR SALE OR DISTRIBUTION. 150 Chapter 5: Microbial Growth and Nutrition

ASEXUAL CELL CYCLE © Jones & Bartlett Learning, LLC (nutrients © Jones & Bartlett Learning, LLC NOT FOR SALE BOR period DISTRIBUTION plentiful) NOTC FORperiod SALE OR DISTRIBUTION

D Germination. A Asymmetric cell division. Under favorable A transverse membrane © Jones & Bartlettnutrient Learning, conditions, LLC © Jones & Bartlett Learning,septum LLC separates one the aged spore NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTIONDNA molecule and a germinates to small section of cytoplasm. produce a new vegetative cell. Cell DNA Cell wall membrane Vegetative cell © JonesSpore & coatBartlett Learning, LLC © Jones & Bartlett Learning, LLC DNA NOT FORCortex SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Free spore Transverse septum © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC SPORULATION NOT FOR SALE OR DISTRIBUTIONCYCLE NOT FOR SALE OR DISTRIBUTION (carbon/nitrogen Cortex limitation) Mother cell Prespore

B Early sporulation. A peptidoglycan cortex Spore coat forms between the two C Late sporulation. © Jones & Bartlett Learning,segments LLC © Jonesmembranes & Bartlett of the prespor Learning,e. LLC The spore coat is completed and the NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION mother cell ruptures, freeing the spore.

FIGURE 5.8 The Formation of a Bacterial Spore by Bacillus subtilis . ( A–E ) When nutrient conditions become limiting (e.g., carbon, nitrogen), endospore formers, such as B. subtilis, enter the sporulation cycle shown here. ❯ ❯ Hypothesize how a vegetative cell “knows” © Jones &nutrient Bartlett conditions Learning, are limiting. LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

978128057782_CH05_140_171.indd 150 10/10/14 2:26 PM © Jones & Bartlett Learning. NOT FOR SALE OR DISTRIBUTION. Optimal Microbial Growth Is Dependent on Several Physical Factors 151

Germination of 25 Million-Year-Old© Jones & Bartlett Endospores? Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Endospores have been discovered in various archaeological sterilized the amber sample before cracking it open. He also sites and environments around the world. Living spores have carried out all the procedures in a class II laminar flow hood, been recovered and germinated from the intestines of Egyptian which prevents outside contamination from entering the working mummies© Jones several thousand& Bartlett years Learning, old. In 1983, LLC archaeologists area. In addition,© Jones the hood & Bartlett had never Learning, been used for LLC any other found NOT viable FORThermoactinomyces SALE OR DISTRIBUTION spores in sediment lining bacterial extractionNOT FOR processes. SALE Several OR DISTRIBUTION other precautions were Minnesota’s Elk Lake. The sediment was over 7,500 years old. added to eliminate any chance that the spores were modern-day All these reports, though, pale in comparison to the contaminants from an outside source. controversial discovery reported in 1995 by researcher Raul The major question remaining is whether DNA can remain Cano of California Polytechnic State University, San Luis Obispo. intact and functional a er so long a period of dormancy. Does © JonesCano & Bartlett found bacterial Learning, spores LLC in the gut of a fossilized © bee Jones the DNA & Bartlett really have Learning, a capability LLC of replication and producing NOT FORtrapped SALE in amber—aOR DISTRIBUTION hardened resin—produced from a treeNOT in newFOR vegetative SALE OR growth? DISTRIBUTION Granted, the DNA presumably was the Dominican Republic. The amber with the fossilized bee was protected in a resistant spore, but could DNA remain intact for dated as being about 25 million years old. When the amber was 25 million years? Research on bacterial DNA suggests the cracked open and the material from the gut of the bee extracted maximum survival time is about 400,000 to 1.5 million years. If and placed in nutrient medium, the equally ancient endospores true, then the 25 million-year-old spores could not be viable. But germinated. With microscopy,© Jones the cells & fromBartlett a colony Learning, were very LLCthat is based on current predictions,© Jones and they& Bartlett may be subject Learning, to LLC similar to Bacillus sphaericusNOT , which FOR is foundSALE today OR in DISTRIBUTIONbees in the change as more research is carriedNOT out FOR with SALE ancient DNA.OR DISTRIBUTION Dominican Republic. Is it possible for an endospore to survive for The verdict? It seemed unlikely such ancient endospores 25 million years—even if it is encased in amber? could germinate a er 25 million years. Then, in 2000, another Critics were quick to claim the bacterial species may research group using Cano’s techniques revived 250 million-year- represent a modern-day species that contaminated the amber old bacterial cells from spores trapped in salt crystals. Maybe it sample© being Jones examined. & Bartlett However, Learning, Professor Cano LLC had carried is possible.© Jones & Bartlett Learning, LLC out appropriateNOT FOR and SALErigorous ORdecontamination DISTRIBUTION procedures and NOT FOR SALE OR DISTRIBUTION

the agent of the 2001 anthrax bioterror attack In hospital and healthcare facilities, C. di - © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC through the mail. is potentially deadly cile (o en referred to as C . d i ) can cause seri- NOT FORdisease, SALE originally OR DISTRIBUTION studied by Koch and Pasteur, ous NOTinfections FOR of SALE the colon OR (colitis) DISTRIBUTION that are of- develops when inhaled spores germinate in ten resistant to antibiotic treatment. More than the lower respiratory tract and the resulting three million C . d i c i l e infections occur in U.S. vegetative cells secrete two deadly toxins. Other hospitals each year, partly coming from the tis- human diseases result© fromJones the & germination Bartlett Learning, sue germination LLC of endospores originally© Jones pres- & Bartlett Learning, LLC of Clostridium endospores—gasNOT FOR gangrene SALE OR (C . DISTRIBUTIONent on improperly cleaned medical NOTequipment FOR SALE OR DISTRIBUTION perfringens ) and tetanus or lockjaw (C . tetani ). and on personal items. ( ese diseases will be Clostridial endospores o en are found in soil covered in more detail in the appropriate body and, should they nd their way into human system chapter). tissue ©through Jones a & puncture Bartlett wound, Learning, the spores LLC © Jones & Bartlett Learning, LLC will germinateNOT FOR in theSALE wound’s OR DISTRIBUTIONdead tissue and Optimal MicrobialNOT Growth FOR SALE Is OR DISTRIBUTION produce deadly toxins. Botulism, caused by Dependent on Several Physical C . botulinum, can be a deadly form of food Factors poisoning. Many foods improperly preserved can be contaminated with endospores that Having examined the bacterial cell cycle and © Joneson & germination,Bartlett Learning, the vegetative LLC cells produce dormancy,© Jones let’s & examine Bartlett the Learning, essential physical LLC NOT FORdeadly SALE toxins. OR DISTRIBUTION and NOTchemical FOR factors SALE in ORuencing DISTRIBUTION cell growth.

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONTemperature. Temperature is oneNOT of FOR the SALE Another OR DISTRIBUTION group of “cold-loving” microbes most important factors governing growth. are the psychrotrophs or psychrotolerant Every microbial species has an optimal microorganisms. ese species have a slightly growth temperature. Each species also has higher optimal growth temperature as well as an approximate© Jones & 30°C Bartlett operating Learning, range, LLCfrom a broader operating© Jonesrange. Psychrotrophs & Bartlett Learning, can LLC minimumNOT FORto maximum, SALE ORover DISTRIBUTION which the cells be found in water andNOT soil FOR in temperate SALE OR regions DISTRIBUTION will grow although they will have a slower of the world but are perhaps most commonly generation time ( FIGURE 5.9 ). In general, encountered on spoiled refrigerated foods most microbes can be assigned to one of (4°C). Some bacterial and archaeal species three temperature groups—psychrophiles, are psychrotolerant as are several microbial © Jones &mesophiles, Bartlett Learning,or thermophiles—based LLC on their eukaryotic© Jones species, & Bartlett including Learning, fungi (molds).LLC NOT FORoptimal SALE growth OR DISTRIBUTION temperature. WhenNOT such FOR contaminated SALE OR foods DISTRIBUTION are consumed Microbes that have their optimal growth without heating, the toxins the microbes rates near 15°C but can still grow at 0°C to 20°C produce may cause food poisoning. One are called psychrophiles ( psychro = “cold”). example is Campylobacter, the most frequently © Jones & Bartlett Learning,Because aboutLLC 70% of the Earth is covered© Jones by & identi Bartlett ed causeLearning, of infective LLC diarrhea ( Clinical NOT FOR SALE OR DISTRIBUTIONoceans having deep-water temperaturesNOT below FOR SALECase 5 ).OR DISTRIBUTION 5°C, psychrophiles make up a large portion of At the opposite extreme are the the global microbial community. In fact, many thermophiles (thermo = “heat”) that multiply psychrophiles can grow as fast at 4°C as E. best at temperatures around 60°C but still coli does at 37°C. On the other hand, at these multiply over a range from 40°C to 70°C. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC low temperatures, psychrophiles would not be ermophiles are present in compost heaps and humanNOT pathogens FOR SALE because OR they DISTRIBUTION cannot grow at hot springs, and canNOT be contaminantsFOR SALE ORin dairy DISTRIBUTION the warmer 37°C body temperature. products because they survive pasteurization temperatures. However, thermophiles pose little threat to human health because they do not © Jones & Bartlett Learning, LLC grow© Joneswell at the & coolerBartlett temperature Learning, of the LLC body. NOT FOR SALE OR DISTRIBUTION NOT ere FOR also SALEare many OR archaeal DISTRIBUTION species that Archaea grow optimally at temperatures exceeding Bacteria 80°C and having optima near 95°C. ese Eukarya hyperthermophiles have been isolated from seawater near hot-water vents along ri s on © Jones & Bartlett Learning, LLC Thermophiles © Jones & Bartlett Learning, LLC the oor of the Paci c Ocean. Because the high NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Hyperthermophiles pressure keeps the water from boiling, some Mesophiles owth archaeal species can grow at an astonishing Psychrotrophs 121°C (see Table 5.1 ). of gr Psychrophiles te Most of the best-characterized microbial © Jones & Bartlett Learning, LLC species are mesophiles © Jones (meso = & “middle”), Bartlett which Learning, LLC NOT FOR SALE OR DISTRIBUTION thrive at the middleNOT temperature FOR SALE range ORof 10°C DISTRIBUTION easing ra to 45°C. is includes the pathogens that grow Incr in warm-blooded animals, including humans, as well as those species found in aquatic and soil environments in temperate and tropical regions ©0–10 Jones10 20& Bartlett30 40 Learning,50 60 70LLC80 90 100110 © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION of NOTthe world. FOR E. SALE coli is aOR typical DISTRIBUTION mesophile. e Temperature (˚C) vast majority of microbes discussed in this text are mesophiles. FIGURE 5.9 Growth Rates for Di erent Microorganisms in Response to Temperature. Temperature optima and ranges define the growth rates for Oxygen. e growth of many microbes Bacteria, Archaea, and Eukarya. Notice that the growth rates decline quite rapidly to depends on a plentiful supply of oxygen gas, © Joneseither & side Bartlett of the optimal Learning, growth temperature. LLC ❯ ❯ Propose what adaptations© Jones are & andBartlett in this Learning, respect, such LLC obligate aerobes must NOT FORneeded SALE for microbes OR toDISTRIBUTION survive at the psychrophilic or thermophilicNOT extremes. FOR SALEuse the ORgas asDISTRIBUTION a nal electron acceptor to make

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION CLINICAL CASE 5 An Outbreak of Food Poisoning Caused by Campylobacter jejuni © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 1 On August 15, a cook began his day by cutting up raw chickens to be roasted for dinner.

2 He also cut up lettuce, tomatoes, cucumbers, and other salad ingredients on the same © countertop.Jones & The Bartlett countertop Learning, surface where LLChe © Jones & Bartlett Learning, LLC NOTworked FOR was unusuallySALE ORsmall. DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 3 For lunch, the cook prepared sandwiches on the same countertop. Most were garnished with lettuce.

4 Restaurant patrons enjoyed sandwiches for © Jones & Bartlettlunch andLearning, roasted chicken LLC for dinner. Many © Jones & Bartlett Learning, LLC NOT FOR SALEpatrons OR alsoDISTRIBUTION had a portion of salad with their NOT FOR SALE OR DISTRIBUTION meal. None of the foods had been refrigerated.

5 During the next 3 days, 14 people experienced stomach cramps, nausea, and vomiting.

6 Public health o® icials© Jones learned that& Bartlett all the Learning, LLC © Jones & Bartlett Learning, LLC a® ected patrons NOThad eaten FOR salad SALE with lunch OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION or dinner. Campylobacter jejuni, a bacterial Campylobacter jejuni agar culture. pathogen of the intestines, was isolated from Courtesy of Sheila Mitchell/CDC. their stools (see figure).

7 On inspection, microbiologists concluded that the chicken was probably contaminated with C. jejuni. © JonesHowever, the& microbiologistsBartlett Learning, suspected the LLC cooked chicken was not the cause© ofJones the illness. & Rather,Bartlett Learning, LLC NOT C. jejuni FOR from SALE the raw chickenOR DISTRIBUTION was the source. NOT FOR SALE OR DISTRIBUTION Questions: (Answers can be found in Appendix E .) A. Why would the cooked chicken not be the source for the illness? © Jones & BartlettB. Why was Learning, the raw chicken LLC identified as the source? © Jones & Bartlett Learning, LLC NOT FOR SALEC. How, OR in fact,DISTRIBUTION did the patrons become ill? NOT FOR SALE OR DISTRIBUTION

For additional information see http://www.cdc.gov/mmwr/preview/mmwrhtml/00051427.htm .

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC cellular energy (ATP).NOT Other FOR species, SALE such OR as DISTRIBUTIONoxygen. Many bacterial and archaealNOT species, FOR as SALE OR DISTRIBUTION Treponema pallidum, the agent of syphilis, are well as a few fungal and protistan species, are termed microaerophiles because they survive obligate anaerobes, which are inhibited or killed in environments where the concentration of if oxygen is present. is means they need other oxygen© isJones relatively & Bartlett low. In theLearning, body, certain LLC ways to make ATP.© Some Jones anaerobic & Bartlett bacterial Learning, LLC microaerophilesNOT FOR causeSALE disease OR DISTRIBUTION of the oral species use sulfur inNOT their FOR metabolic SALE activities OR DISTRIBUTION cavity, urinary tract, and gastrointestinal tract. instead of oxygen, and therefore they produce

Conditions can be established in the laboratory hydrogen sul de (H2 S) rather than water (H2 O) to study these microbes ( FIGURE 5.10A ). as a waste product of their metabolism. Others we e anaerobes , by contrast, are microbes have already encountered, such as the ruminant © Jonesthat & Bartlettdo not or Learning, cannot use LLC oxygen. Some are archaeal© Jones organisms & Bartlett that produce Learning, methane LLC as NOT FORaerotolerant SALE OR, meaning DISTRIBUTION they are insensitive to the NOTbyproduct FOR of SALE the energy OR DISTRIBUTION conversions. In

978128057782_CH05_140_171.indd 153 10/10/14 2:26 PM © Jones & Bartlett Learning. NOT FOR SALE OR DISTRIBUTION. 154 Chapter 5: Microbial Growth and Nutrition

Screw Candle clamp Gasket © Jones & Bartlett HLearning,2 + O2 LLC © Jones & Bartlett Learning, LLC Palladium Cork NOT FOR SALE OR DISTRIBUTIONH2O catalyst NOT FOR SALE OR DISTRIBUTION

Hydrogen gas generator Liquid media © Jones & Bartlettin tubes Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION Petri dishes NOT FOR SALE OR DISTRIBUTION

Solid media in inverted petri dishes (A) (B) (C)

© JonesFIGURE & Bartlett 5.10 Bacterial Learning, Cultivation LLC in Di erent Gas Environments© Jones. Two types & of Bartlett cultivation methodsLearning, are shown LLC for bacterial species NOT FORgrowing SALE poorly in OR an oxygen-rich DISTRIBUTION environment. ( A) A candle jar, in whichNOT microaerophilic FOR SALE bacterial OR species DISTRIBUTION grow in an atmosphere where the oxygen is reduced by the burning candle. ( B, C) An anaerobic jar, in which hydrogen is released from a generator and then combines with oxygen through a palladium catalyst to form water and creates an anaerobic environment. ❯ ❯ In which jar would a facultative anaerobe grow? ( C ) © Scott Coutts/Alamy.

fact, ©life Jones originated & Bartlett on Earth Learning, in an anaerobic LLC A common way© to Jones test an organism’s& Bartlett oxygen Learning, LLC environmentNOT FOR consisting SALE ofOR methane DISTRIBUTION and other sensitivity is to useNOT a FORthioglycollate SALE OR broth DISTRIBUTION , gases. MicroFocus 5.3 recounts the events which binds free oxygen so that only fresh leading to an oxygen-rich atmosphere. oxygen entering at the top of the tube would be Some anaerobic bacterial species cause available ( FIGURE 5.11 ). © Jones &disease Bartlett in humans. Learning, For example, LLC the Clostridium © Finally, Jones there & Bartlett are bacterial Learning, species said LLC to be NOT FORspecies SALE mentioned OR DISTRIBUTION above causing tetanus and gas capnophilicNOT FOR ( capno SALE = “smoke”); OR DISTRIBUTION they require an gangrene multiply in the dead, anaerobic tissue atmosphere low in oxygen but rich in carbon of a wound and produce toxins causing tissue dioxide gas. Members of the genera Neisseria damage. Botulism, caused by another species and Streptococcus are capnophiles. of Clostridium multiplies in the oxygen-free pH. e cytoplasm of most microorganisms © Jones & Bartlett Learning,environment LLC of an improperly sealed can© Jones of food, & hasBartlett a pH nearLearning, 7.0. is LLC means the majority of NOT FOR SALE OR DISTRIBUTIONwhere it produces the lethal toxin of botulism.NOT FOR SALEspecies ORare DISTRIBUTION neutrophiles , growing optimally Among the most widely used methods to near neutral pH but having a growth range establish anaerobic conditions in the laboratory that covers 2 to 3 pH units (1,000-fold change is the GasPak system, in which hydrogen reacts in H + concentration). However, some bacterial with ©oxygen Jones in &the Bartlett presence Learning, of a catalyst LLC to species, such as Vibrio© Jones cholerae, & Bartlett can tolerate Learning, LLC formNOT water, FOR thereby SALE creating OR DISTRIBUTION an oxygen-free acidic conditions asNOT low asFOR pH 2.0SALE and ORalkaline DISTRIBUTION atmosphere ( FIGURE 5.10B, C ). conditions as high as pH 9.5. Many microbes are neither strictly aerobic Acid-tolerant bacteria called acidophiles nor anaerobic but rather can grow in either the grow best at pHs below 5 and are valuable in the © Jones &presence Bartlett of or Learning, a reduced concentration LLC of oxygen. food© Jonesand dairy & industries.Bartlett Learning,For example, LLC certain NOT FOR SALE is group OR includes DISTRIBUTION E. coli, many staphylococci speciesNOT FORof Lactobacillus SALE OR DISTRIBUTIONand Streptococcus and streptococci, members of the genus Bacillus, produce the acid that converts milk to buttermilk and the fungal yeasts. us, we o en refer to these and cream to sour cream. ese species pose no organisms as facultative anaerobes because threat to good health even when consumed in they can grow best in the presence of oxygen gas large amounts. e “active cultures” in a cup © Jones & Bartlett Learning,but will switchLLC to anaerobic metabolism© Jones when & ofBartlett yogurt alsoLearning, are acidophilic LLC bacterial species. NOT FOR SALE OR DISTRIBUTIONoxygen gas is absent. NOT FOR SALEExtreme OR acidophiles DISTRIBUTION , preferring pHs of 1 to 2,

978128057782_CH05_140_171.indd 154 10/10/14 2:26 PM © Jones & Bartlett Learning. NOT FOR SALE OR DISTRIBUTION. Optimal Microbial Growth Is Dependent on Several Physical Factors 155

“It’s Not Toxic to Us!”© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

It’s hard to think of oxygen as a poisonous gas considering how Among the survivors of these first communities were gigan- many organisms need it to survive. Yet billions of years ago, tic, shallow-water colonies called “stromatolites.” In fact, these oxygen was extremely toxic. One whi® by an organism and a rock-like looking structures are still found in a few places on cascade© ofJones highly & destructive Bartlett cellular Learning, reactions LLC was set into Earth, such© as Jones Shark Bay & o®Bartlett the western Learning, coast of Australia LLC (see motion.NOT Death FOR followed SALE quickly. OR DISTRIBUTION figure). TheseNOT structures FOR formedSALE from OR ocean DISTRIBUTION sediments and cal- Di® icult to believe? Not if you realize that the ancient cium carbonate that became trapped in the microbial commu- members of the Bacteria and Archaea relied on anaerobic nity as a biofilm. The top few inches in the crown of a stromato- chemistry for their energy needs. The atmosphere was full of lite contain the oxygen-evolving, photosynthetic cyanobacteria, methane, carbon dioxide, and other gases that they could use while below the crown are other bacterial species that can also © Jonesto & generate Bartlett energy. Learning, But no oxygen. LLC And it stayed that way© for Jones tolerate & oxygenBartlett and Learning, sunlight. Buried LLC yet beneath these organ- NOT FORaround SALE 1 billion OR years. DISTRIBUTION NOT ismsFOR are SALE other bacterialOR DISTRIBUTION species surviving the anaerobic, dark Then, about 3 billion years ago, along came the niche of the stromatolite interior where neither oxygen nor sun- cyanobacteria with their ability to perform photosynthesis. light can reach. A couple of billion years would pass before one Floating on the surface of the oceans, the cyanobacteria trapped particularly well-known species of oxygen-breathing creature sunlight and converted it to chemical energy in carbohydrates; evolved: Homo sapiens. the process was photosynthesis.© Jones But & there Bartlett was a downsideLearning, to LLC © Jones & Bartlett Learning, LLC photosynthesis: oxygen wasNOT a waste FOR product SALE of the OR process—and DISTRIBUTION NOT FOR SALE OR DISTRIBUTION – • it was deadly because the oxygen radicals (O2 , OH ) produced could disrupt cellular metabolism by “tearing away” electrons from other molecules. Over the next few hundred million years, oxygen gas remained© Jonesscarce. Then, & Bartlett about 2.4 Learning, billion years ago,LLC there was a © Jones & Bartlett Learning, LLC suddenNOT and dramatic FOR SALE increase OR in oxygen DISTRIBUTION gas. Unable to cope with NOT FOR SALE OR DISTRIBUTION the toxic conditions, enormous numbers of microbial species became extinct. Others “escaped” to oxygen-free environments, such as lake and deep ocean sediments where they still exist today. The cyanobacteria survived in the open oceans because © Jonesthey & Bartlett evolved the Learning, enzymes to safelyLLC tuck away oxygen atoms ©in aJones Stromatolites,& Bartlett Shark Learning, Bay, Western LLC Australia. NOT FORnontoxic SALE form—that OR DISTRIBUTION form was water. NOT FOR © JonSALE Nightingale/ShutterStock, OR Inc. DISTRIBUTION

Type of Both aerobic Aerobic growth Aerobic growth Growth is Growth occurs growth and anaerobic only at low only in the insensitive to only in the growth concentrations presence of O2 O2 absence of O2 © Jones of& O Bartlett2 Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Bacterial +O2 ©growth Jones in & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOTthioglycollate FOR SALE OR–O2 DISTRIBUTION NOT FOR SALE OR DISTRIBUTION broth

(A) (B) (C) (D) (E)

FIGURE 5.11 The E ect of Oxygen on Microbial Growth. Each tube contains a thioglycollate broth into which © Jones & Bartlett Learning, LLC ❯ ❯ © Jones & Bartlett Learning, LLC was inoculated a di® erent bacterial species. Identify the O2 requirement in each thioglycollate tube based on the NOT FORgrowth SALE density OR [example: DISTRIBUTION ( A ) represents facultative anaerobe species].NOT FOR SALE OR DISTRIBUTION

978128057782_CH05_140_171.indd 155 10/10/14 2:26 PM © Jones & Bartlett Learning. NOT FOR SALE OR DISTRIBUTION. 156 Chapter 5: Microbial Growth and Nutrition

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONare found among the Archaea. At theNOT opposite FOR SALE eOR fact DISTRIBUTIONthat some microbial life can survive extreme are the alkaliphiles , which grow best under extreme environmental conditions is at pHs above 9. clear from reading MicroFocus 5.4 . e majority of known bacterial species, FIGURE 5.12 summarizes the physical however,© Jones do not& Bartlett grow well Learning, under LLCacidic factors in uencing© microbial Jones growth.& Bartlett Learning, LLC conditions.NOT FOR us, SALEthe acidic OR environment DISTRIBUTION of the NOT FOR SALE OR DISTRIBUTION stomach helps deter disease, while providing a natural barrier to the organs beyond. In addition, CONCEPT AND REASONING CHECKS 2 Bacteria: © NIAID you may have noted certain acidic foods such as a. In a broth tube, describe the status lemons, oranges, and other citrus fruits as well of the bacterial cell population in © Jones &as tomatoesBartlett andLearning, many vegetables LLC are hardly ever © Joneseach phase& Bartlett of the growthLearning, curve. LLC NOT FORcontaminated SALE OR DISTRIBUTIONby bacterial growth. However, NOTb. Explain FOR SALE how the OR trigger DISTRIBUTION to persister such damaged produce may be subject to fungal cell survival is di erent from the trigger to endospore formation. growth because many fungi are acidophiles and c. Identify what would be grow well at a pH of 5 or lower. extremophile-type conditions © Jones & Bartlett Learning, Other LLC Factors. Further environmental© Jones & Bartlettfor Learning, each of the physicalLLC factors NOT FOR SALE OR DISTRIBUTIONfactors can in uence the growth ofNOT microbial FOR SALE ORdescribed DISTRIBUTION in this section. cells. Psychrophiles in deep ocean waters and Hydrostatic hydrostatic pressure: T h e sediments are under extremely high pressure exerted by pressure. In some deep marine trenches the hydrostatic pressure can be as high as 16,000 the weight of water. CHAPTER CHALLENGE pounds© Jones per square & Bartlett inch (psi) Learning, and only LLCsome © Jones & Bartlett B Learning, LLC extremophilesNOT FOR may SALE be adapted OR DISTRIBUTION to the pressure. NOT FOR SALE OR DISTRIBUTION Such barophiles ( baro = “pressure”) in fact will You have now studied many of the major physical rupture quite quickly at normal atmospheric factors (temperature, oxygen, pH, and salt) that pressures (14.7 psi). influence microbial growth and reproduction © Jones & Bartlett Other microbes Learning, have LLC adapted to saline or ©and Jones you have & heard Bartlett about many Learning, that can survive LLC NOT FORhighly SALE saline OR environments DISTRIBUTION such as the Great Salt NOTthese FORphysical SALE extremes OR (see TableDISTRIBUTION 5.1 ). Lake in Utah, the Dead Sea, and in evaporation Question B: ponds. ese halophiles ( halo = “salt”) are Could a microbe survive the physical extremes characterized by their need for hypersaline on Mars? What kind of earthly microbe might conditions for growth. ey include species, this Martian microbe be like? Here are some © Jones & Bartlett Learning, LLC © Jones & Bartlettuseful facts Learning, about Mars. LLC such as Vibrio cholerae, that grow optimally NOT FOR SALE OR DISTRIBUTIONat 2% to 5% NaCl and other generaNOT FORthat SALE Surface OR temperature:DISTRIBUTION Estimated to be from a warm 27°C (81°F) to –143°C (–225°F) at the grow optimally at 5% to 20% NaCl. Extreme winter polar caps. halophiles, like Halobacterium salinarium and Atmosphere: 95% carbon dioxide, but also the eukaryotic green alga Dunaliella salina, present are nitrogen (2.7%), argon (1.6%), grow© optimally Jones at& 20% Bartlett to 30% Learning, NaCl. In contrast, LLC and oxygen (0.13%;© Jones 21% on& Earth)Bartlett gases; Learning, LLC speciesNOT like FOR E. coli SALE are nonhalophiles OR DISTRIBUTION because mean surface NOT pressure FOR is much SALE lower OR than DISTRIBUTION they grow optimally at less than 2% NaCl and Earth’s. UV radiation is 3× Earth’s (note: there genera, such as Staphylococcus, are halotolerant are earthly bacterial species able to survive 5,000× the dose that would kill human cells). because they can grow in saline (up to 8% NaCl) Soil: Existence of water ice confirmed; soil pH = © Jones &as Bartlettwell as nonsaline Learning, environments. LLC © Jonesabout 7.7; & severalBartlett chemicals Learning, found that couldLLC NOT FOR SALE e hypersalineOR DISTRIBUTION conditions can be deadly NOTserve FOR as SALE nutrients OR for life DISTRIBUTION forms, including to microbial cells because water will be lost to magnesium, potassium, and chloride. the hypertonic external medium. To prevent Salt: Dark, finger-like features could be the flow the loss of cellular water, halophiles accumulate of salty water perhaps equivalent in salinity to Earth’s oceans. high cytoplasmic concentrations of solutes, © Jones & Bartlett Learning,such as sugars, LLC amino acids, or ions, ©to Jonesbalance & Bartlett Answers Learning, can be found in LLC Appendix F . NOT FOR SALE OR DISTRIBUTIONout the osmotic di erence. NOT FOR SALE OR DISTRIBUTION

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Drilling for Microbes© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

There are some 400 known subglacial lakes beneath the Note: An American team in 2012 reported the discovery of Antarctic ice sheet. One of these, Lake Vostok, lies at a depth diverse bacterial life in another Antarctic lake (Lake Vida) that of more than 3 kilometers below Vostok Station, a Russian lies under just 27 meters of ice. This highly saline lake with a research© outpostJones some & Bartlett 1,300 kilometers Learning, southeast LLC of the South water temperature© Jones of & –13°C Bartlett was distinct Learning, from that LLC in other Pole. ManyNOT scientists FOR SALE believe OR Lake DISTRIBUTION Vostok, which is estimated saline lakesNOT in the FORsame area. SALE And ORyet another DISTRIBUTION American team in to be 260 kilometers long and 48 kilometers across at its widest 2013 reported preliminary evidence for microbial life growing in point, similar in area to Lake Ontario, has been isolated from culture dishes from samples of water taken from Lake Whillans the atmosphere for 15 million years. Most interesting from a that lies 800 meters below the ice sheet. microbiological perspective is the presence of liquid water, which Should we be surprised with these discoveries? Perhaps © Jonesmeans & Bartlett there is theLearning, possibility ofLLC microbial life existing within ©the Jones not as & we Bartlett know microbes Learning, will eke LLCout an existence anywhere an NOT FORancient SALE lake. OR DISTRIBUTION NOT energyFOR sourceSALE is ORavailable. DISTRIBUTION For more than two decades and 57 Antarctic expeditions, Russian scientists have been slowly and carefully drilling down through the ice toward the lake to examine the water contents. Then, on Sunday, February 5, 2012, just a day before the expedition would end© its Jones season, &they Bartlett hit the lake! Learning, However, LLC © Jones & Bartlett Learning, LLC when the drill contactedNOT the lake, FOR the SALE lake water, OR being DISTRIBUTION under NOT FOR SALE OR DISTRIBUTION such great pressure from the ice above, shot up the bore hole and froze. At this writing, the scientists have not reported if the frozen water contains any microbes. Meanwhile, an American research group has collected ice cores from© Jones frozen water & Bartlett at the bottom Learning, (underside) LLC of the glacier © Jones & Bartlett Learning, LLC ice coveringNOT Lake FOR Vostok. SALE These OR cores DISTRIBUTION should represent a record NOT FOR SALE OR DISTRIBUTION of the lake surface water that froze at the glacier interface, and correspond to what is in the liquid at the surface of the lake. In 2013, the group reported they discovered a diverse microbial population in these cores. DNA sequence analyses © Jonesfrom & Bartlett four cores Learning, identified more LLC than 3,500 unique sequences,© Jones & Bartlett Learning, LLC NOT FORwhich SALE presumably OR DISTRIBUTION equates to that number of species or strains.NOT FOR SALE OR DISTRIBUTION About 95% of the sequences were associated with bacterial species (two additional sequences were related with archaeal species) and 5% liked with eukaryotic species, especially the fungi. Therefore, Lake Vostok itself might contain a complex web of organisms that have evolved© Jones and developed& Bartlett over Learning, the tens of LLC © Jones & Bartlett Learning, LLC millions of years of its existence.NOT FOR SALE OR DISTRIBUTION Courtesy of Michael Studinger/Lamont-Doherty EarthNOT Observatory. FOR SALE OR DISTRIBUTION

KEY© Jones CONCEPT 5.3& BartlettBacteria: © NIAID Learning, LLC nutrients dissolved ©in Joneswater. A & erBartlett sterilization Learning, LLC and specimen inoculation, the broth will CultureNOT MediaFOR AreSALE Used OR to Grow DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Microbes and Measure Their Growth eventually become turbid or cloudy as the microbial population grows in the container. Microorganisms can be grown (cultured) in a Solid media consist of the liquid media to liquid medium (pl. media) or on a solid medium. which has been added a solidifying agent called © JonesLiquid & Bartlett media, Learning, called broths LLC, are contained in agar© . Agar,Jones a polysaccharide & Bartlett Learning, derived from LLC red NOT FORtubes SALE and ORbottles, DISTRIBUTION and consist of the growth seaweed,NOT contains FOR SALE no nutrients, OR DISTRIBUTION and like gelatin,

978128057782_CH05_140_171.indd 157 10/10/14 2:26 PM © Jones & Bartlett Learning. NOT FOR SALE OR DISTRIBUTION. 158 Chapter 5: Microbial Growth and Nutrition

include

which includes microbes microbes microbes growing tolerating requiring three groups microbes microbes not NaCl are NaCl preferring using O2 are using O2 are © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONAerobic Anaerobic NOT FOR SALEHalotolerant OR DISTRIBUTION

microbes “Cold” “Hot” microbes microbes temperature temperature not requiring sensitive tolerating O2 are to O2 are NaCl are © Jones & Bartlett“Warm” Learning, LLC © Jones & Bartlett Learning, LLC temperature NOT FOR SALE OR DISTRIBUTIONObligate NOT FORObligate SALE OR DISTRIBUTION aerobes anaerobes Nonhalophiles with temperature optima near up to microbes microbes 15% are 37°C requiring tolerating reduced levels O are 2 Halophiles © Jones & Bartlettof O2 are Learning, LLC © Jones & Bartlett Learning, LLC Mesophiles at NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR15–30% DISTRIBUTION Aerotolerant are

Extreme halophiles 15°C 25°C © Jones & Bartlett Learning,Microaerophiles LLC © Jones & Bartlett Learning, LLC Psychrophiles Psychrotrophs NOT FOR SALE OR DISTRIBUTIONmicrobes NOT FOR SALE OR DISTRIBUTION growing with or without below near above O2 are 5 7 9 60°C 95°C

© Jones & BartlettThermophiles Learning, HyperthermophilesLLC Facultative© Jones &Acidophiles Bartlett Learning,Neutrophiles LLC Alkaliphiles NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 5.12 Types of Microbes Based on Physical Factors. This concept map shows four major physical factors controlling microbial growth. ❯ ❯ Escherichia coli is a mesophilic, facultative, nonhalophilic neutrophile. What would be the makeup of the environment where the organism would optimally grow?

melts© when Jones heated & Bartlett and then Learning, solidi es LLCwhen is, in nutrients designed© Jones to mimic & Bartlett the natural Learning, LLC cooled.NOT A FORer sterilization SALE OR of theDISTRIBUTION nutrient agar environment. Today,NOT di FOR erent SALEculture OR media DISTRIBUTION medium, it is poured into a culture (Petri) plate recipes are used in the clinical microbiology lab or tube where it will solidify on cooling. A er (CML) and research lab because species vary specimen inoculation, the specimen will grow in their nutritional requirements and no single © Jones &as Bartlettcolonies (bacterialLearning, organisms LLC and yeasts) or medium© Jones will grow& Bartlett all microorganisms. Learning, LLC NOT FORas SALE laments OR (molds) DISTRIBUTION on the surface of the agar. NOT For theFOR isolation SALE and OR identi DISTRIBUTION cation of most microorganisms, two types of culture media are commonly used. A chemically unde ned Culture Media Are of Two Basic Types medium, called a complex medium , contains Since the time of Pasteur and Koch, nutrients in which the exact components or © Jones & Bartlett Learning,microbiologists LLC have been growing bacterial© Jones and & theirBartlett quantity Learning, is not completely LLC known. Such NOT FOR SALE OR DISTRIBUTIONother microbial species in arti cial media;NOT FORthat SALEmedia typicallyOR DISTRIBUTION contain animal or plant digests

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION preciseNOT chemical FOR SALE composition OR DISTRIBUTION and amount Composition of a Complex and TABLE 5.3 of all components are known ( TABLE 5.3B ). a Synthetic Growth Medium Chemically de ned media are useful when trying to determine an organism’s speci c Nutrient growth requirements. Ingredient Supplied© Jones & Bartlett Amount Learning, LLC © Jones & Bartlett Learning, LLC A. Complex Agar MediumNOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Culture Media Can Be Modified to Peptone Amino acids, 5.0 g peptides Select For or Di erentiate Between Beef extract Vitamins, minerals, 3.0 g Microbial Species © Jones & otherBartlett nutrients Learning, LLC In the CML, the basic© Jones ingredients & Bartlett of growth Learning, LLC SodiumNOT chloride FOR SALE Sodium andOR DISTRIBUTION 8.0 g media can be modi NOT ed in FOR one SALEof three OR ways DISTRIBUTION (NaCl) chloride ions to provide fast and critical information about Agar 15.0 g a pathogen causing an infection or disease TABLE 5.4 Water 1.0 liter ( ). © Jones & Bartlett Learning, LLC © A Jones selective & medium Bartlett contains Learning, ingredients LLC NOT FORB. SALEChemically OR Defined DISTRIBUTION Broth Medium to inhibitNOT FORthe growth SALE of OR certain DISTRIBUTION microbes in Glucose Simple sugar 5.0 g a mixture while allowing (selecting for) the Ammonium Nitrogen, 1.0 g growth of others. e basic growth medium phosphate phosphate may contain extra salt (NaCl) or a dye to inhibit ((NH ) HPO ) 4 2 4 © Jones & Bartlett Learning,the growth LLC of intolerant or sensitive© organisms Jones & Bartlett Learning, LLC but permit the growth of those species or Sodium chloride SodiumNOT and FOR SALE 5.0 g OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION (NaCl) chloride ions pathogens one wants to isolate. Magnesium sulfate Magnesium ions, 0.2 g Another modi cation to a basic growth

(MgSO 4 · 7H2 O) sulphur medium is the addition of one or more Potassium Potassium ions, 1.0 g ingredients that allow the observer to © Jones & Bartlett Learning, LLC di erentiate between© Jones very &similar Bartlett species Learning, LLC phosphate (K2 HPO4 ) phosphate NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Water 1.0 liter growing in or on a culture medium based on speci c biochemical or physiological properties. i s di erential medium contains speci c (e.g., beef extract, soybean extract) or yeast chemicals to indicate which species possess and © Jonesextracts & Bartlett of an Learning,unde ned nature LLC ( TABLE 5.3A ). which© Joneslack a particular& Bartlett biochemical Learning, process. LLC Complex media are commonly used in the Such indicators make it easy to distinguish NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION teaching laboratory because most of the visually colonies of one organism from colonies organisms used will grow in these nutrient-rich of other similar organisms on the same culture conditions. plate. Look at FIGURE 5.13 and see if you can e second type of medium is a chemically determine which medium was used in each de ned medium . In© Jonesthis medium,& Bartlett the Learning, example. LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

TABLE 5.4 A Comparison of Special Culture Media

Name© Jones & Bartlett Components Learning, UsesLLC Examples© Jones & Bartlett Learning, LLC SelectiveNOT medium FOR SALE Growth ORstimulants DISTRIBUTION Selecting certain microbes MannitolNOT salt FORagar for SALE staphylococci OR DISTRIBUTION Growth inhibitors out of a mixture Di® erential medium Dyes, growth stimulants, Distinguishing di® erent MacConkey agar for gram-negative growth inhibitors microbes in a mixture bacteria © Jones Enriched& Bartlett medium Learning, Growth stimulantsLLC Cultivating fastidious© Jones & Blood Bartlett agar for streptococci;Learning, chocolate LLC NOT FOR SALE OR DISTRIBUTION microbes NOT FORagar SALE for Neisseria OR DISTRIBUTIONspecies

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1 11 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION(A) NOT FOR SALE(B) OR DISTRIBUTION FIGURE 5.13 Special Media Formulations. A ) Four ( di® erent bacterial species (1–4), two gram-positive and two gram-negative, were streaked onto separate sections of a MacConkey agar plate and allowed to incubate for 48 hours. MacConkey agar only supports the growth of gram-negative species. ( B ) Because the two gram-negative species cannot be visually distinguished from one another, they were then streaked into an eosin methylene blue agar (EMB) plate and incubated© Jones for 48 & hours. Bartlett EMB allows Learning, one to distinguish LLC between human enteric bacteria,© Jones where Escherichia & Bartlett coli Learning, LLC producesNOT a green FOR metallic SALE sheen OR while DISTRIBUTION other enteric species produce a pink color. ❯ ❯ NOT Which specialFOR SALEmedium isOR DISTRIBUTION selective and which is di erential? Explain your reasoning. Courtesy of Dr. Je rey Pommerville.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALEMicroInquiry OR DISTRIBUTION 5 presents another example areNOT said FORto be SALEin a viable OR DISTRIBUTION but noncultured using these two approaches to identify and ( VBNC ) state. Procedures for identifying di erentiate between similar bacterial species. VBNC organisms include direct microscopic Although many microorganisms grow well examination (see below) and, most commonly, in nutrient broth and nutrient agar, certain ampli cation of diagnostic gene sequences or © JonesFastidious: & Bartlett Having Learning, LLC © Jones & Bartlett Learning, LLC special nutritional so-called fastidious organisms may require an 16S rRNA gene sequences. NOT FORrequirements. SALE OR DISTRIBUTIONenriched medium containing extraNOT vitamins, FOR SALEWhy OR DISTRIBUTIONdo these organisms remain amino acids, or other nutrients to promote uncultured? Microbiologists believe part of growth. For example, the causative agent of the reason may be due to their presence in gonorrhea, Neisseria gonorrhoeae, requires a “foreign” environment. ese species have the addition© Jones of & powdered Bartlett Learning,hemoglobin. LLC e adapted to their ©own Jones familiar & Bartlett and speci Learning, c LLC CentersNOT for FOR Disease SALE Control OR DISTRIBUTIONand Prevention environment; an arti NOT cial FOR medium SALE is not OR their DISTRIBUTION (CDC) o en is involved with particularly usual home. erefore, these species go into vexing identi cations of such fastidious a type of dormancy state and do not divide; species, as MicroFocus 5.5 details. in other words, they are viable, but cannot be © Jones & BartlettIdentification Learning, of Nongrowing LLC Microbes. cultured.© Jones Investigating & Bartlett the Learning, Microbial LLCWorld 5 NOT FORMany SALE bacterial OR DISTRIBUTION and archaeal species are examinesNOT FOR this SALEanomaly. OR StudiesDISTRIBUTION on VBNC impossible to cultivate in any arti cial culture bacterial and archaeal species present a vast and medium yet devised. In fact, less than 2% of the as yet unexplored eld (they are o en referred to species in nature can be cultured. Consequently, as “microbial dark matter”), which is important it is impossible to estimate accurately microbial not only for detection of human pathogens, but © Jones & Bartlett Learning,diversity LLCin an environment based ©solely Jones on & alsoBartlett to reveal Learning, the tremendous LLC diversity in the NOT FOR SALE OR DISTRIBUTIONculturability. Such “uncultured” NOTorganisms FOR SALEmicrobial OR world. DISTRIBUTION

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Identification of a Bacterial© Jones Species & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

It o en is necessary to identify a bacterial microorganisms (no carbon = no energy You can identify each bacterial species or be able to tell the di® erence source). So, only marine photosynthetic species by taking a complex broth between similar-looking species in a microbes should be present. medium, such as nutrient broth, and mixture.© InJones microbial & Bartlett ecology, itLearning, might ■ LLC As an infectious disease o® icer© in Jones a lo- adding & Bartlett salt andLearning, mannitol LLC (mannitol be necessaryNOT FOR to isolate SALE certain OR naturally DISTRIBUTION cal hospital, you routinely swabNOT critical FOR salt SALE broth) OR and DISTRIBUTION phenol red. Next, you growing species from others in a mixture. care areas to determine if there are any inoculate a sample of each colony into a In the clinical and public health setting, antibiotic-resistant bacteria present. separate tube. You inoculate the 10 tubes microbes might be pathogens associated You are especially concerned about and incubate them for 48 hours at 37°C. with disease or poor sanitation. In methicillin-resistant Staphylococcus 5e. The broth tubes are shown be- © Jonesaddition, & Bartlett some Learning, may be resistantLLC to © Jones & Bartlett Learning, LLC aureus (MRSA), as it frequently can low. What do the results signify? NOT FORstandard SALE antibiotics OR DISTRIBUTION normally used to NOT FOR SALE OR DISTRIBUTION cause disease outbreaks in a hospital Which tubes contain which spe- treat an infection. In all these cases, setting. One swab you put in a broth cies of Staphylococcus ? identification can be accomplished by tube showed turbidity a er 48 hours. modifying the composition of a complex 1 2345 or chemically defined growth medium. 5b. Knowing Staphylococcus s p e - Let’s go through two scenarios.© Jones & Bartlett Learning,cies are halotolerant, LLC how could © Jones & Bartlett Learning, LLC you devise an agar medium to ■ Suppose you are anNOT undergraduate FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION visually determine if any of the student in a marine microbiology growth is due to Staphylococcus course. On a field trip, you collect aureus ? some seawater samples and, now 6 78910 back in the lab, you want to grow only Again, a selective medium would photosynthetic© Jones & marine Bartlett microbes. Learning, be LLCused. It would be prepared by© adding Jones & Bartlett Learning, LLC NOT How FOR would SALE you OR select DISTRIBUTION for 7.5% salt to a complex agar medium.NOT AFOR SALE OR DISTRIBUTION photosynthetic microbes? First, you sample from the broth tube would be know the photosynthetic organisms streaked on the plate and incubated at manufacture their own food, so their 37°C for 48 hours. Results from di® erential broth tubes. energy source will be sunlight and not 5c. What would you expect to find © Jones & Bartlettthe organic Learning,compounds typically LLC found © Jones & Bartlett Learning, LLC on the agar plate a er 48 hours? This method is an example of a NOT FOR SALEin culture OR media DISTRIBUTION (see Table 5.3 ). So, you NOT FOR SALE OR DISTRIBUTIONdi® erential medium because it allowed would need to use a chemically-defined Your selective medium contained 10 you to visually di® erentiate or distinguish medium but leave out the glucose. discrete colonies. You do a Gram stain and between two very similar bacterial species. Also, knowing the salt is typically found discover all the colonies contain clusters Knowing which colonies on the original in ocean waters, you would want to of purple spheres; they are gram-positive. selective medium plate are S. aureus, add it to the medium.© You Jones would &then Bartlett However, Learning, other species LLC of Staphylococcus you need© Jonesto determine & Bartlett which, if Learning,any, are LLC inoculate a sample NOT of the FOR collected SALE do notOR cause DISTRIBUTION disease. One is S. epidermidis, resistantNOT to the FOR antibiotic SALE methicillin. OR DISTRIBUTION material into a broth tube, place the a common skin bacterium. A Gram stain tube in the light, and incubate for one therefore is of no use to di® erentiate S. 5f. How could you design an agar week at a temperature typical of where aureus from S. epidermidis. medium to identify any MRSA colonies? the organisms were collected. 5d. Knowing that only S. aureus will © Jones & Bartlett Learning, LLC © Jones & Bartlett 5g. If the Learning, plates are LLC devoid of 5a.NOT What FOR would SALE you expect OR toDISTRIBUTION find produce acid in the presenceNOT FOR SALE OR DISTRIBUTION in the broth tube a er one of the sugar mannitol, how growth, what can you conclude? week’s incubation? could you design a di erential Again, you have used a selective medium; the addition of methicillin will What you have used in this scenario broth medium to determine permit the growth of any MRSA bacteria is a selective medium; that is, one that will if any of the colonies are S. and suppress the growth of staphylococci © Jonesencourage & Bartlett the growthLearning, of photosynthetic LLC aureus© ? Jones ( Hint: phenol& Bartlett red is Learning, a LLC susceptible to methicillin. NOT FORmicrobes SALE (light OR andDISTRIBUTION sea salt) and sup- pH indicator;NOT FOR it is red SALE at neutral OR DISTRIBUTION Answers can be found in Appendix E . press the growth of nonphotosynthetic pH and yellow at acid pH).

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“Enriching” Koch’s Postulates© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

On July 21–23, 1976, some 5,000 Legionnaires attended the by Koch’s postulates. L. pneumophila then was recovered from Bicentennial Convention of the American Legion in Philadelphia, infected guinea pigs, verifying the organism as the causative PA. About 600 of the Legionnaires stayed at the Bellevue Stratford agent of Legionnaires’ disease. Hotel. As © the Jones meeting & was Bartlett ending, Learning, several Legionnaires LLC who Today, we© know Jones L. pneumophila & Bartlett is foundLearning, in many LLC aquatic stayed at NOTthe hotel FOR complained SALE ofOR flu-like DISTRIBUTION symptoms. Four days environments,NOT both FOR natural SALE and artificial.OR DISTRIBUTION At the Bellevue a er the convention ended, an Air Force veteran who had stayed Stratford Hotel, epidemiological studies indicated guests were at the hotel died. He would be the first of 34 Legionnaires over exposed to L. pneumophila as a fine aerosol emanating from several weeks to succumb to a lethal pneumonia, which became the air-conditioning system. Through some type of leak, the known as Legionnaires’ disease. organism gained access to the system from the water cooling © Jones & As Bartlettwith any new Learning, disease, epidemiological LLC studies look for© Jonestowers. & Bartlett Learning, LLC NOT FORthe source SALE of theOR disease. DISTRIBUTION The Centers for Disease Control andNOT FOR Koch’s SALE postulates OR are DISTRIBUTION still useful—it’s just hard sometimes to Prevention (CDC) had an easy time tracing the source back to satisfy the postulates without an isolated pathogen. the Bellevue Stratford Hotel. Epidemiological studies also try to identify the causative agent. Using Koch’s postulates, CDC sta® collected tissues from lung biopsies and sputum samples. However, no microbes could© beJones detected & Bartlett on slides ofLearning, stained LLC © Jones & Bartlett Learning, LLC material. By December 1976,NOT they FORwere no SALE closer ORto identifying DISTRIBUTION NOT FOR SALE OR DISTRIBUTION the infectious agent. How can you verify Koch’s postulates if you have no infectious agent? It was almost like being back in the times of Pasteur and Koch. Why was this bacterial species so di® icult to culture on© bacteriological Jones & Bartlett media? Perhaps Learning, it was a LLCvirus. © Jones & Bartlett Learning, LLC A er NOT trying FOR 17 di® SALEerent culture OR DISTRIBUTION media formulations, the NOT FOR SALE OR DISTRIBUTION infectious agent was finally cultured. It turns out it was a bacterial species, but one with fastidious growth requirements. The initial agar medium contained a beef infusion, amino acids, and starch. When this medium was enriched with 1% hemoglobin © Jonesand & 1% Bartlett isovitalex, Learning, small, barely LLC visible colonies were seen© Jones & Bartlett Learning, LLC NOT FORa er five SALE days ofOR incubation DISTRIBUTION at 37°C. Investigators then realizedNOT FOR SALE OR DISTRIBUTION the hemoglobin was supplying iron to the bacterium and the isovitalex was a source of the amino acid cysteine. Using these two chemicals in pure form, along with charcoal to absorb

bacterial waste, a pH of 6.9, and an atmosphere of 2.5% CO2 , bacterial growth was significantly© Jones enhanced & Bartlett (see figure). Learning, From LLC © Jones & Bartlett Learning, LLC a microscope examination NOT of these FOR cultures, SALE a gram-negative OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION rod was confirmed and the organism was appropriately named Legionella pneumophila. With an enriched medium to pure culture the organism, Colonies of L. pneumophila on an enriched medium. susceptible animals (guinea pigs) could be injected as required Courtesy of Dr. Jim Feeley/CDC. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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The Great Plate Count© Jones Anomaly & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION About 98% of bacterial species from the environment cannot be grown using known culture media. ■ OBSERVATION: Take a sediment sample from an environmental water source, mix it with saline solution (or water), and wait for the sediment to settle. Now take a drop from the liquid and place it on a culture dish with nutrient agar (or any type of complex medium). Place another drop on a slide and add stain. On the stained slide, you will undoubtedly be© able Jones to count & hundredsBartlett of Learning, cells and find LLCdozens of di® erent bacterial morphologies.© Jones & On Bartlett the plate, Learning, if you are lucky, LLC maybeNOT one FOR or two SALE colonies OR will DISTRIBUTION appear in a few days. The majority of theNOT cells willFOR not SALE grow even OR though DISTRIBUTION they are inundated in nutrients. This is the so-called “great plate count anomaly.” Standard laboratory culture techniques fail to support the growth of these viable but noncultured (VBNC) species residing in environmental sediments. ■ QUESTION: Why won’t 98% of the bacterial species grow in laboratory culture media? © Jones■ & Bartlett HYPOTHESIS: Learning, VBNCs need LLC some essential “nutrient” from© Jones their neighbor & Bartlett species Learning, in the natural LLC environment. If so, NOT FOR SALEthen growing OR DISTRIBUTION the VBNCs in their natural environment willNOT supply FOR the needed SALE nutrient OR DISTRIBUTIONand the uncultured should grow. ■ EXPERIMENTAL DESIGN: A di® usion apparatus is designed to sandwich a microbial sediment sample in agar between two semipermeable membranes that allow for the free di® usion of “nutrients” and waste products through the chamber (see figure). © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC ■ EXPERIMENT: Sediment from a freshwater pond sample is mixed with agar and placed within the di® usion chamber. The chamber is thenNOT placed FOR back SALE in its OR natural DISTRIBUTION environment. Another pond sediment NOT sample FOR from SALE the same OR DISTRIBUTION environment is cultured on standard agar culture medium in a culture dish. Both the di® usion chamber culture and the lab culture are le undisturbed for 4 weeks. A er the incubation period, the colonies isolated on the culture plate and on the agar in the di® usion chamber (but not the biofilm growing on the semipermeable membrane) are identified. Phylum identification is carried out by sequencing each isolate’s 16S ribosomal RNA gene. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC ■ RESULTS:NOT FOR See Table SALE . OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION ■ CONCLUSIONS: QUESTION Was1 : the hypothesis supported? Explain using the table. QUESTION 2 Are : the majority of the isolates representative of gram-positive or gram-negative organisms? Explain. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE QUESTION OR DISTRIBUTION What3 : might have been the result if the biofilmNOT organisms FOR SALE growing OR on DISTRIBUTION the membranes were included in the analysis? Answers can be found in Appendix E . ■ FURTHER QUESTION: What is it that the previously uncultured colonies in the di® usion chamber recognize? New evidence suggests one© Jones“nutrient” & is Bartlett iron, which Learning, is needed for LLC ATP generation and other biochemical© Jones processes. & Bartlett To Learning, LLC grow, the unculturedNOT must “capture”FOR SALE the iron OR (presumably DISTRIBUTION from their neighbors) in the boundNOT form FOR that is SALE otherwise OR DISTRIBUTION unavailable in a culture dish “environment.”

Diffusion chamber Top view © Jones & Bartlett Learning, LLCSide view © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Semipermeable Microbial sample membrane in agar © Jones & Bartlett Learning, LLC Adapted from: Bollman, A., Lewis, K., and© JonesEpstein, S. S.& Bartlett Learning, LLC (2007) Appl. Environ. Microbiol. 73 (20): 6386–6390. NOT FOR SALE OR DISTRIBUTION. NOT FOR SALE OR DISTRIBUTION(Continues)

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC TABLE PhylogeneticNOT FOR SALE Strains OR Obtained DISTRIBUTION by Culture Dish and Di usion-ChamberNOT FOR SALEMethods OR DISTRIBUTION

Number of Strains Isolated By Di usion Total number of © Jones Phylum & Bartlett Learning, Culture LLC dish only chamber only© Jones Both & methods Bartlett Learning,isolated strains LLC NOT Alphaproteobacteria FOR SALE OR DISTRIBUTION 20 36 NOT FOR SALE 6 OR DISTRIBUTION 62 Betaproteobacteria 3 63 3 69 Gammaproteobacteria 2 4 1 7 Deltaproteobacteria 1 1 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Bacteroidetes 5 6 1 12 NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Spirochaetes 4 4 Firmicutes 5 1 6 Actinobacteria 5 1 6 Total © Jones & Bartlett 40 Learning, LLC 116 11 © Jones & Bartlett 167 Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Population Bartlett Learning, Measurements LLC Are Made © A Jones second, & more Bartlett commonly Learning, used technique, LLC called the streak-plate method , uses a single NOT FORUsing SALE Pure OR DISTRIBUTION Cultures NOT FOR SALE OR DISTRIBUTION plate of nutrient agar ( FIGURE 5.15A-D ). An Aseptic Microorganisms rarely occur in nature as a inoculum from a mixed culture is removed with technique: T h e a sterile loop using aseptic technique , and a series practice of single species as Pasteur discovered in trying to prove his germ theory of disease. Rather, they of streaks is made on the surface of one area of © Jonestransferring & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC microorganisms are mixed with other species, in a so-called the plate. e loop is amed, touched to the rst NOT FORto a sterile SALE culture OR DISTRIBUTION“mixed culture”—most o en as aNOT bio FOR lm . SALEarea, and OR a DISTRIBUTIONsecond series is made in a second medium without erefore, to study a species, microbiologists area. Similarly, streaks are made in the third and introducing other and laboratory technologists must use a pure fourth areas, thereby spreading out the individual contaminating culture—that is, a population consisting of only cells so they grow as separated colonies. A er a organisms. one species.© Jones It was & BartlettKoch’s ability Learning, to pure culture LLC 24 hour to 48 hour ©incubation, Jones & discrete Bartlett colonies Learning, LLC pathogensNOT thatFOR provided SALE theOR proof DISTRIBUTION for the germ will be present on theNOT plate FOR ( FIGURE SALE 5.15E OR ). DISTRIBUTION theory. In both methods, each colony is a pure If one has a mixed broth culture of bacterial culture because the colony is derived from an Subculturing: T h e original single cell that underwent numerous process of species, how can the organisms be isolated as binary ssions. e researcher, medical transferring© bacterial Jones &pure Bartlett colonies? Learning, Two established LLC methods are © Jones & Bartlett Learning, LLC available. e rst is the pour-plate method . technologist, or student can select samples of cells from oneNOT culture FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION medium to another. Here, diluted samples of the mixed culture in the colonies for further testing and subculturing . molten nutrient agar tubes are each poured into a sterile Petri plate and allowed to harden. Population Growth Can Be During a 24 hour to 48 hour incubation, the Measured in Several Ways © Jones & Bartlett Learning,cells divide LLC to form discrete colonies© Joneson and & MicrobialBartlett Learning,growth in a LLCculture medium can be NOT FOR SALE OR DISTRIBUTIONwithin the agar ( FIGURE 5.14 ). NOT FOR SALEmeasured OR by DISTRIBUTION direct and indirect methods.

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOTDirect FOR Counting SALE Methods.OR DISTRIBUTION ere are a number of ways to directly measure cell numbers. Scientists may wish to perform a direct microscopic count using a known © Jones & Bartlett Learning,volume of LLC the liquid sample that has been© Jones placed & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONon a specially designed countingNOT chamber FOR SALE OR DISTRIBUTION ( FIGURE 5.16 ). However, this procedure will count both live and dead cells. In the most probable number test , microbial samples diluted 10× and 100× © Jones & Bartlett Learning, LLC are added to a set© Jonesof lactose & Bartlettbroth tubes Learning, LLC NOT FOR SALE OR DISTRIBUTIONand the presence orNOT absence FOR ofSALE gas formedOR DISTRIBUTION in fermentation gives a rough statistical estimation of the cell number, referred to as the most probable number. is technique has © Jones & Bartlett Learning, LLC been© used Jones for measuring & Bartlett water Learning, quality. LLC NOT FOR SALE OR DISTRIBUTION NOTIn the FOR standard SALE plate OR countDISTRIBUTION procedure , FIGURE 5.14 A Pour Plate. The dispersed bacterial a sample of a broth culture is placed in a cells grow as individual, discrete colonies. ❯ ❯ By looking sterile Petri dish and melted nutrient agar is at this plate, how would you know the original broth added (pour-plate method) ( FIGURE 5.17 ).

culture was a mixture of bacterial species? e assumption is that each cell will undergo Courtesy of Dr. Je rey Pommerville. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

(A) (B) Mixed culture (C)

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOTFirst FOR set SALESecond OR DISTRIBUTIONset Third set Fourth setNOT FOR SALE OR DISTRIBUTION of streaks of streaks of streaks of streaks (D) (E)

FIGURE 5.15 The Streak-Plate Method. A ) A( loop is sterilized, (B ) a sample of cells is obtained from a mixed culture, and ( C) streaked near one sector of the agar plate. ( D ) Successive streaks are performed, and the plate is incubated. (E ) Well-isolated and defined colonies illustrate a © Jonessuccessful & Bartlett isolation. Learning, ❯ ❯ Justify theLLC need to streak a mixed sample© overJones four areas & Bartlett on a culture Learning, plate. LLC NOT FOR Courtesy SALE of James Gathany/CDC. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONCounting CoverslipNOT FORA SALE The counting OR chamberDISTRIBUTION is a specially chamber marked slide containing a grid of 25 large squares of known area. The total volume of liquid held is 0.00002 ml (2 x10–5 ml). Sample © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC B The counting chamber is placed on NOT FOR SALE OR DISTRIBUTION the stage of a light microscopeNOT FOR. The number SALE OR DISTRIBUTION of cells are counted in several of the large squares to determine the average number. One of the 25 large squares © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

FIGURE 5.16 Direct Microscopic Count. This procedure can be used to estimate the total number of live and dead cells in a culture sample. ❯ ❯ Suppose the average number of cells per large square was 14. Calculate the number © Jones & Bartlett Learning,of cells in a LLC 10 ml sample. © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

multiple rounds of cell division to produce Indirect Measurement Methods. One separate colonies on the plate. Because two or of the simplest indirect methods of population more cells could clump together on a plate and measurement is to determine the cloudiness, or grow© as Jones a single colony,& Bartlett the standard Learning, plate LLCcount turbidity, in a broth© culture Jones using & Bartlett a spectropho- Learning, LLC is expressedNOT FOR as the SALE number OR of DISTRIBUTION colony-forming tometer. is instrumentNOT FORdetects SALE the amount OR DISTRIBUTION of units ( CFUs). A er incubation, the number of light scattered by a suspension of cells placed in CFUs will be used to estimate the number of the spectrophotometer such that the amount of viable cells originally plated. light scatter (optical density, OD) is a function © Jones & Bartlett Learning, LLC of ©the Jones cell number; & Bartlett therefore, Learning, the more LLC cells NOT FOR SALE OR DISTRIBUTION present,NOT theFOR more SALE light ORis scattered DISTRIBUTION or absorbed, resulting in a higher absorbance reading on the spectrophotometer ( FIGURE 5.18 ). A standard curve can be generated to serve as a measure of cell numbers. However, because more than 10 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC million cells are needed to make a reading on NOT FOR SALE OR DISTRIBUTION NOT FOR SALEthe spectrophotometer, OR DISTRIBUTION turbidity is not a useful way to study the growth of small populations of bacterial cells. In conclusion , we examined the major © Jones & Bartlett Learning, LLC physical factors and© nutrient Jones media & Bartlett formulations Learning, LLC NOT FOR SALE OR DISTRIBUTION a ecting the rate at NOTwhich FORmicrobial SALE populations OR DISTRIBUTION grow. In particular, the generation time for a bacterial species is dependent on the physical factors (temperature, oxygen need, etc.) and © Jones & Bartlett Learning, LLC nutrients© Jones available & Bartlett in the Learning, environment, LLC be it NOT FOR SALE OR DISTRIBUTION theNOT soil, FORa laboratory SALE culture OR DISTRIBUTION dish, or a human FIGURE 5.17 The Standard Plate Count. Individual host. In addition, many pathogenic bacteria bacterial colonies have grown on this blood agar plate. are fastidious at least in a laboratory setting, Each colony represents a colony-forming unit (CFU). ❯ ❯ If a 0.1 ml sample of a 104 dilution contained so that if they are taken from di erent parts 250 colonies, how many bacterial cells were in 10 ml of the body, the clinical microbiology lab may © Jones & Bartlett Learning,of the original LLC broth culture? © Jones & haveBartlett to use Learning, di erent growth LLC media for isolation NOT FOR SALE OR DISTRIBUTION © R.A. Longuehaye/Science Source. NOT FOR SALEand identi OR cation.DISTRIBUTION is brings up an interesting

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(A) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC LightNOT FOR SALE OR DISTRIBUTIONLight NOT FOR SALE OR DISTRIBUTION source detector

Bacterial© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC (B) cells NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 5.18 Using Turbidity to Measure Population Growth. As ( A light ) passes through a sterile broth tube in the spectrophotometer, the instrument is standardized at 0 absorbance. (B ) As a bacterial population in a broth tube grows, the cells will scatter more of the light, which on the spectrophotometer is detected as an increase in absorbance. ❯ ❯ Why do turbidity measurements represent an indirect method to measure population growth? Courtesy of Dr. Je © rey Pommerville.Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

On November 13, 1971, Mariner 9 went into orbit around the Red Planet. Before its arrival, we thought Mars was a dead world. Then Mariner 9 sent back pictures of a landscape where it appeared water once flowed. But the cra found no signs of biological material in the Martian soil. As more spacecra have gone to Mars, the evidence for life on the Red Planet© hasJones seesawed—yet & Bartlett some areLearning, hopeful that LLCmicrobial life may exist near the surface© Jones or & Bartlett Learning, LLC deeper underground. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Question C: As a result, several missions to Mars are being planned by American, European, Russian, and Chinese space agencies. As an exomicrobiologist (one who looks and searches for microbial life beyond Earth), what© Jones types of experiments& Bartlett would Learning, you design LLC (based on this chapter) to see if© microbial Jones life & does Bartlett exist on Learning, LLC Mars? The spacecra will not return to Earth, so the experiments need to be completed on the Red Planet. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Answers can be found in Appendix F .

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC problem when considering the diagnosis NOT FOR SALE OR DISTRIBUTION Bacteria:NOT © NIAID FOR SALE OR DISTRIBUTION CONCEPT AND REASONING CHECKS 3 and treatment of an infectious disease. Such a. Compare and contrast complex and pathogens, including Treponema pallidum, have chemically defined media. to be identi ed from actual clinical samples b. Explain how microbiologists © Jones & Bartlett Learning, LLC using direct microscopic© Jones observation. & Bartlett So, Learning, one LLC have figured that some 98% of has to wonder how many human diseases NOTthe FOR microbial SALE world OR has DISTRIBUTION not been NOT FOR SALE OR DISTRIBUTION may go undiagnosed today because they are “seen.” c. Why might it be more di icult to VBNC? In fact, microbiologists have argued isolate a colony from a pour-plate whether members of the Archaea can cause than from a streak-plate culture? human disease. None are currently known to © Jones & Bartlettd. Distinguish Learning, between LLC direct and cause© Jones disease, & yet Bartlett we know Learning, from microscopic LLC NOT FOR SALEindirect OR DISTRIBUTION methods to measure observationNOT FOR that SALE archaeal OR organisms DISTRIBUTION are present population growth. in the human body. Could some of them be VBNC pathogens?

5.1 Microbial Reproduction Is Part of the Cell Cycle 5.3 Culture Media Are Used to Grow Microbes and 1. The© Jonesbacterial cell & cycleBartlett involves Learning, cell growth, DNA LLC replication, Measure© Their Jones Growth & Bartlett Learning, LLC andNOT binary FOR fission SALE to produce OR genetically DISTRIBUTION identical daughter 6. ComplexNOT and FOR chemically SALE defined OR media DISTRIBUTION contain the cells. ( Fig. 5.2 A) nutrients for microbial growth. 2. Binary fissions occur at intervals called the generation time , 7. Growth media can be modified to select for a desired which may be as short as 20 minutes. ( Fig. 5.3 ) microbial species, to di® erentiate between two similar species, or to enrich for species requiring special nutrients. 5.2 Microbial Growth Progresses Through Distinct ( Fig. 5.13 ) Phases © Jones & Bartlett Learning, LLC © Jones8. &Pure Bartlett cultures canLearning, be produced LLC from a mixed culture by the NOT FOR SALE3. The dynamics OR DISTRIBUTION of the bacterial growth curve show how a NOT FORpour-plate SALE OR method DISTRIBUTION or the streak-plate method . In both cell population grows logarithmically, reaches a certain peak cases, discrete colonies can be identified that represent only and levels o® , and then may decline. ( Fig. 5.4 ) one microbial species. (Figs. 5.14, 5.15) 4. Dormancy is a response to potential or actual environmental 9. Microbial growth can be measured by direct microscopic change. (Figs. 5.6, 5.8) count , the most probable number test , and the standard 5. Temperature, oxygen,© pH, Jones and salt concentration& Bartlett are Learning, physical LLC plate count procedure. Indirect© Jones methods & o Bartlett en use Learning, LLC factors that influence microbial growth. Away from the turbidity measurements. (Fig. 5.18 ) optimal condition, growthNOT slows FOR within SALE a set range. OR (Figs. DISTRIBUTION 5.9, NOT FOR SALE OR DISTRIBUTION 5.11)

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For STEPS A–D , answers© to Jonesquestions & and Bartlett problems Learning, can be found LLC in Appendix D . © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

STEP© A: Jones REVIEW & Bartlett OF FACTS Learning, AND LLCTERMS © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Multiple Choice 5. A microbe that is a microaerophilic mesophile would grow optimally at and . Read each question carefully, then select the one answer that A. high O 2 ; 30°C

best fits the question or statement. B. low O2 ; 20°C C. no O ; 30°C © Jones & 1.Bartlett Which one Learning, of the following LLC statements does NOT apply to © Jones & Bartlett2 Learning, LLC D. low O ; 37°C NOT FOR SALEbacterial OR reproduction? DISTRIBUTION NOT FOR SALE2 OR DISTRIBUTION A. A fission ring apparatus is present. 6. If the carbon source in a growth medium is beef extract, the B. Septum formation occurs. medium must be an example of a/an medium. C. A spindle apparatus is used. A. complex D. Symmetrical cell division occurs. B. chemically defined 2. If a bacterial cell in a ©broth Jones tube has & a Bartlettgeneration time Learning, of LLC C. enriched © Jones & Bartlett Learning, LLC D. di® erential 40 minutes, how manyNOT cells willFOR there SALE be a er 6OR hours DISTRIBUTION of optimal NOT FOR SALE OR DISTRIBUTION growth? 7 . A medium would involve the addition of the antibiotic A. 1 8 streptomycin to identify streptomycin-resistant bacteria. B. 6 4 A. di® erential C. 1 2 8 B. selective D.© 5Jones 1 2 & Bartlett Learning, LLC C. thioglycollate© Jones & Bartlett Learning, LLC D. V B N C 3. ANOT bacterial FOR species SALE generation OR time DISTRIBUTION would be determined during NOT FOR SALE OR DISTRIBUTION the phase. 8. Which one of the following is NOT part of the streak-plate A. decline method? B. l a g A. Making four sets of streaks on a plate C. l o g B. Diluting a mixed culture in molten agar © Jones & BartlettD. stationary Learning, LLC © Jones &C. UsingBartlett a mixed Learning, culture LLC D. Using a sterilized loop NOT FOR SALE 4. Which ORone of DISTRIBUTION the following is NOT an example of dormancy? NOT FOR SALE OR DISTRIBUTION A. Log phase 9. Direct methods to measure bacterial growth would include all B. Endospore formation the following except . C. VBNCs A. total bacterial count D. Persister cells B. direct microscopic count © Jones & Bartlett Learning, LLC C. turbidity measurements© Jones & Bartlett Learning, LLC D. most probable number NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORTrue–False SALE OR DISTRIBUTION NOT14. FOR SALE In attempting OR DISTRIBUTION to culture a fastidious bacterial pathogen, a di®erential medium would be used. Each of the following statements is true (T) or false (F). If the 15. Acidophiles grow best at pHs greater than 9. statement is false, substitute a word or phrase for the underlined word or phrase to make the statement true. 16. Mesophiles have their optimal growth near 37°C. 10. Endospores are produced© Jones by some & Bartlett gram-negative Learning, LLC17. Bacterial and archaeal© cells Jones lack a mitotic & Bartlett spindle to Learning, LLC bacterial species. NOT FOR SALE OR DISTRIBUTIONseparate chromosomes. NOT FOR SALE OR DISTRIBUTION 11. Obligate aerobes use oxygen gas as a final electron 18. The fastest doubling time would be found in the lag acceptor in energy production. phase of a bacterial growth curve. 12. The most common growth medium used in the teaching 19. If E. coli cells are placed in distilled water, they will burst. laboratory is a complex medium. © Jones & Bartlett Learning, LLC 20. Halophiles© Jones would & dominate Bartlett in marineLearning, environments. LLC 13. NOT The majorityFOR SALEof bacterial OR and DISTRIBUTION archaeal organisms can be NOT FOR SALE OR DISTRIBUTION cultured in growth media.

21. Describe the three phases of a bacterial cell cycle. (Key 26. Explain the procedures used in the pour-plate and streak- Concept 1) plate methods. (Key Concept 3) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 22. Compare the events of each phase of a bacterial growth 27. Construct a concept map for Growth Measurements using the curve. (Key Concept 2)NOT FOR SALE OR DISTRIBUTIONfollowing terms. (Key ConceptNOT 3) FOR SALE OR DISTRIBUTION 23. Explain the importance of bacterial dormancy. (Key Concept 2) CO2 production Indirect measurement 24. Identify the four major physical factors governing microbial methods growth, and describe how microorganisms have adapted to Cell mass Metabolic activity these© physical Jones environments. & Bartlett (Key Learning, Concept 2) LLC © Jones & Bartlett Learning, LLC Colony-forming unit Most probable number test 25. ExplainNOT how FOR selective SALE and dierential OR DISTRIBUTION media are each NOT FOR SALE OR DISTRIBUTION constructed. (Key Concept 3) Direct-counting method Oxygen uptake Direct microscopic count Standard plate count Turbidity © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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28. Use the log phase growth curves (1, 2, or 3) below to answer 29. Consumers are advised to avoid stu®ing a turkey the night each of the following questions (a–c). before cooking, even though the turkey is refrigerated. A homemaker questions this advice and points out that the © Jones & Bartlett Learning, LLC bacterial species of human© disease Jones grow & mainly Bartlett at warm Learning, LLC Growth curves NOT FOR SALE1 OR DISTRIBUTIONtemperatures, not in the refrigerator.NOT FOR What SALE explanation OR might DISTRIBUTION you o®er to counter this argument? 30. Public health o®icials found the water in a Midwestern town was contaminated with sewage bacteria. The o®icials suggested 2 homeowners boil their water for a couple of minutes before © Jones & Bartlett Learning, LLC drinking© it.Jones (a) Would & this Bartlett treatment Learning, sterilize the water? LLC Why? (b) NOT FOR SALE OR DISTRIBUTION Is it importantNOT FOR for the SALEwater to beOR sterile? DISTRIBUTION Explain. Number of Cells 3

Time (min)

© Jones & Bartletta. Which curve Learning, (1, 2, or 3) best LLC represents the growth curve for© a Jones & Bartlett Learning, LLC NOT FOR SALEmesophile OR incubated DISTRIBUTION at 60°C? NOT FOR SALE OR DISTRIBUTION b. Which curve (1, 2, or 3) best represents a nonhalophile growing in 5% salt? c. Which curve (1, 2, or 3) best represents an acidophile growing at pH 4? © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

STEP D: QUESTIONS FOR THOUGHT AND DISCUSSION 31. To© preventJones decay & Bartlettby bacterial speciesLearning, and to display LLC the 33. During© the Jones filming of& the Bartlett 1997 movie Learning, Titanic, researchers LLC mummifiedNOT FOR remains SALE of ancient OR peoples,DISTRIBUTION museum o®icials place discoveredNOT at FOR least 20 SALE di®erent ORbacterial DISTRIBUTION and archaeal species the mummies in glass cases where oxygen has been replaced literally consuming the ship, especially a rather large piece of with nitrogen gas. Why do you think nitrogen is used? the midsection. What type of environmental conditions are these bacterial and archaeal species subjected to at the wreck’s 32. Extremophiles are of interest to industrial corporations, who see depth of 12,600 feet? these organisms as important sources of enzymes that function © Jones & Bartlettat temperatures Learning, of 100°C and LLC pH levels of 10 (the enzymes have© Jones 34. & Every Bartlett year news Learning, media report cases LLC of skin and lung infections been dubbed “extremozymes”). What practical uses can you in people sitting in hot tubs. How might such infections occur in NOT FOR SALEforesee OR for these DISTRIBUTION enzymes? NOT FORhot SALE tubs? OR DISTRIBUTION

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