Microbial Growth and Nutrition: Chapter Preview

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5
The Martian surface as seen by NASA’s Curiosity Mars rover.
CHAPTER PREVIEW
Microbial Growth and Nutrition
Bacteria: © NIAID
5.1 Microbial Reproduction Is Part
B
© Jones
of the & Bartlett Learning,
Cell Cycle LLC © Jones & Bartlett Learning, LLC
NOT FOR SALE OR DISTRIBUTIONooks have been written
NOTabout
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movies OR
haveDISTRIBUTION
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 Schiaparelli, 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 Learning,
Campylobacter jejuni LLC century that © this
Jones & Bartlett
notion Learning,
was disproved. LLC we gaze at the red
Still, when
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5.3 Culture Media Are Used to NOT FOR SALE OR DISTRIBUTION
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 scientific colleagues, wondering if
microbial
© Jones & Bartlett
of a Bacterial Species life once
Learning, LLCexisted on the Red Planet©or,Jones
for that &
matter, elsewhere
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in our solar
Investigating the Microbial 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
surface? Researchers, using a device to simulate the Martian soil
environment, placed in the soil microbes known to survive extremely
NOT FOR SALE OR DISTRIBUTION cold environments here onNOT Earth.FOR
TheirSALE
results OR DISTRIBUTION
suggested that members
of the Archaea, specifically the methanogens, could grow in the cold,
low-oxygen atmosphere, especially if protected from UV light by being
buried just under the soil surface.
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Microbial Growth and Nutrition 141
TABLE 5.1 Some Microbial Record Holders (and Their Taxonomic Domain)
Hottest environment (Juan de Fuca ridge)—121°C: Strain 121 (Archaea)

Coldest environment (Antarctica)— –15°C: Planococcus cryohalophilus (Bacteria)
Highest radiation survival—5 MRad, or 5000× what kills humans: Deinococcus radiodurans (Bacteria)
Deepest—3.2 km underground: Many bacterial and archaeal species
Most acid environment (Iron Mountain, CA)—pH 0.0: Ferroplasma acidarmanus (Archaea)
© Jones
Most alkaline & Bartlett
environment Learning,
(Lake Calumet, LLC
IL)—pH 12.8: Proteobacteria (Bacteria) © Jones & Bartlett Learning, LLC
NOT
Longest FOR
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satellite)—6 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(SouthLearning, LLC below Earth’s surface: Desulforudis
Africa)—3.3 kilometers © Jones & Bartlett Learning, LLC
audaxviator
Source: http://www.astrobio.net/news/
So, microbiologists have joined the search In 2004, NASA sent two spacecraft to Mars
for extraterrestrial life. NOT ThFOR SALE
is seems OR DISTRIBUTION
a valid NOT
to look for indirect signs of past life. FOR SALE
Scientists OR DISTRIBUTION
Extremophile: A
microorganism that
pursuit because the extremophiles 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© Jones different&from
Bartlett
MarsLearning,
( FIGURE 5.1LLC © Jones
). existed on the planet. Some fi&ndings
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suggest extremely LLC
high/low
If life (as NOT we FOR
know it) did orOR
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DISTRIBUTION NOTsalty
FORseasSALE
once washed
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temperatures, high
it almost certainly was or is bacterial/archaeal. over the plains of Mars, creating a life-friendly acidity, or high salt
environments.
environment.
Opportunity found evidence for ancient
shores on what once was believed to be a sea.
Scientists reported in 2008 that a more recent
spacecraft, 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,CuriosityLLC © Jones
is searching to see if there are any& Bartlett Learning, LLC
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chemical signs suggesting life may have FOR SALE OR DISTRIBUTION
existed
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 physical
© Jones and chemical
& Bartlett Learning, LLC
FIGURE NOT 5.1 FOR SALESurface?
The Martian OR DISTRIBUTION
This barren-
requirements they must possess to survive,
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© Photodisc/age
& Bartlett fotostock. Learning, LLC © Jones
microbial & Bartlett
growth Learning, LLC
can be measured.
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142 Chapter 5: Microbial Growth and Nutrition
NOT FOR SALE OR DISTRIBUTION BinaryOR
NOT FOR SALE Fission Is Part of the
DISTRIBUTION
CHAPTER CHALLENGE Cell Cycle
The series of events involving growth, DNA
replication, and cell division is called the cell
The United States
© Jones has sent several
& Bartlett spacecraft
Learning, LLC cycle. For a bacterial ©cell
Jones
to Mars since the first Viking landers in 1976.
like E.&coli
Bartlett
, the cycleLearning,
can LLC
NOT FOR SALE OR DISTRIBUTION be divided into three
Recently, an international team of scientists
NOT FOR
periods SALE5.2A
( FIGURE OR).DISTRIBUTION
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
craft, and they could even survive the journey. The of metabolic “growth,” called the B period,
team believes most spacecraft that have touched where the cell&increases
© Jones & Bartlett Learning, LLC
down on Mars were not thoroughly sterilized, so
© Jones BartlettinLearning,
cell mass and LLCcell
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DISTRIBUTION size. During this period, the
NOT FOR SALE OR DISTRIBUTION cell is preparing for
they could carried living microbes from
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 size, DNA replication starts. During this C
carriedLLC
on the spacecraft. However, in laboratory© Jones & Bartlett Learning, LLC
period, DNA synthesis occurs and the bacterial
tests, here on Earth the scientists cameNOT acrossFOR SALE OR DISTRIBUTION
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
© Jones survive
extremophile & Bartlett
on Mars, Learning, LLC cytoskeleton but, unlike
or could there © Jones & Bartlett
eukaryotic Learning, LLC
cells, lacks
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actually be Martian microbes on the planet? NOT to FOR
segregate
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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 Jones & binary
©undergoes Bartlettfission . DuringLLC
Learning, this D
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( FIGURE 5.2B ). The splitting of the cell in half
(cytokinesis) is coordinated by cytoskeletal
KEY CONCEPT 5.1 Bacteria: © NIAID proteins organized into a “fission ring
apparatus,” which ensures two nearly identical
Microbial Reproduction Is Part of the
Cell Cycle
© Jones & Bartlett cells
daughter Learning, LLCDepending on the
are formed.
NOT FOR SALE OR DISTRIBUTION NOT FOR SALE growthOR DISTRIBUTION
conditions, 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
Jonesin&the population
Bartlett size withLLC
Learning, each species. With other©species,
Joneslike & E. coli, the Learning,
Bartlett cells LLC
cell carrying the identical
NOT FOR SALE OR DISTRIBUTION genetic instructions completely separate from one another aft
NOT FOR SALE OR DISTRIBUTION er the
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 Bacterial and Archaeal Cells Can
chromosomes in the cell LLC nucleus allows for © Jones & Bartlett Learning, LLC
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NOTExponentially
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precise of mitosis and cytokinesis.
Bacterial and archaeal cells divide without The interval of time between successive binary
this elaborate division apparatus but still fissions of a cell or population of cells is known as
possess similar protein filaments to use in the the generation time (or doubling time). Under
reproduction
© Jones & Bartlett Learning, LLCprocess. © Jones & optimal
Bartlettconditions,
Learning,some LLC species, especially
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Bacterial and Archaeal Cells Can Grow Exponentially 143
Bacterial
chromosome
Cell wall
Cell membrane
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, LLCD period © Jones & Bartlett Learning, LLC
(Binary fission)
NOT FOR SALE OR DISTRIBUTION C periodNOT FOR SALE OR DISTRIBUTION
(Chromosome
replication)
Fission ring Replicated NOT FOR SALE OR DISTRIBUTION
apparatus DNA molecules
DNA synthesis and
replication generate two
daughter chromosomes.
(A)
(B)
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
differ for aLearning, LLC having cells arranged in©chains
prokaryotic organism Jones & Bartlett
and another forming Learning,
single cells? LLC
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many pathogens,
NOT FOR SALE OR DISTRIBUTION have a very fast generation for 36 OR
NOT FOR SALE hours, the bacterial cells would cover
DISTRIBUTION
time; for others, including many environmental the surface of the Earth! Thankfully, 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. for growth. The majority of the bacterial cells
One enterprising mathematician calculated would starve to death or die in their own waste.
that NOT FOR
if E. coli SALE
binary OR were
fissions DISTRIBUTION
to continue The generationNOT
time isFOR
usefulSALE OR DISTRIBUTION
in determining
at their optimal generation time (15 minutes) how much time passes before disease symptoms
Bacteria: © NIAID
MICROFOCUS
MICROFOCUS 5.1: Evolution/Environmental Microbiology
A Microbe’s
NOT FOR SALE ORLife—Zombie Style
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What do World War Z, Zombieland, Dawn of the Dead and some rock-dwelling microbial zombies could be beneficially affecting
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 Learning,
big screen flicks LLC
Scientifically, these bacterial © Jones & Bartlett
communities offerLearning,
an LLC
NOTdied
identifies “creatures” that have FOR andSALE OR DISTRIBUTION
been reanimated as opportunity to learn how cells can NOT FORonSALE
survive so few OR DISTRIBUTION
nutrients
unconscious, mindless monsters, often 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.
© already
As you Jonesknow,& Bartlett
microbesLearning,
can be found LLC just about © Jones & Bartlett Learning, LLC
anywhereNOT on andFOR SALE
in planet EarthOR DISTRIBUTION
where 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
© Jones
ocean &floor.
Bartlett Learning,
More impressive, theseLLC
organisms are up to 100 million© Jones & Bartlett Learning, LLC
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years old, and theOR DISTRIBUTION
researchers 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© the
Jones & Bartlett
infinitesimally Learning,
small amount of LLC © Jones & Bartlett Learning, LLC
energy and nutrients available NOT FOR
in their rockySALE
homes. AtOR DISTRIBUTION
these 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
microbes]© Jones
alive & Bartlett
when [they’re] Learning,
doubling LLCof years?
every thousands © Jones & Bartlett Learning, LLC
Dr. Beth Orcutt (front row, second from left) and researchers
It’s almostNOT FOR SALE
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to understand OR DISTRIBUTION
how life below the 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.
978128057782_CH05_140_171.indd 144 10/10/14 2:26 PM

Bacterial and Archaeal Cells Can Grow Exponentially 145
NOT FOR SALE ORExamples
DISTRIBUTION
of Generation NOT FOR SALE OR DISTRIBUTION
1,000,000
Times—Common Bacterial
TABLE 5.2
Species Growing Under Time Number of
of day cells
Optimal Conditions1 900,000
8:00 PM 100
© Jones
Growth & Bartlett
Generation Time Learning, LLC 8:30 200© Jones & Bartlett Learning, LLC
Species NOT FOR
Medium SALE OR DISTRIBUTION
(minutes)
800,000
9:00 400NOT FOR SALE OR DISTRIBUTION
9:30 800
Escherichia coli Synthetic 17 10:00 1,600
10:30 3,200
Bacillus Synthetic 25
11:00 6,400
Actual number of bacterial cells

megaterium 700,000
11:30 12,800
© JonesComplex
Staphylococcus & Bartlett Learning,
27–30 LLC © Jones
12:00 AM & Bartlett Learning, LLC
25,600
aureusNOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION
12:30 51,200
600,000 1:00 102,400
Streptococcus Milk 26 1:30 204,800
lactis 2:00 409,600
2:30 819,200
Streptococcus Complex 48 500,000
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
400,000
acidophilus
Mycobacterium Synthetic 792–932
tuberculosis (= 12–15.5 hours) 300,000
Treponema © Jones
Rabbit testes & (=Bartlett
1,980 Learning, LLC
33 hours) © 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
0
8
Incubation9 10 11 12 1 2 3
appear in an infected individual; faster division PM Time (hours) AM period: The time
times often mean a shorter incubation period from entry of a
for a disease. For example, suppose you eat an FIGURE 5.3 A Skyrocketing Bacterial Population. pathogen into the
© Jonesimproperly
& Bartlettrefrigerated
Learning,chocolate
LLC © Jones
éclair that The number & Bartlett
of Staphylococcus Learning,
aureus LLC
cells progresses body until the first
NOT FORwasSALE OR DISTRIBUTION
contaminated with Staphylococcus aureus from NOT 100 cellsFOR SALE
to almost OR
2 million DISTRIBUTION
cells in a mere 7 hours. symptoms appear.
( FIGURE 5.3 ). If you ingested 100 cells at 8:00 The J-shaped growth curve gets steeper and steeper as the
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 by midnight.
© Jones By 3:00Learning,
& Bartlett figure? LLC © Jones & Bartlett Learning, LLC
AM, the exponential growth
NOT will
FOR have produced
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more than 1.6 million cells. Depending on the
response of the immune system, it is quite likely CONCEPT AND REASONING CHECKS 1 Bacteria: © NIAID
that sometime during the night you would a. Propose an explanation as to how
know ©you have food poisoning. a bacterial cell “knows” when to
Jones & Bartlett Learning, LLC divide. © Jones & Bartlett Learning, LLC
Bacterial and archaeal organisms are subject
NOT FOR SALE OR DISTRIBUTION b. If it takes anNOT E. coliFOR
cell 7 SALE
hours ofOR DISTRIBUTION
to the same controls on growth as all other
growth to reach some 2 million
organisms on Earth. Let’s examine the most
cells, how long would it take a T.
important growth factors conferring optimal pallidum cell to reach the same
generation times. number under optimal conditions?
978128057782_CH05_140_171.indd 145 10/10/14 2:26 PM

NOT FOR SALE OR DISTRIBUTION A Bacterial
NOT FOR SALE Growth Curve Illustrates
OR DISTRIBUTION
CHAPTER CHALLENGE A the Dynamics of Growth
A typical bacterial growth curve for a
population illustrates the events occurring over
We already know
© Jones some bacterial
& Bartlett and archaeal
Learning, LLC time ( FIGURE 5.4 ©). Jones & Bartlett Learning, LLC
organisms can survive in hostile environments.
If a sample of bacterial
NOT FOR SALE OR DISTRIBUTION cells is transferred NOT
In fact, these extremophiles not only survive in
FOR
to a tube SALE
of fresh ORfour
broth, DISTRIBUTION
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.
population numbers. TheLearning,
first portionLLCof the
© Jones & Bartlett Learning, LLC ©The
JonesLag & Phase.
Bartlett
NOT FOR SALE QuestionOR A:DISTRIBUTION growth curve, during which
NOT FOR SALE 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, LLC extremophile would have
an Earthly © been
Jones & Bartlett Learning, LLC
on a spacecraft to Mars? And if it did,NOT transfer or the inability to adapt to the new
wouldFOR SALE
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environment. The actual length of the lag phase
it survive?
Answers can be found in Appendix F. (B period of the cell cycle) depends on the
metabolic activity of the microbial population.
They must grow in size, take up nutrients,
© Jones & Bartlett Learning, LLC and replicate their© DNA Jones (C&period
Bartlett
)—allLearning,
in LLC
NOT FOR SALE OR DISTRIBUTION preparation for binary NOT FOR(D
fission SALE
periodOR
). DISTRIBUTION
KEY CONCEPT 5.2 Bacteria: © NIAID The Log Phase. The 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 are undergoing
Jones & Bartlettbinary fission LLC
Learning, and the
In the previous
NOT FOR SALE OR DISTRIBUTION section, we discovered how generation
NOT FOR timeSALE
is dependent on the species and
OR DISTRIBUTION
fast some bacterial cells can grow under ideal environmental conditions present. The 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 & Bartlett10Learning, LLC © Jones & Bartlett Learning, LLC

NOT FOR SALE OR 9 DISTRIBUTION NOT FOR SALE OR DISTRIBUTION
8 (C) Stationary phase
(D) Decline
Logarithm (10n) of viable cells
7 phase
6
5
(B) Log
4 © Jonesphase
3
2 (A) Lag
© Jones &phase
Bartlett Learning, LLC © Jones & Bartlett Learning, LLC
0
NOT FOR0 SALE 1 OR DISTRIBUTION
2 3 4 5 NOT FOR6 SALE OR7 DISTRIBUTION
8
Time (hours)
Total cells in population:
Few cells Viable cells Nonviable/dead cells

FIGURE 5.4 TheLLC © JonesA&typical
Growth Curve for a Bacterial Population. Bartlett
bacterialLearning, LLC
population goes sequentially through four
NOT FOR SALEgrowth
OR DISTRIBUTION
phases. ❯❯ Why would antibiotics work bestNOT FOR
to kill or SALE
inhibit ORlogDISTRIBUTION
cells in the phase?
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Some Bacterial Cells Can Exist In a Metabolically Inactive State 147
As each generation time
NOT FOR SALE OR DISTRIBUTION passes, the number of logarithmically.
NOT FORFor manyOR
SALE species, the history of
DISTRIBUTION
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 cell numbers. escape death by entering a state of dormancy.
If plated on solid growth medium, bacterial
NOTthat
growth will be so vigorous FOR SALE
visible OR DISTRIBUTION
colonies NOT FOR SALE OR DISTRIBUTION
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 © Jones
stage & Bartlett
of growth becauseLearning, LLC and often they might©not
many antibiotics Jones & Bartlett
be favorable Learning, LLC
to active
affect metabolic
NOT FOR processes
SALE like
ORprotein synthesis population growth. Such
DISTRIBUTION NOTunfavorable
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). Therefore, throughout the cell cycle
© Jonesare not available
& Bartlett Learning, LLCTherefore, after microbes
indefinitely. © Jones must&constantly monitor orLLC
Bartlett Learning, sense
NOT FORsome
SALEhoursOR broth tube, available nutrients theirNOT
in aDISTRIBUTION surroundings
FOR SALE to ensure
OR the conditions will
DISTRIBUTION
become scarce and waste products accumulate. support continued growth. For some bacterial
This 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. The vigor of the population changes and (nondividing) state. When environmental
© Jones
the cells enter a plateau, & Bartlett
called the stationary Learning, LLC
conditions © Jones
improve, the cells “revive” and& Bartlett Learning, LLC
phase. Now the number NOT of FOR
viableSALE OR DISTRIBUTION
cells equals once again start dividing. Two suchNOT FOR SALE OR DISTRIBUTION
dormancy
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
© Jonesbecome exceedingly
& Bartlett Learning, the produce a subset of
low,LLC © cells
Jonesthat&normally
Bartlett stop
Learning, LLC
population enters a decline phase
NOT FOR SALE OR DISTRIBUTION (or death dividing and remainNOT FOR SALE ORthe
in the B period of 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. (A) Bacterial colonies cultured on blood agar in a culture dish. Blood
agar is a mixture of nutrient agar and blood cells. It is often 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)&orBartlett
(B) start? Learning, LLC © Jones & Bartlett Learning, LLC
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(A) Courtesy OR(B) Courtesy
of Dr. J. J. Farmer/CDC. DISTRIBUTION
of CDC. NOT FOR SALE OR DISTRIBUTION
978128057782_CH05_140_171.indd 147 10/10/14 2:26 PM

cells maintain
NOT FOR SALE OR DISTRIBUTION a very low rate of metabolism. dormant
NOT FOR SALE structures called
OR DISTRIBUTION 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 differentiation
die. For example, such “altruistic-like” behavior process specifically tied to nutrient limitation
benefits pathogens like M. tuberculosis. If (starvation) in the environment and is initiated
NOT FOR
antibiotics SALE
are used ORthe
to treat DISTRIBUTION
infection, any by quorum sensing NOTwithin
the “starving”
persister cells will survive because they are not population.
affected 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 & Bartlett
Should Learning,beLLC
the antibiotics © Jones
withdrawn at some hundreds of & Bartlett
genes. Learning,
The process LLCthe
involves
NOT FORlaterSALEdate,OR
the persister cells become active again, replication
DISTRIBUTION NOT FOR of the
SALEbacterial chromosome and
OR DISTRIBUTION
forming a new actively growing population binary fission that produces an asymmetric cell
with some persister cells present. division ( FIGURE 5.8 ). The smaller cell, called
Endospore Formation. A few medically- the prespore, will become the mature endospore,
signifi
© Jones & Bartlett Learning,cantLLC Jones & while
gram-positive genera, ©especially the larger
Bartlett motherLLC
Learning, cell will commit itself to
Bacillus and Clostridium, undergo aNOT
NOT FOR SALE OR DISTRIBUTION different maturation
FOR SALE of the endospore before rupturing.
OR DISTRIBUTION
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
© Jones
botulism) enter &
theBartlett
stationaryLearning,
phase and LLC
begin for species identifi© Jones
cation & Bartlett
purposes). Learning, LLC
The entire
sporeNOT FOR or
formation SALE OR DISTRIBUTION
sporulation that produces process takes aboutNOT 6 to 8FOR
hours.SALE OR DISTRIBUTION
© Jones & Bartlett Learning, LLC A © Jones & Bartlett Learning, LLC
During log phase, FOR SALE OR DISTRIBUTION
persister cell number Antibiotic
increases. treatment
Persister cell
Antibiotic sensitive cell NOT FOR SALE OR DISTRIBUTION
C B
© Jones & Bartlett Learning, LLC Sensitive cells © Jones & Bartlett Learning, LLC
Binary fission
of persister cells
killed; persister NOT FOR SALE OR DISTRIBUTION
cells survive.
resumes.
Killed cell
Antibiotic
treatment
© Jones & Bartlett Learning, LLC stopped
FIGURE 5.6 Persister Cells. The presence of persister cells within a bacterial cell population allows the species
to survive environmental changes, in this case©
theJones
presence & Bartlett
of an antibiotic.Learning, LLC
❯❯ What other way could a susceptible
NOT FOR SALE OR DISTRIBUTION NOTofFOR
bacterial cell population survive in the presence SALE OR DISTRIBUTION
an antibiotic?
978128057782_CH05_140_171.indd 148 10/10/14 2:26 PM

Some Bacterial Cells Can Exist In a Metabolically Inactive State 149
Coat
layers
Core Cortex

Endospores
(A) (B)
© Jones & Bartlett Learning, LLC © Jones

FIGURE 5.7 & Bartlett
Three Different Learning, LLC
Views of Bacterial Endospores. (A) A light
microscope image of Clostridium cells 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
coat dividesLLC
equatorially along the long axis, ©
andJones & Bartlett
as it separates, Learning, LLC
the vegetative
cell emerges.
NOT FOR SALE OR DISTRIBUTION (Bar = 2.0 µm.) ❯❯ If an endospore is resistant to so many
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.
The 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 related to biological
© JonesDNA.
& Bartlett Learning,
Thick layers LLC
of peptidoglycan form the reveals.© Jones & Bartlett
No wonder Learning,
some scientists LLC
believe they effect.
NOT FOR SALE OR DISTRIBUTION NOT FOR SALE
cortex and some 70 proteins form the layers of the could survive a trip 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” produced
© Jones in responseLearning,
& Bartlett rapidly germinates
LLC and grows out as a©vegetative
Jones & Bartlett Learning, LLC
to environmental “hardNOT times.FOR
” cell (see Figure 5.7C).
SALE OR DISTRIBUTION 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 effect on the spore. By containing little pressure will do the trick. If they contaminate
water,©endospores
Jones & also are heat
Bartlett resistantLLC
Learning, and machinery, such as © theJones
mail-sorting equipment
undergo very few chemical reactions. These contaminated in the 2001 anthrax attacks,
properties make them difficult 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
They Learning, LLC
can survive in © Jones
70% ethyl alcohol diseases & Bartlett
in humans Learning,
are caused LLC
by spore formers.
NOT FORforSALE OR
20 years. DISTRIBUTION
Humans can barely withstand 500 The NOT most FOR SALE has
newsworthy OR been
DISTRIBUTION
B. anthracis,
978128057782_CH05_140_171.indd 149 10/10/14 2:26 PM

D period
ASEXUAL
CELL CYCLE
© Jones & Bartlett Learning, LLC (nutrients © Jones & Bartlett Learning, LLC
plentiful)
NOT FOR SALE BOR period
DISTRIBUTION NOTC FOR
period SALE OR DISTRIBUTION
D Germination. A Asymmetric cell division.

Under favorable A transverse membrane
© Jones & Bartlett
nutrientLearning,
conditions, LLC © Jones & Bartlett Learning,
septumLLC
separates one
the aged spore DNA molecule and a
germinates to NOT FOR SALE OR DISTRIBUTION
small section of cytoplasm.
produce a new
vegetative cell. Cell Cell
DNA membrane
wall
Vegetative cell
Spore
© Jones & coat
Bartlett Learning, LLC © Jones & Bartlett Learning, LLC
DNA
Cortex NOT FOR SALE OR DISTRIBUTION
Free spore
Transverse
septum
SPORULATION
NOT FOR SALE OR DISTRIBUTION CYCLE 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 © Jones & Bartlett
membranes LLC Learning,
of the prespore.
The spore coat is
completed and the NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION
mother cell ruptures,
freeing the spore.

The spore coat assembles
around the cortex. NOT FOR SALE OR DISTRIBUTION
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”
nutrient conditions are limiting.
978128057782_CH05_140_171.indd 150 10/10/14 2:26 PM

Optimal Microbial Growth Is Dependent on Several Physical Factors 151
Bacteria: © NIAID NOT FOR SALE OR DISTRIBUTION
MICROFOCUS
MICROFOCUS 5.2: Being Skeptical
Germination of 25 Million-Year-Old Endospores?

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 & Bartlett
several thousand yearsLearning,
old. In 1983,LLC © Jones
archaeologists area. In addition, & Bartlett
the hood had neverLearning, LLC
been used for any other
found NOT
viable FOR
Thermoactinomyces spores
SALE OR DISTRIBUTION in sediment lining bacterial extraction
NOT FOR SALE OR DISTRIBUTIONwere
processes. Several other precautions
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 after so long a period of dormancy. Does
© JonesCano
found bacterial sporesLLCin the gut of a fossilized © beeJones
the DNA& Bartlett
really haveLearning,
a capability LLC
of replication and producing
trapped in amber—a hardened resin—produced from a NOT FOR SALE OR DISTRIBUTION
tree in new vegetative growth? 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
© Jones
germinated. With microscopy, & from
the cells Bartlett Learning,
a colony were very LLC © Jones
that is based on current predictions, & Bartlett
and they Learning,
may be subject to LLC
similar to Bacillus sphaericus , which is found today in bees in the change as more research is NOT FOR SALE OR DISTRIBUTION
carried out with ancient DNA.
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 after 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 & Bartlett
examined. However, Learning,
Professor CanoLLChad carried is possible.© Jones & Bartlett Learning, LLC
out appropriate and rigorous decontamination procedures and NOT FOR SALE OR DISTRIBUTION
the agent of the 2001 anthrax bioterror attack In hospital and healthcare facilities, C. diffi-
© Jonesthrough
& Bartlettthe Learning,
mail. This LLC © Jones
potentially deadly cile (oft & Bartlett
en referred Learning,
to as C. diff LLC
) can cause seri-
disease, originally studied by Koch and Pasteur, ous infections of the colon (colitis) 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. difficile infections occur in U.S.
vegetative cells secrete two deadly toxins. Other hospitals each year, partly coming from the tis-
human diseases result© fromJonesthe&germination sue germination
Bartlett Learning, LLC of endospores originally pres-& Bartlett Learning, LLC
© Jones
of Clostridium endospores—gas
NOT FORgangrene
SALE OR (C. DISTRIBUTION
ent on improperly cleaned medical NOT equipment
perfringens) and tetanus or lockjaw (C. tetani). and on personal items. (These diseases will be
Clostridial endospores often are found in soil covered in more detail in the appropriate body
and, should they find their way into human system chapter).
tissue ©through
Jonesa&puncture
Bartlettwound, the spores
Learning, LLC © Jones & Bartlett Learning, LLC
will germinate in the wound’s dead tissue and Optimal Microbial Growth Is
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, LLC cells produce dormancy,
the vegetative © Jones
let’s&examine
Bartlett theLearning, LLC
essential physical
NOT FOR SALE
deadly OR DISTRIBUTION
toxins. and NOT FOR
chemical SALE
factors inflOR DISTRIBUTION
uencing cell growth.
978128057782_CH05_140_171.indd 151 10/10/14 2:26 PM

Temperature.
NOT FOR SALE OR DISTRIBUTION Temperature is one
NOTofFOR Another
the SALE group of “cold-loving” microbes
OR DISTRIBUTION
most important factors governing growth. are the psychrotrophs or psychrotolerant
Every microbial species has an optimal microorganisms. These species have a slightly
growth temperature. Each species also has higher optimal growth temperature as well as
an approximate 30°C operating range, from a broader operating range. Psychrotrophs can
minimum to maximum, over which the cells be found in water and soil in temperate regions
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
mesophiles, LLC
or thermophiles—based © Jonesspecies,
on their eukaryotic & Bartlett Learning,
including LLC
fungi (molds).
NOT FORoptimal SALEgrowth temperature.
OR DISTRIBUTION When
NOT such
FOR contaminated
SALE ORfoods are consumed
DISTRIBUTION
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 about
LLC 70% of the Earth is covered © Jones by & identified cause
Bartlett Learning,of infective
LLC diarrhea (Clinical
NOT FOR SALE OR DISTRIBUTION oceans having deep-water temperatures NOTbelowFOR 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
low temperatures, Learning,
psychrophiles would not LLC be Thermophiles are present © Jones & Bartlett
in compost heapsLearning,
and LLC
human NOT FOR SALE
pathogens becauseOR DISTRIBUTION
they cannot grow at hot springs, and can NOT FOR SALE OR
be contaminants DISTRIBUTION
in dairy
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 well at the
© Jones & cooler
Bartletttemperature
Learning,of the
LLCbody.
NOT FOR SALE OR DISTRIBUTION Th ere also are many
NOT FOR SALE OR DISTRIBUTION archaeal species that
Archaea grow optimally at temperatures exceeding
Bacteria 80°C and having optima near 95°C. These
Eukarya hyperthermophiles have been isolated from
seawater near hot-water vents along rifts on
© Jones & Bartlett Learning, LLC Thermophiles © Jones & the Bartlett
floor ofLearning, LLC Because the high
the Pacific Ocean.
pressure keeps the water from boiling, some
Hyperthermophiles
Increasing rate of growth
Mesophiles archaeal species can grow at an astonishing

Psychrotrophs
121°C (see Table 5.1).
Psychrophiles Most of the best-characterized microbial
© Jones & Bartlett Learning, LLC species are mesophiles (meso =&“middle”),
© Jones Bartlettwhich
Learning, LLC
NOT FOR SALE OR DISTRIBUTION thrive at the middleNOT temperature
FOR SALE range OR
of 10°C
DISTRIBUTION
to 45°C. This includes the pathogens that grow
in warm-blooded animals, including humans, as
well as those species found in aquatic and soil
©0 Jones & Bartlett Learning, LLC80 90 100 110 environments © Jones &inBartlett
temperate and tropical regions
Learning, LLC
–10 10 20 30 40 50 60 70 of the world. E. coli is a typical mesophile. The
NOT FOR SALE Temperature OR DISTRIBUTION
(˚C) NOT FOR SALE OR DISTRIBUTION
vast majority of microbes discussed in this text
are mesophiles.
FIGURE 5.9 Growth Rates for Different Microorganisms in Response
to Temperature.Temperature optima and ranges define the growth rates for Oxygen. The 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,
© Jones
either&side
Bartlett Learning,
of the optimal LLC ❯❯ Propose what adaptations
growth temperature. © Jones are & and
Bartlett
in thisLearning,
respect, suchLLCobligate aerobes must
NOT FOR
neededSALE OR to
for microbes DISTRIBUTION
survive at the psychrophilic or thermophilicNOT FOR SALE
extremes. use theORgas asDISTRIBUTION
a final electron acceptor to make
978128057782_CH05_140_171.indd 152 10/10/14 2:26 PM

CLINICAL CASE 5
An Outbreak of Food Poisoning Caused by Campylobacter jejuni
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
© Jones &The
countertop. Bartlett
countertopLearning,
surface where LLC
he © Jones & Bartlett Learning, LLC
worked was unusually small.
3 For lunch, the cook prepared sandwiches on
the same countertop. Most were garnished with
lettuce.
4 Restaurant patrons enjoyed sandwiches for
lunch and LLC
roasted chicken for dinner. Many © Jones & Bartlett Learning, LLC
NOT FOR SALEpatrons
OR also
DISTRIBUTION
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 © Jones
Public health officials & Bartlett
learned that all the Learning, LLC © Jones & Bartlett Learning, LLC
affected patrons NOT
had eaten salad with lunch
FOR SALE 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.
© Jones
However, the & microbiologists
suspected the © of
cooked chicken was not the cause Jones & Rather,
the illness. Bartlett Learning, LLC
C. jejuni from the raw chicken
NOT FOR SALE OR 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?
B. Why was LLC
the raw chicken identified as the source? © Jones & Bartlett Learning, LLC
NOT FOR SALE OR
C. How, DISTRIBUTION
in fact, did the patrons become ill? NOT FOR SALE OR DISTRIBUTION
For additional information see http://www.cdc.gov/mmwr/preview/mmwrhtml/00051427.htm.
cellular energy (ATP).NOTOther
FORspecies,
SALE suchOR oxygen. Many bacterial and archaealNOT
as DISTRIBUTION 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. This means they need other
oxygen is relatively low. In the body, certain ways to make ATP. Some anaerobic bacterial
microaerophiles cause disease of the oral species use sulfur in their metabolic activities
cavity, urinary tract, and gastrointestinal tract. instead of oxygen, and therefore they produce
Conditions can be established in the laboratory hydrogen sulfide (H2S) rather than water (H2O)
to study these microbes ( FIGURE 5.10A ). as a waste product of their metabolism. Others we
The anaerobes, by contrast, are microbes have already encountered, such as the ruminant
© Jonesthat
& Bartlett
do not orLearning,
cannot useLLCoxygen. Some are archaeal © Jones & Bartlett
organisms Learning,
that produce LLCas
methane
NOT FORaerotolerant
, meaning they are insensitive to the NOT FORofSALE
byproduct OR DISTRIBUTION
the energy conversions. In
978128057782_CH05_140_171.indd 153 10/10/14 2:26 PM

Screw
Candle clamp
Gasket
© Jones & Bartlett HLearning,
2 + O2 LLC
Palladium
Cork
H2O catalyst NOT FOR SALE OR DISTRIBUTION
Hydrogen gas
generator
Liquid
media
in tubes
© Jones & Bartlett Learning, LLC Petri dishes
Solid media in inverted
petri dishes
(A) (B) (C)
© Jones & Bartlett

FIGURE Learning,
5.10 Bacterial LLC
Cultivation in Different Gas Environments. © Jones &ofBartlett
Two types Learning,
cultivation methods LLC
are shown for bacterial species
growing poorly in an oxygen-rich
NOT FOR SALE OR DISTRIBUTION environment. ( A) A candle jar, in which
NOT FOR SALE OR DISTRIBUTION where the
microaerophilic bacterial species grow in an atmosphere
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, ©
lifeJones & Bartlett
originated on Earth Learning, LLC
in an anaerobic A common way©toJones & Bartlett
test an organism’s Learning, LLC
oxygen
NOT FOR
environment SALE of
consisting OR DISTRIBUTION
methane and other sensitivity is to use NOT a FOR SALE OR
thioglycollate DISTRIBUTION
broth ,
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 in humans.
Bartlett Learning, LLC the Clostridium
For example, ©Finally,
Jonesthere are bacterial
& Bartlett species said
Learning, LLC to be
species mentioned
NOT FOR SALE OR DISTRIBUTION above causing tetanus and gas capnophilic ( capno = “smoke”);
NOT FOR SALE 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. The cytoplasm of most microorganisms
© Jones & Bartlett Learning, LLC © Jones & Bartlett
environment of an improperly sealed can of food, has a pH near Learning,
7.0. ThisLLCmeans the majority of
where it produces the lethal toxin of botulism. FOR SALE OR DISTRIBUTION
species are 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 presence
Bartlett of a catalyst
Learning, LLC to species, such as Vibrio© Jones cholerae, can tolerate
formNOT water,FOR
thereby
SALE creating an oxygen-free acidic conditions asNOT
OR DISTRIBUTION low asFORpH 2.0
SALEand OR
alkaline
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
presence of or a reduced concentration of oxygen. food and dairy industries. For example, certain
This group includes E. coli, many staphylococci species of Lactobacillus and Streptococcus
and streptococci, members of the genus Bacillus, produce the acid that converts milk to buttermilk
and the fungal yeasts. Thus, we often refer to these and cream to sour cream. These 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. The “active cultures” in a cup
© Jones & Bartlett Learning, LLC to anaerobic metabolism
but will switch © Jones
when & of
Bartlett Learning,
yogurt also LLC bacterial species.
are acidophilic
oxygen gas is absent. NOT FOR SALE ExtremeOR DISTRIBUTION
acidophiles , preferring pHs of 1 to 2,
978128057782_CH05_140_171.indd 154 10/10/14 2:26 PM

MICROFOCUS
MICROFOCUS 5.3: EVOLUTION
“It’s Not Toxic to Us!”
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 whiff by an organism and a rock-like looking structures are still found in a few places on
cascade © ofJones
highly & Bartlettcellular
destructive Learning, LLC
reactions was set into Earth, such© asJones
Shark Bay &offBartlett Learning,
the western LLC (see
coast of Australia
motion.NOT DeathFOR
followed quickly.
SALE OR DISTRIBUTION figure). These structures formed
NOT FOR SALE OR DISTRIBUTION from ocean sediments and cal-
Difficult 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 Learning,
energy. LLC And it stayed that way©forJones
But no oxygen. & oxygen
tolerate Bartlett andLearning, LLC
sunlight. Buried yet beneath these organ-
around 1 billion years. NOT FOR SALE OR DISTRIBUTION the anaerobic, dark
isms are other bacterial species surviving
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.
© Jones
the process was photosynthesis. But&there
Bartlett Learning,
was a downside to LLC © Jones & Bartlett Learning, LLC
photosynthesis: oxygen was NOT FOR SALE OR DISTRIBUTION
a waste product of the process—and 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© Jones & Bartlett
scarce. Then, about 2.4Learning, LLCthere was a
billion years ago, © Jones & Bartlett Learning, LLC
suddenNOT FOR SALE OR DISTRIBUTION
and dramatic increase in oxygen 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 theLearning, LLCtuck away oxygen atoms ©
enzymes to safely in aJones Stromatolites,
& BartlettShark Learning, LLC
Bay, Western Australia.
nontoxic form—that
NOT FOR SALE OR DISTRIBUTION form was water. NOT FOR© JonSALE ORInc. DISTRIBUTION
Nightingale/ShutterStock,
Type of Both aerobic Aerobic growth Aerobic growth Growth is Growth occurs
growth and anaerobiconly at low only in the insensitive to only in the
growth concentrations presence of O2 O2 absence of O2
© Jones of&OBartlett
2 Learning, LLC © Jones & Bartlett Learning, LLC
+O
©Bacterial
growth in
2
LLC © Jones & Bartlett Learning, LLC
NOT FOR SALE OR
thioglycollate –O2DISTRIBUTION NOT FOR SALE OR DISTRIBUTION
broth
(A) (B) (C) (D) (E)
FIGURE 5.11 The Effect of Oxygen on Microbial Growth. Each tube contains a thioglycollate broth into which
© Joneswas
& inoculated
Bartletta Learning, LLC
different bacterial © Jonesin &
species. ❯❯ Identify the O2 requirement Bartlett
each Learning,
thioglycollate tube basedLLC
on the
NOT FOR SALE
growth OR
density DISTRIBUTION
[example: NOT FOR SALE OR DISTRIBUTION
(A) represents facultative anaerobe species].
978128057782_CH05_140_171.indd 155 10/10/14 2:26 PM

are found
NOT FOR SALE OR DISTRIBUTION among the Archaea. At the opposite
NOT TheOR
FOR SALE fact DISTRIBUTION
that 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.
The majority of known bacterial species, FIGURE 5.12 summarizes the physical
however, do not grow well under acidic factors influencing microbial growth.
conditions. Thus, the acidic environment of the
NOThelps
stomach FOR SALE
deter OR while
disease, DISTRIBUTION
providing a NOT FOR SALE OR DISTRIBUTION
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
as tomatoes and many vegetables are hardly ever © each phase
Jones of the growth
& Bartlett Learning, curve.LLC
NOT FORcontaminated by bacterial growth. However,
SALE OR DISTRIBUTION b. Explain how the
NOT FOR SALE OR DISTRIBUTION trigger to persister
cell survival is different from the
such damaged produce may be subject to fungal
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, OtherLLC Factors. Further environmental
© Jones & Bartlett forLearning,
each of the physical
LLC factors
factors
NOT FOR SALE OR DISTRIBUTION can influence the growth of microbial described
NOT FOR SALE OR DISTRIBUTION in this section.
cells. Psychrophiles in deep ocean waters and
Hydrostatic
pressure: The
sediments are under extremely high hydrostatic
pressure exerted by pressure. In some deep marine trenches the
the weight of water. hydrostatic pressure can be as high as 16,000
pounds© Jones & Bartlett
per square Learning,
inch (psi) and only LLCsome
CHAPTER
© Jones CHALLENGE B
NOT FOR
extremophiles maySALE OR DISTRIBUTION
be adapted 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
Other microbes
© Jones & Bartlett Learning, haveLLC
adapted to saline or ©and you have
Jones &heard about many
Bartlett that can survive
Learning, LLC
highly saline environments such as the Great Salt these physical extremes (see Table 5.1).
Lake in Utah, the Dead Sea, and in evaporation Question B:
ponds. These 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. They include species, this Martian microbe be like? Here are some
© Jones & Bartlett Learning, LLC cholerae, that grow ©optimally
such as Vibrio Jones & Bartlett Learning,
useful facts about Mars. LLC
at 2% to 5% NaCl and other genera that NOT FOR SALE OR
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temperature: 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 at 20% to 30% NaCl.
Jones & Bartlett Learning, LLC In contrast, © Jones
and oxygen (0.13%; 21% on& Earth)
Bartlett
gases;Learning, LLC
species
NOTlikeFOR are nonhalophiles
E. coliSALE OR DISTRIBUTION because mean surface pressure is much
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they grow optimally at less than 2% NaCl and Earth’s. UV radiation is 3× Earth’s (note: there
are earthly bacterial species able to survive
genera, such as Staphylococcus, are halotolerant
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 =
as well as nonsaline environments. about 7.7;
© Jones & Bartlett Learning, LLC © Jones & several chemicals
Bartlett found that could
Learning, LLC
The hypersaline conditions can be deadly serve as nutrients for life forms, including
NOT FOR SALE OR DISTRIBUTION NOTmagnesium,
to microbial cells because water will be lost to potassium, and chloride.
the hypertonic external medium. To prevent Salt: Dark, finger-like features could be the flow
of salty water perhaps equivalent in salinity
the loss of cellular water, halophiles accumulate
to Earth’s oceans.
high cytoplasmic concentrations of solutes,
© Jones & Bartlett Learning, LLC amino acids, or ions, © AnswersLearning,
can be found inLLCAppendix F.
such as sugars, to Jones
balance & Bartlett
out the osmotic difference. NOT FOR SALE OR DISTRIBUTION
978128057782_CH05_140_171.indd 156 10/10/14 2:26 PM

MICROFOCUS
MICROFOCUS 5.4: Environmental Microbiology
Drilling for Microbes© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC
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© outpost
Jonessome & Bartlett Learning,
1,300 kilometers LLC
southeast © Jones
of the South water temperature of & Bartlett
–13°C was distinct Learning, LLC
from that in other
Pole. Many scientists believe Lake
NOT FOR SALE OR DISTRIBUTION Vostok, which is estimated saline lakes in the same area. And
NOT FOR SALE OR DISTRIBUTION yet another 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
there is the LLC
possibility of microbial life existing within© theJones
not as&weBartlett Learning,
know microbes will eke LLCout an existence anywhere an
ancient lake. NOT FOR SALE OR DISTRIBUTION
energy source is available.
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©itsJonesseason, &
theyBartlett Learning,
hit the lake! However, LLC © Jones & Bartlett Learning, LLC
when the drill contactedNOT FOR SALE OR DISTRIBUTION
the lake, the lake water, being 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
© Jones
cores from & Bartlett
frozen water Learning,
at the bottom LLC
(underside) of the glacier © Jones & Bartlett Learning, LLC
ice covering Lake Vostok. These cores 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 coresLearning,
identified moreLLCthan 3,500 unique sequences, © Jones & Bartlett Learning, LLC
which presumably
NOT FOR SALE OR DISTRIBUTIONequates 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
© Jones
of organisms that have evolved & Bartlett
and developed overLearning,
the tens of LLC © Jones & Bartlett Learning, LLC
millions of years of its existence. NOT
NOT FOR SALE OR DISTRIBUTION Courtesy of Michael Studinger/Lamont-Doherty Earth Observatory.
KEY CONCEPT 5.3

© Jones & Bartlett
Bacteria: © NIAID
Learning, LLC nutrients dissolved ©
in Jones
water. Aft
&erBartlett
sterilization
Learning, LLC
and specimen inoculation, the broth will
NOT Media
Culture FOR Are
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UsedOR DISTRIBUTION
to Grow 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, LLC
called broths , are contained in agar© Jones
. Agar, & Bartlett Learning,
a polysaccharide derived from LLCred
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tubes and OR DISTRIBUTION
bottles, and consist of the growth NOTcontains
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no nutrients, and like gelatin,
978128057782_CH05_140_171.indd 157 10/10/14 2:26 PM

PHYSICAL FACTORS FOR SALE OR DISTRIBUTION
CONTROLLING
MICROBIAL GROWTH
include
Temperature NOT FOR SALE OR DISTRIBUTION
Oxygen pH NOT FOR SALE
Osmotic OR DISTRIBUTION
conditions
which includes microbes microbes microbes

three groups growing tolerating requiring
microbes microbes not
preferring NaCl are NaCl
using O2 are using O2 are
Aerobic Anaerobic NOT FOR SALE OR DISTRIBUTION
Halotolerant
“Cold” “Hot” microbes

microbes microbes not
temperature temperature requiring sensitive tolerating
O2 are to O2 are NaCl are
“Warm” © Jones & Bartlett Learning, LLC
temperature
NOT FOR SALE OR DISTRIBUTIONObligate NOT FOR SALE OR DISTRIBUTION
Obligate
aerobes anaerobes Nonhalophiles
with temperature optima near
up to
microbes microbes 15% are
37°C requiring tolerating
reduced levels O2 are
Halophiles
of O2 are © Jones & Bartlett Learning, LLC
Mesophiles at
15–30%
Aerotolerant are
Extreme
halophiles
15°C 25°C
Microaerophiles
Psychrophiles Psychrotrophs
NOT FOR SALE OR DISTRIBUTION microbes
growing
with or without below near above
O2 are 5 7 9
60°C 95°C
Thermophiles Facultative
© Jones & Bartlett Learning,Hyperthermophiles
LLC © Jones &Acidophiles Neutrophiles LLC Alkaliphiles
Bartlett Learning,
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 and then
& Bartlett solidifies LLC
Learning, when
is, in nutrients designed
© Jonesto mimic the natural
cooled. AftFOR
NOT er sterilization
SALE OR of the nutrient agar
DISTRIBUTION environment. Today, NOTdiffFOR
erent SALE
cultureORmedia
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. After
(CML) and research lab because species vary
specimen inoculation, the specimen will growin their nutritional requirements and no single
as colonies (bacterial organisms and yeasts) or
medium will grow all microorganisms.
as filaments (molds) on the surface of the agar. For the isolation and identification of most
microorganisms, two types of culture media
Culture Media Are of Two Basic Types are commonly used. A chemically undefined
medium, called a complex medium, contains
Since the time of Pasteur and Koch, nutrients in which the exact components or
© Jones & Bartlett Learning, LLC have been growing bacterial
microbiologists © Jonesand & their
Bartlett Learning,
quantity LLC
is not completely known. Such
NOT FOR SALE OR DISTRIBUTION NOT FOR
other microbial species in artificial media; that SALE OR DISTRIBUTION
media typically contain animal or plant digests
978128057782_CH05_140_171.indd 158 10/10/14 2:26 PM

Culture Media Can Be Modified to Select For or Differentiate Between Microbial Species 159
NOT FOR SALE OR DISTRIBUTION precise
NOTchemical
FOR SALEcomposition and amount
OR DISTRIBUTION
Composition of a Complex and of all components are known ( TABLE 5.3B ).
TABLE 5.3
a Synthetic Growth Medium Chemically defined media are useful when
trying to determine an organism’s specific
Nutrient
growth requirements.
Ingredient © Jones
& Bartlett
Amount Learning, LLC
Supplied © 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 Differentiate Between
Microbial Species
Beef extract Vitamins, minerals, 3.0 g
© Jones &other
Bartlett Learning, LLC In the CML, the basic
nutrients © Jones & Bartlett
ingredients Learning, LLC
of growth
NOT
Sodium FOR SALE
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Sodium and 8.0 g NOT
media can be modified inFOR
one SALE
of threeOR DISTRIBUTION
ways
(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 © selective&medium
A Jones Bartlettcontains ingredients
Learning, LLC
B. Chemically Defined Broth Medium to inhibit the growth of certain microbes in
Glucose Simple sugar 5.0 g a mixture while allowing (selecting for) the
Ammonium Nitrogen, 1.0 g growth of others. The basic growth medium
phosphate phosphate may contain extra salt (NaCl) or a dye to inhibit
((NH4)2HPO4) the growth of intolerant or sensitive organisms
but permit the growth of those species or
Sodium chloride Sodium and 5.0 g
(NaCl) NOT
chloride FOR SALE OR DISTRIBUTION
ions pathogens one wants to isolate. NOT FOR SALE OR DISTRIBUTION
Magnesium sulfate Magnesium ions, 0.2 g Another modification to a basic growth
(MgSO4 · 7H2O) sulphur medium is the addition of one or more
Potassium Potassium ions, 1.0 g
ingredients that allow the observer to
© Jones & Bartlett
phosphate (K2HPO4) phosphate Learning, LLC differentiate between very &
© Jones similar species
NOT FOR SALE OR DISTRIBUTION growing in or on aNOT culture
FORmedium
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OR onDISTRIBUTION
Water 1.0 liter
specific biochemical or physiological properties.
This differential medium contains specific
(e.g., beef extract, soybean extract) or yeast chemicals to indicate which species possess and
extracts of an undefined nature ( TABLE 5.3A ). which lack a particular biochemical process.
Complex media are commonly used in the Such indicators make it easy to distinguish
teaching laboratory because most of the visually NOT FOR SALE
colonies OR DISTRIBUTION
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
The second type of medium is a chemically determine which medium was used in each
defined medium. In © Jones & BartletttheLearning,
this medium, example.LLC © Jones & Bartlett Learning, LLC
TABLE 5.4 A Comparison of Special Culture Media
Name© Jones & Bartlett

Components Learning, Uses
Examples
NOT
Selective FOR SALE
medium Growth OR DISTRIBUTION
stimulants NOT
Selecting certain microbes Mannitol salt FOR
agar forSALE OR DISTRIBUTION
staphylococci
Growth inhibitors out of a mixture
Differential medium Dyes, growth stimulants, Distinguishing different MacConkey agar for gram-negative
growth inhibitors microbes in a mixture bacteria
© Jones Enriched
medium LLC
Growth stimulants © Jones &
Cultivating fastidious Bartlett
Blood Learning,
agar for streptococci; LLC
chocolate
NOT FOR SALE OR DISTRIBUTION microbes agar for Neisseria species
978128057782_CH05_140_171.indd 159 10/10/14 2:26 PM

1
11
2 4
3
3 3
(A) (B)
FIGURE 5.13 Special Media Formulations. (A) Four different 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
© Jones
and incubated for 48&hours.
Bartlett Learning,
EMB allows LLC between human enteric bacteria,
one to distinguish © Jones & Bartlett
where Escherichia coli Learning, LLC
NOT
produces FOR
a green SALE
metallic sheenOR other enteric species produce a pink color. ❯❯ NOT
whileDISTRIBUTION FOR SALE
Which special medium is OR DISTRIBUTION
selective and which is differential? Explain your reasoning.
Courtesy of Dr. Jeffrey Pommerville.
MicroInquiry
NOT FOR SALE 5 presents another example
OR DISTRIBUTION areNOT
said FOR in a viable
to beSALE but noncultured
OR DISTRIBUTION
using these two approaches to identify and (VBNC) state. Procedures for identifying
differentiate between similar bacterial species. VBNC organisms include direct microscopic
Although many microorganisms grow well examination (see below) and, most commonly,
Fastidious: Having in nutrient broth and nutrient agar, certain amplification of diagnostic gene sequences or
so-called LLC
fastidious organisms may © Jones
require an & 16S
Bartlett
rRNA Learning, LLC
gene sequences.
special nutritional
NOT FOR SALE
requirements. OR DISTRIBUTION NOT
enriched medium containing extra vitamins, FOR SALE OR DISTRIBUTION
Why do 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. These species have
the addition
© Jonesof&powdered hemoglobin.LLC
Bartlett Learning, The adapted to their © own familiar
Jones and specifi
& Bartlett c
Learning, LLC
CentersNOTforFOR
Disease
SALE Control and Prevention environment; an artifi
OR DISTRIBUTION NOT cialFOR
medium
SALEis not
ORtheir
DISTRIBUTION
(CDC) often is involved with particularly usual home. Therefore, these species go into
vexing identifications 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
Identification of Nongrowing Microbes. cultured. Investigating the Microbial World 5
Many bacterial and archaeal species are examines this anomaly. Studies on VBNC
impossible to cultivate in any artificial culture bacterial and archaeal species present a vast and
medium yet devised. In fact, less than 2% of the as yet unexplored field (they are often 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 LLC
in an environment based©solelyJones on & also
Bartlett Learning,
to reveal LLC diversity in the
the tremendous
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organisms ORworld.
microbial DISTRIBUTION
978128057782_CH05_140_171.indd 160 10/10/14 2:26 PM

MICROINQUIRY
MICROINQUIRY 5
Identification of a Bacterial
© JonesSpecies
It often is necessary to identify a bacterial microorganisms (no carbon = no energy You can identify each bacterial
species or be able to tell the difference 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 & Bartlett
microbial ecology, itLearning,
might LLC © Jones
■ As an infectious disease officer in a lo- & Bartlett
adding salt andLearning,
mannitolLLC (mannitol
be necessary to isolate certain
NOT FOR SALE OR DISTRIBUTION naturally cal hospital, you routinely swabNOT criticalFOR SALE OR DISTRIBUTIONyou
salt broth) and phenol red. Next,
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
© Jonesaddition,
& Bartlett 5e. TheLLC broth tubes are shown be-
someLearning, LLC to
may be resistant © Jones
aureus (MRSA), & Bartlett
as it frequently can Learning, low. What do the results signify?
NOT FOR SALEantibiotics
standard OR DISTRIBUTION
normally used to cause diseaseNOT FOR inSALE
outbreaks a hospitalOR DISTRIBUTIONWhich 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 after 48 hours.
modifying the composition of a complex 1 2 3 4 5
or chemically defined growth medium. 5b. Knowing Staphylococcus spe-
Let’s go through two scenarios. cies are halotolerant,
LLC how could © Jones & Bartlett Learning, LLC
you devise an agar medium to
■ Suppose you are anNOT FOR SALE OR DISTRIBUTION
undergraduate 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 7 8 9 10
back in the lab, you want to grow only Again, a selective medium would
© Jones &marine
photosynthetic Bartlett Learning,
microbes. be LLC
used. It would be prepared by©adding Jones & Bartlett Learning, LLC
NOT
How FOR would SALEyou OR selectDISTRIBUTION
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 differential broth tubes.
energy source will be sunlight and not 5c. What would you expect to find
the organic compounds LLC found
typically © Jones & Bartlett Learning, LLC is an example of a
This method
on the agar plate after 48 hours?
NOT FOR SALE in cultureOR DISTRIBUTION
media (see Table 5.3). So, you NOT FOR SALE OR DISTRIBUTION differential medium because it allowed
would need to use a chemically-defined Your selective medium contained 10 you to visually differentiate 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 & Bartlett
would then Learning,
However, other speciesLLCof Staphylococcus you need © Jones & Bartlett
to determine which, if Learning,
any, are LLC
inoculate a sample NOT of the FOR SALE
collected do not
ORcause disease. One is S. epidermidis, resistant
DISTRIBUTION NOTto theFOR SALE
antibiotic OR DISTRIBUTION
methicillin.
material into a broth tube, place the a common skin bacterium. A Gram stain
5f. How could you design an agar
tube in the light, and incubate for one therefore is of no use to differentiate S.
medium to identify any MRSA
week at a temperature typical of where aureus from S. epidermidis.
colonies?
the organisms were collected. 5d. Knowing that only S. aureus will
5g. If theLearning,
plates are LLC devoid of
5a. What would you expect to find produce acid in the presence
NOT FOR SALE OR
in the broth tube after one
DISTRIBUTION of the sugar mannitol, how
NOT FOR SALE OR
growth, DISTRIBUTION
what can you conclude?
week’s incubation? could you design a differential Again, you have used a selective
broth medium to determine medium; the addition of methicillin will
What you have used in this scenario
is a selective medium; that is, one that will if any of the colonies are S. permit the growth of any MRSA bacteria
aureus© ? (Jones
Hint: phenol and suppress the growth of staphylococci
red is a Learning,
© Jonesencourage
the growth LLC
of photosynthetic & Bartlett LLC
pH indicator; it is red at neutral susceptible to methicillin.
microbes (light
NOT FOR SALE OR DISTRIBUTIONand sea salt) and sup- NOT FOR SALE OR DISTRIBUTION
pH and yellow at acid pH). Answers can be found in Appendix E.
press the growth of nonphotosynthetic
978128057782_CH05_140_171.indd 161 10/10/14 2:26 PM

MICROFOCUS
MICROFOCUS 5.5: Public Health
“Enriching” Koch’s Postulates

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 © theJones
meeting&was
Bartlett
ending,Learning, LLC who
several Legionnaires Today, we©know Jones & Bartlett
L. pneumophila Learning,
is found in manyLLC
aquatic
stayed at NOT
the hotelFOR SALE ofOR
complained DISTRIBUTION
flu-like symptoms. Four days environments,NOT both FOR naturalSALE OR DISTRIBUTION
and artificial. At the Bellevue
after 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 Bartlett
with any newLearning, LLC
disease, epidemiological studies look for© Jones
towers. & Bartlett Learning, LLC
NOT FOR SALE
the source ORdisease.
of the DISTRIBUTION
The Centers for Disease Control andNOT FOR Koch’sSALE postulates ORareDISTRIBUTION
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
staff collected tissues from lung biopsies and sputum samples.
However, no microbes could © be
Jones
detected& Bartlett
on slides ofLearning,
stained LLC © Jones & Bartlett Learning, LLC
material. By December 1976, NOT
they FOR
were no SALE
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to identifying 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 difficult to
culture on©bacteriological
media? Perhaps it was a LLC
virus. © Jones & Bartlett Learning, LLC
After NOT
trying FOR
17 diffSALE OR DISTRIBUTION
erent culture 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
© Jones
and & 1%Bartlett
isovitalex,Learning, LLC
small, barely visible colonies were seen© Jones & Bartlett Learning, LLC
after five
NOT FOR days of
SALE ORincubation at 37°C. Investigators then realizedNOT FOR SALE OR DISTRIBUTION
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,
© Jones
bacterial growth was significantly & Bartlett
enhanced Learning,
(see figure). From LLC © Jones & Bartlett Learning, LLC
a microscope examination NOT of these
FOR cultures,
SALE a gram-negative
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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.
978128057782_CH05_140_171.indd 162 10/10/14 2:26 PM

INVESTIGATING THE MICROBIAL WORLD 5
© Jones
The Great Plate Count & Bartlett Learning, LLC
Anomaly © Jones & Bartlett Learning, LLC
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© Jones
able &hundreds
to count BartlettofLearning,
cells and find LLC © Jones &OnBartlett
dozens of different bacterial morphologies. the plate,Learning, LLC
if you are lucky,
NOTone
maybe FOR SALE
or two ORwillDISTRIBUTION
colonies appear in a few days. The majority of theNOT FOR
cells will notSALE ORthough
grow even 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 Learning,
HYPOTHESIS: LLC
VBNCs need © Jones
some essential “nutrient” from & Bartlett
their neighbor speciesLearning,
in the naturalLLC
environment. If so,
then growing the VBNCs in their natural environment willNOT
supplyFOR SALE
the needed OR DISTRIBUTION
nutrient and the uncultured should grow.
■ EXPERIMENTAL DESIGN: A diffusion apparatus is designed to sandwich a microbial sediment sample in agar between
two semipermeable membranes that allow for the free diffusion of “nutrients” and waste products through the
chamber (see figure).
■ EXPERIMENT: Sediment © Jones & Bartlett
from a freshwater Learning,
pond LLCwith agar and placed within
sample is mixed © the
Jones & Bartlett
diffusion chamber. Learning, LLC
The chamber is then NOT FOR
placed backSALE
in its OR
natural environment. Another pond sediment NOT
DISTRIBUTION sampleFORfromSALE OR DISTRIBUTION
the same
environment is cultured on standard agar culture medium in a culture dish. Both the diffusion chamber culture and the
lab culture are left undisturbed for 4 weeks. After the incubation period, the colonies isolated on the culture plate and
on the agar in the diffusion 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.
■ RESULTS:
NOT FORSee Table
SALE .
OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION
■ CONCLUSIONS:
QUESTION 1: Was the hypothesis supported? Explain using the table.
QUESTION 2: Are the majority of the isolates representative of gram-positive or gram-negative organisms? Explain.
QUESTION 3: What might have been the result if the biofilm organisms growing on 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 diffusion chamber recognize? New
evidence suggests one© Jones
“nutrient”&isBartlett
iron, whichLearning,
is needed forLLC © Jonesprocesses.
ATP generation and other biochemical & BartlettTo Learning, LLC
grow, the unculturedNOT
must “capture” the iron (presumably
FOR SALE OR DISTRIBUTION from their neighbors) in the bound form that is otherwise
unavailable in a culture dish “environment.”
Diffusion chamber
Top view
Side view
Semipermeable Microbial sample

membrane in agar
Adapted from: Bollman, A., Lewis, K., and Epstein, S. S.
(2007) Appl. Environ. Microbiol. 73(20): 6386–6390.
NOT FOR SALE OR DISTRIBUTION . NOT FOR SALE OR DISTRIBUTION (Continues)
978128057782_CH05_140_171.indd 163 10/10/14 2:26 PM

(CONTINUED)
TABLE Phylogenetic
NOT FOR SALE Strains
ORObtained by Culture Dish and Diffusion-Chamber
DISTRIBUTION Methods
Number of Strains Isolated By
Diffusion Total number of
Phylum Culture dish only chamber only Both methods isolated strains
Alphaproteobacteria
NOT 20
FOR SALE OR DISTRIBUTION 36 6
NOT FOR SALE 62
OR DISTRIBUTION
Betaproteobacteria 3 63 3 69
Gammaproteobacteria 2 4 1 7
Deltaproteobacteria 1 1
Bacteroidetes 5
© Jones 6& Bartlett Learning,
1
LLC 12
Spirochaetes 4 4
Firmicutes 5 1 6
Actinobacteria 5 1 6
Total © Jones & Bartlett
40 Learning, LLC116 11 © Jones & Bartlett
167 Learning, LLC
Population
© Jones & Measurements
Bartlett Learning, LLC Are Made ©AJones
second,&more commonly
Bartlett used technique,
Learning, LLC
NOT FORUsing
SALEPure
OR DISTRIBUTION called the streak-plate method
NOT FOR SALE OR DISTRIBUTION , uses a single
Cultures 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: The
single species as Pasteur discovered in trying to a sterile loop using aseptic technique, and a series
practice of
transferring prove his germ theory of disease. Rather, they of streaks is made on the surface of one area of
© Jones & Bartlett
microorganisms
Learning,
are mixedLLC with other species, in a©so-called
Jones & the
Bartlett Learning,
plate. Th LLC touched to the first
e loop is flamed,
NOT FOR SALE OR
to a sterile culture DISTRIBUTION
“mixed culture”—most often as a biofilm. NOT FOR SALE
area, OR
and a DISTRIBUTION
second series is made in a second
medium without Therefore, 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. After a
organisms.
one species.
© Jones It was Koch’s ability
& Bartlett LLC 24 hour to 48 hour©
to pure culture
Learning, incubation,
Jones &discrete
Bartlettcolonies
Learning, LLC
pathogens that provided the proof
NOT FOR SALE OR DISTRIBUTION for the germ will be present on the plate ( FIGURE 5.15E ).
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: The
species, how can the organisms be isolated as original single cell that underwent numerous
process of
transferring©bacterial pure colonies? Two established methods are binary fissions. The researcher, medical
Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC
cells from one culture available. The first is the pour-plate method. technologist, or student can select samples of
NOT FORHere,
medium to another.
diluted samples of the mixed culture in theNOT FOR
colonies forSALE
further OR DISTRIBUTION
testing and subculturing.
molten nutrient agar tubes are each poured
Population Growth Can Be
into a sterile Petri plate and allowed to harden.
During a 24 hour to 48 hour incubation, the Measured in Several Ways
cells divide to form discrete colonies © Jones
on and & Microbial
Bartlett Learning,
growth in a LLCculture medium can be
NOT FOR SALE OR DISTRIBUTION within the agar ( FIGURE 5.14 ). NOT FOR SALE measured ORbyDISTRIBUTION
direct and indirect methods.
978128057782_CH05_140_171.indd 164 10/10/14 2:26 PM

Population Growth Can Be Measured in Several Ways 165
NOT FOR SALE OR DISTRIBUTION DirectFOR
NOT Counting
SALE Methods. There are
OR DISTRIBUTION
a number of ways to directly measure cell
numbers. Scientists may wish to perform a
direct microscopic count using a known
volume of the liquid sample that has been placed
on a specially designed counting chamber
( 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© Jones & Bartlett
of lactose Learning, LLC
broth tubes
NOT FOR SALE OR DISTRIBUTIONand the presence or absence
NOT FOR of gas formed
in fermentation gives a rough statistical
estimation of the cell number, referred to as
the most probable number. This technique has
© Jones & Bartlett Learning, LLC been©used for measuring
Jones & Bartlettwater quality. LLC
Learning,
NOT FOR SALE OR DISTRIBUTION In theFOR
NOT standard
SALE plate
OR count procedure,
DISTRIBUTION
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?
The assumption is that each cell will undergo
Courtesy of Dr. Jeffrey Pommerville. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC
(A) (B) Mixed culture (C)
First set
NOT FOR SALESecond
of streaks
set
of streaks
Third set
OR DISTRIBUTION
of streaks
Fourth set
NOT
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
isolation. LLC
❯❯ Justify the need to streak a mixed sample © over
Jones & Bartlett
four areas Learning,
on a culture plate. LLC
NOT FOR
CourtesySALE OR DISTRIBUTION
of James Gathany/CDC. NOT FOR SALE OR DISTRIBUTION
978128057782_CH05_140_171.indd 165 10/10/14 2:27 PM

Counting
NOT FOR SALE OR DISTRIBUTION chamber
NOT FORA SALE
Coverslip OR chamber
The counting DISTRIBUTION
is a specially
marked slide containing a grid of 25
large squares of known area. The total
volume of liquid held is 0.00002 ml
(2 x 10–5 ml).
Sample
NOT FOR SALE OR DISTRIBUTIONB The counting chamber is placed on
NOT FOR
the stage of a light microscope. SALE
OR DISTRIBUTION
The number
of cells are counted in several
of the large squares to determine the
average number.
One of the 25
large squares
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 of cells in aLLC
Learning, 10 ml sample. © Jones & Bartlett Learning, LLC
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©asJones & Bartlett
a single colony, Learning,
the standard plate LLC
count turbidity, in a broth©culture
Jonesusing& Bartlett Learning, LLC
a spectropho-
NOT FOR
is expressed SALE
as the number ORofDISTRIBUTION
colony-forming NOT FOR
tometer. This instrument detectsSALE OR DISTRIBUTION
the amount of
units (CFUs). After 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 ©theJones
cell number; therefore,
& Bartlett the more
Learning, LLCcells
NOT FOR SALE OR DISTRIBUTION present, the more light is scattered
NOT FOR SALE OR 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 & million
Bartlett Learning,
cells are neededLLC to make a reading on
the spectrophotometer, 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
Jonesmedia formulations
NOT FOR SALE OR DISTRIBUTION affecting the rate at NOT whichFORmicrobial
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
nutrients available in the environment, be it
the soil, a laboratory culture dish, or a human
NOT FOR SALE OR The DISTRIBUTION
Standard Plate Count. Individual NOT FOR SALE OR DISTRIBUTION
FIGURE 5.17 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 different parts
250 colonies, how many bacterial cells were in 10 ml of the body, the clinical microbiology lab may
of the original LLC broth culture? © Jones & haveBartlett
to useLearning, LLCmedia for isolation
different growth
© R.A. Longuehaye/Science Source. NOT FOR SALE OR cation.
and identifi DISTRIBUTION
This brings up an interesting
978128057782_CH05_140_171.indd 166 10/10/14 2:27 PM

Population Growth Can Be Measured in Several Ways 167
No NOT FOR SALE OR DISTRIBUTION
bacterial
cells
(A) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC
LightNOT FOR SALE OR DISTRIBUTION
Light NOT FOR SALE OR DISTRIBUTION
source detector
Bacterial
(B) cells
FIGURE 5.18 Using Turbidity to Measure Population Growth. (A) As 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. Jeffrey Pommerville. © Jones & Bartlett Learning, LLC
CHAPTER CHALLENGE
© Jones & Bartlett Learning, LLC C © Jones & Bartlett Learning, LLC
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
craft found no signs of biological material in the Martian soil. As more spacecraft have gone to Mars, the evidence
© has
for life on the Red Planet Jones & Bartlett
seesawed—yet Learning,
some are hopeful that LLC © Jones
microbial life may exist near the surface 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 types of experiments would you design (based on this chapter) to see if microbial life does exist on
© Jones
Mars? & Bartlett
The spacecraft Learning,
will not return LLC
to Earth, so © Jones
the experiments need to be completed & Red
on the Bartlett
Planet. Learning,
LLC
NOTcan
Answers FOR SALE
be found OR DISTRIBUTION
in Appendix F. NOT FOR SALE OR DISTRIBUTION
978128057782_CH05_140_171.indd 167 10/10/14 2:27 PM

NOT FOR SALE OR DISTRIBUTION NOT FOR SALE problemORwhen considering the diagnosis
DISTRIBUTION
CONCEPT AND REASONING CHECKS 3 Bacteria: © NIAID
and treatment of an infectious disease. Such
a. Compare and contrast complex and pathogens, including Treponema pallidum, have
chemically defined media. to be identified from actual clinical samples
b. Explain how microbiologists using direct microscopic observation. So, one
© Jones & Bartlett
have figured Learning,
that some 98% of LLC © Jones & Bartlett Learning, LLC
has to wonder how many human diseases
theFOR
NOT microbial
SALE world
ORhas not been
DISTRIBUTION NOTtoday
FORbecause
SALE they
OR DISTRIBUTION
“seen.”
may go undiagnosed are
c. Why might it be more difficult 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
d. Distinguish LLC
between direct and © Jones
cause disease,&yet
Bartlett
we know Learning, LLC
from microscopic
NOT FOR SALEindirect methods to measure
OR DISTRIBUTION observation
NOT FOR thatSALE
archaeal
OR organisms are present
DISTRIBUTION
population growth. in the human body. Could some of them be
VBNC pathogens?
SUMMARY
NOT FOROF KEY
SALE OR CONCEPTS
DISTRIBUTION NOT FOR SALE OR DISTRIBUTION
Bacteria: © NIAID
5.1 Microbial Reproduction Is Part of the Cell Cycle 5.3 Culture Media Are Used to Grow Microbes and
bacterial cell Measure Their Growth
1. The
© Jones & cycle involves
Bartlett cell growth, DNA
Learning, LLCreplication, © Jones & Bartlett Learning, LLC
and binary fission to produce genetically identical daughter 6. Complex and chemically defined media contain the
cells. (Fig. 5.2A)
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 differentiate between two similar
5.2 Microbial Growth Progresses Through Distinct species, or to enrich for species requiring special nutrients.
(Fig. 5.13)
© Jones &Phases
Bartlett Learning, LLC © Jones8. & Bartlett
Pure Learning,
cultures can be producedLLCfrom a mixed culture by the
3. The dynamics
NOT FOR SALE of the bacterial growth curve show how a NOT FORpour-plate
OR DISTRIBUTION SALE OR method or the streak-plate method. In both
DISTRIBUTION
cell population grows logarithmically, reaches a certain peak cases, discrete colonies can be identified that represent only
and levels off, 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, and salt concentration are physical
factors that influence© Jones
microbial & Bartlett
growth. Away from Learning,
the LLC plate count procedure. Indirect© Jonesmethods often use
turbidity measurements. (Fig. 5.18)
NOTslows
optimal condition, growth FOR SALE
within OR(Figs.
a set range. DISTRIBUTION
5.9, NOT FOR SALE OR DISTRIBUTION
5.11)
978128057782_CH05_140_171.indd 168 10/10/14 2:27 PM

Chapter Self-Test 169
CHAPTER SELF-TEST
Bacteria: © NIAID
For STEPS A–D, answers©

to Jones
questions&and
Bartlett
problemsLearning,
can be foundLLC
in Appendix D. © Jones & Bartlett Learning, LLC
STEP©A: REVIEW
Jones OF FACTS
& Bartlett AND LLC
Learning, TERMS © Jones & Bartlett Learning, LLC
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 O2; 30°C
best fits the question or statement. B. low O2; 20°C
© Jones &1.Bartlett
Which oneLearning, statements does NOT apply to © Jones & no
of the followingLLC C. O2; 30°C
bacterial reproduction? D. low O2; 37°C
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 © Jones
broth tube has&a Bartlett Learning,
generation time of LLC C. enriched © Jones & Bartlett Learning, LLC
40 minutes, how many cells will there be after 6 hours of optimal D. differential
growth? 7. A medium would involve the addition of the antibiotic
A. 18 streptomycin to identify streptomycin-resistant bacteria.
B. 64 A. differential
C. 128 B. selective
D. 512 C. thioglycollate
3. A bacterial species generation time would be determined during D. VBNC
the phase. 8. Which one of the following is NOT part of the streak-plate
A. decline method?
B. lag A. Making four sets of streaks on a plate
C. log B. Diluting a mixed culture in molten agar
D. stationary C. Using a mixed culture
D. Using Learning,
a sterilized loop LLC
4. Which one of the following is NOT an example of dormancy?
9. Direct methods to measure bacterial growth would include all
A. Log phase
B. Endospore formation the following except .
C. VBNCs A. total bacterial count
D. Persister cells B. direct microscopic count
C. turbidity measurements
© Jones & Bartlett Learning, LLC D. most probable number
978128057782_CH05_140_171.indd 169 10/10/14 2:27 PM

True–False
NOT FOR SALE OR DISTRIBUTION NOT14. In attempting
FOR SALE to culture a fastidious bacterial pathogen,
OR DISTRIBUTION
a differential 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. © Jones
Endospores are produced & Bartlett
by some Learning, LLC
gram-negative 17. Bacterial and archaeal©cells
Jones & Bartlett
lack a mitotic spindle toLearning, LLC
bacterial species. NOT FOR SALE OR DISTRIBUTION separate 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. © Jones
Halophiles &dominate
would Bartlett Learning,
in marine LLC
environments.
13. The majority
NOT FOR SALE of bacterial
OR andDISTRIBUTION
archaeal organisms can be NOT FOR SALE OR DISTRIBUTION
cultured in growth media.
NOT FOR
STEPSALE OR DISTRIBUTION
B: CONCEPT REVIEW NOT FOR SALE OR DISTRIBUTION
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)
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 DISTRIBUTION NOT
following terms. (Key Concept 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
©physical
these Jones & Bartlett
environments. (KeyLearning,
Concept 2) LLC © Jones & Bartlett Learning, LLC
NOT Colony-forming unit SALE
Most probable number test
25. Explain howFOR SALE
selective OR DISTRIBUTION
and differential media are each NOT FOR OR DISTRIBUTION
constructed. (Key Concept 3) Direct-counting method Oxygen uptake
Direct microscopic count Standard plate count
Turbidity
978128057782_CH05_140_171.indd 170 10/10/14 2:27 PM

Chapter Self-Test 171
STEP
NOT FOR C: OR
SALE APPLICATIONS
DISTRIBUTION AND PROBLEMSNOT FOR SALE OR DISTRIBUTION
28. Use the log phase growth curves (1, 2, or 3) below to answer
29. Consumers are advised to avoid stuffing 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 &mainly
grow Bartlett
at warmLearning, LLC
Growth
curves NOT FOR SALE1 OR DISTRIBUTION temperatures, not in the NOT FOR SALE ORmight
refrigerator. What explanation DISTRIBUTION
you offer to counter this argument?
Number of Cells
30. Public health officials found the water in a Midwestern town

was contaminated with sewage bacteria. The officials suggested
2 homeowners boil their water for a couple of minutes before
© Jones & Bartlett Learning, LLC © it.
drinking Jones
(a) Would &this
Bartlett
treatmentLearning, LLC
sterilize the water? Why? (b)
Is it important for the water to be sterile? Explain.
3
Time (min)

a. Which curve LLC
(1, 2, or 3) best represents the growth curve for©
a Jones & Bartlett Learning, LLC
NOT FOR SALE ORincubated
mesophile DISTRIBUTIONat 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,
STEP D: QUESTIONS FOR THOUGHT AND DISCUSSION

31. To
©prevent
Jones decay by bacterial species
& Bartlett and to display
Learning, LLCthe 33. During©the filming of
Jones &the 1997 movieLearning,
Bartlett Titanic, researchers
LLC
mummified remains of ancient peoples, museum officials place discovered at least 20 different bacterial 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
at temperatures of 100°C and pH levels of 10 (the enzymes have 34. Every year news media report cases 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
foresee for these enzymes? NOT FOR SALE
hot tubs? OR DISTRIBUTION
978128057782_CH05_140_171.indd 171 10/10/14 2:27 PM

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5.1 Microbial Reproduction Is Part

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Microbial Growth and Nutrition 141

Hottest environment (Juan de Fuca ridge)—121°C: Strain 121 (Archaea)

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142 Chapter 5: Microbial Growth and Nutrition

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Bacterial and Archaeal Cells Can Grow Exponentially 143

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144 Chapter 5: Microbial Growth and Nutrition

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Bacterial and Archaeal Cells Can Grow Exponentially 145

Actual number of bacterial cells

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146 Chapter 5: Microbial Growth and Nutrition

© Jones & Bartlett10Learning, LLC © Jones & Bartlett Learning, LLC

Few cells Viable cells Nonviable/dead cells

© Jones & Bartlett Learning,

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Some Bacterial Cells Can Exist In a Metabolically Inactive State 147

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148 Chapter 5: Microbial Growth and Nutrition

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Some Bacterial Cells Can Exist In a Metabolically Inactive State 149

© Jones & Bartlett Learning, LLC

© Jones & Bartlett Learning, LLC © Jones

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150 Chapter 5: Microbial Growth and Nutrition

D Germination. A Asymmetric cell division.

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Optimal Microbial Growth Is Dependent on Several Physical Factors 151

Germination of 25 Million-Year-Old Endospores?

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Mesophiles archaeal species can grow at an astonishing

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

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KEY CONCEPT 5.3

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which includes microbes microbes microbes

“Cold” “Hot” microbes

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TABLE 5.4 A Comparison of Special Culture Media

Name© Jones & Bartlett

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“Enriching” Koch’s Postulates