Brock Biology of Microorganisms

Sixteenth Edition, Global Edition

Chapter 4
Microbial Growth and Its
Control

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I. Culturing Microbes and Measuring Their
One.

Growth
4.1 Feeding the Microbe: Cell Nutrition
4.2 Growth Media and Laboratory Culture
4.3 Microscopic Counts of Microbial Cell Numbers
4.4 Viable Counting of Microbial Cell Numbers
4.5 Turbidimetric Measures of Microbial Cell Numbers

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4.1 Feeding the Microbe: Cell Nutrition (1 of 8)

• Nutrients
– Supply of elements required by cells for growth
• Macronutrients
– Nutrients required in large amounts
• Micronutrients
– Nutrients required in minute amounts
– Trace metals and growth factors

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4.1 Feeding the Microbe: Cell Nutrition (2 of 8)

• Chemical Makeup of a Cell

– Handful of elements dominate living systems
– C, O, N, H, P, S are ~96% of dry weight of bacterial
cell (Figure 4.1) and required by all life
– K, Na, Ca, Mg, Cl, Fe ~3.7% of dry weight
– 62 total elements can be metabolized
– Macromolecules (proteins, lipids, polysaccharides,
lipopolysaccharides, nucleic acids) ~96% of dry
weight
▪ Mostly proteins and RNA, not DNA

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Figure 4.1 Elemental and Macromolecular
Composition of a Bacterial Cell

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4.1 Feeding the Microbe: Cell Nutrition (3 of 8)

• Carbon, Nitrogen, and Other Macronutrients

– Heterotrophs (require organic carbon (C)) obtain C
from breakdown of organic polymers or uptake of
monomers (amino acids, fatty acids, organic acids,
sugars, nitrogen bases, other organics)
– Autotrophs synthesize organics from carbon dioxide
(CO2)

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4.1 Feeding the Microbe: Cell Nutrition (4 of 8)

• Carbon, Nitrogen, and Other Macronutrients

– Nitrogen (N): mostly proteins, ammonia (NH3), nitrate
(NO3−), or nitrogen gas (N2).
▪ Nearly all microbes can use NH3.

▪ many use nitrate (NO3 )

−

▪ some use organics (e.g., amino acids) or N2

(nitrogen-fixing)
– Oxygen (O) and hydrogen (H): from water

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4.1 Feeding the Microbe: Cell Nutrition (5 of 8)

• Carbon, Nitrogen, and Other Macronutrients

– Phosphorus (P)
▪ nucleic acids and phospholipids

▪ usually inorganic phosphate (PO4

3−)

– Sulfur (S)
▪ sulfur-containing amino acids (cysteine and

methionine)
▪ vitamins (e.g., thiamine, biotin, lipoic acid)

▪ microbes assimilate sulfate (SO4

−2), sulfide (H S),
2
or organics

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4.1 Feeding the Microbe: Cell Nutrition (6 of 8)

• Carbon, Nitrogen, and Other Macronutrients

– Potassium (K)
▪ required by several enzymes

– Magnesium (Mg)
▪ stabilizes ribosomes, membranes, and nucleic acids

▪ also required by many enzymes

– Calcium (Ca) and sodium (Na)

▪ required by some microbes (e.g., marine microbes)

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4.1 Feeding the Microbe: Cell Nutrition (7 of 8)

• Micronutrients: Trace Metals and Growth Factors

– Many enzymes require metal ion or small organic as a
cofactor for catalysis
– Iron (Fe)
▪ cellular respiration, related oxidation-reduction

reactions
– Trace metals (Table 3.1): required in small amounts

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Table 4.1 Micronutrients Needed by
Microorganismsa (1 of 2)
I. Trace elements
Element Function

Boron (B) Autoinducer for quorum sensing in bacteria; also found in some polyketide antibiotics

Cobalt (Co) Vitamin B12; transcarboxylase (only in propionic acid bacteria)

In respiration, cytochrome c oxidase; in photosynthesis, plastocyanin, some superoxide

Copper (Cu)
dismutases

Iron (Fe)b Cytochromes; catalases; peroxidases; iron–sulfur proteins; oxygenases; all nitrogenases
Activator of many enzymes; component of certain superoxide dismutases and of the water-
Manganese (Mn)
II )
splitting enzyme in oxygenic phototrophs (photosystem two
Certain flavin-containing enzymes; some nitrogenases, nitrate reductases, sulfite oxidases, DMS
Molybdenum (Mo)
O–TMAO reductases; some formate dehydrogenases

Nickel (Ni) Most hydrogenases; coenzyme F 430 of methanogens; carbon monoxide dehydrogenase; urease

Selenium (Se) Formate dehydrogenase; some hydrogenases; the amino acid selenocysteine

Tungsten (W) Some formate dehydrogenases; oxotransferases of hyperthermophiles

Vanadium (V) Vanadium nitrogenase; bromoperoxidase

Zinc (Zn) Carbonic anhydrase; nucleic acid polymerases; many DNA-binding proteins

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Table 4.1 Micronutrients Needed by
Microorganismsa (2 of 2)
II. Growth factors
Growth factor Function
PABA (p-aminobenzoic acid) Precursor of folic acid
Folic acid One-carbon metabolism; methyl transfers
Biotin Fatty acid biosynthesis; some CO2 fixation reactions
B12 (Cobalamin) One-carbon metabolism; synthesis of deoxyribose
B1 (Thiamine) Decarboxylation reactions
B6 (Pyridoxine) Amino acid/keto acid transformations
Nicotinic acid (Niacin) Precursor of NAD+
Riboflavin Precursor of FMN, FAD
Pantothenic acid Precursor of coenzyme A
Lipoic acid Decarboxylation of pyruvate and α-ketoglutarate
Vitamin K Electron transport
Coenzymes M and B Methanogenesisc
F420 and F430 Methanogenesisc
aNot all trace elements or growth factors are needed by all organisms, and many growth factors are biosynthesized and
not required from the environment.
bIron is typically needed in larger amounts than the other trace metals shown.
cThe production of methane (CH4) by methanogens (Archaea).

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4.1 Feeding the Microbe: Cell Nutrition (8 of 8)

• Micronutrients: Trace Metals and Growth Factors

– Growth factors: organic micronutrients
▪ vitamins (Table 4.1)

– Most function as coenzymes.

– Most frequently required growth factors
▪ others: amino acids, purines, pyrimidines, other

organics

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4.2 Growth Media and Laboratory
Culture (1 of 5)
• Culture media (Table 4.2)
– Nutrient solutions used to grow microbes in the
laboratory
– Typically sterilized in an autoclave

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Table 4.2 Examples of Culture Media for
Microorganisms with Simple and Demanding
Nutritional Requirementsa

aThe photos are tubes of (a) the defined medium for Escherichia coli, and (b) the complex medium described. Note how
the complex medium is colored from the various organic extracts and digests that it contains. Photo credits: Cheryl L.
Broadie and John Vercillo, Southern Illinois University at Carbondale.

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4.2 Growth Media and Laboratory
Culture (2 of 5)
• Classes of Culture Media
– Defined media: exact chemical composition known
– Complex media: composed of digests of microbial, animal,
or plant products (e.g., yeast and meat extracts)
– Selective medium
▪ contains compounds that selectively inhibit growth of
some microbes but not others
– Differential medium
▪ contains an indicator, usually a dye, that detects

particular metabolic reactions during growth

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4.2 Growth Media and Laboratory
Culture (3 of 5)
• Nutritional Requirements and Biosynthetic Capacity
– Different microorganisms may have vastly different
nutritional requirements
– Necessary to understand physiology & nutritional
requirements and supply with nutrients in proper form
and amount

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4.2 Growth Media and Laboratory
Culture (4 of 5)
• Laboratory Culture
– Either liquid or solid (Figure 4.2)
▪ Solid media are prepared by addition of the gelling

agent agar to liquid media.

▪ When grown on solid media, cells form isolated

masses (colonies).
▪ Colony morphology (visible characteristics)

– Sometimes used to identify microorganisms

– Routinely used to determine if a culture is pure
(one microbe), contaminated, or mixed

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Figure 4.2 Bacterial Colonies

(a) Serratia marcescens, grown on MacConkey

agar.
(b) Close-up of colonies outlined in part a.
(c) Pseudomonas aeruginosa, grown on
trypticase soy agar.
(d) Shigella flexneri, grown on MacConkey
agar.
(e) An agar plate containing many different
bacterial colonies that developed from
plating a dilution of seawater.

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4.2 Growth Media and Laboratory
Culture (5 of 5)
• Laboratory Culture
– Aseptic technique (transfer without contamination)
(Figure 4.3)
– Airborne contaminants everywhere
– Pure cultures containing a single microbe usually
require streak plate technique with inoculating loop

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Figure 4.3 Aseptic Technique

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4.3 Microscopic Counts of Microbial
Cell Numbers (1 of 2)
• Total Cell Count
– Microscopic cell count: observing and enumerating
cells present
– Dried on slides or on liquid samples
– Counting chambers with squares etched on a slide
used for liquid samples (Figure 4.4)
– Several limitations

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Figure 4.4 Direct Microscopic Counting Procedure
Using the Petroff–Hausser Counting Chamber

• Space between coverslip and slide is 0.02 millimeters.

• The whole grid has 25 large squares, a total area of 1 square millimeter and a total volume of 0.02
cubic millimeters.
• Microscopic observation, all cells are counted in a large square, made of 16 small squares
• There are 12 cells in the example.
• In practice, several large squares are counted and the numbers are averaged. To calculate the number
per milliliter of sample, the equation is 12 cells times 25 large squares, times 50, times 10 cubed.

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4.3 Microscopic Counts of Microbial
Cell Numbers (2 of 2)
• Microscopic cell counts in microbial ecology
– Often used on natural samples
– Use stains to visualize and provide phylogenetic or
metabolic properties
– e.g., DAPI (4′,6-diamidino-2-phenylindole) binds DNA.
– Other fluorescent stains differentiate live and dead
cells.
– Phylogenetic stains can determine proportions of
Bacteria and Archaea in a sample.

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4.4 Viable Counting of Microbial Cell
Numbers (1 of 4)
• Viable (alive/living) counts: measurement of living,
reproducing population
– Two main ways to perform viable (plate) counts
(Figure 4.5):
▪ spread-plate method

▪ pour-plate method

– Count colonies on plates with 30–300 colonies

– Reporting in colony-forming units instead of
number of viable cells accounts for clumps.

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Figure 4.5 Two Methods for the Viable
Count

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4.4 Viable Counting of Microbial Cell
Numbers (2 of 4)
• Diluting a Sample
– Samples often contain thousands, millions, billions of
viable cells
– Ten-fold dilutions commonly used (Figure 4.6)
– Serial (successive) dilutions needed for dense cultures

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Figure 4.6 Procedure for Viable Counting Using
Serial Dilutions of the Sample and the Pour-Plate
Method

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4.4 Viable Counting of Microbial Cell
Numbers (3 of 4)
• Applications of the Plate Count
– quick and easy
– used in food, dairy, medical, and aquatic microbiology
– high sensitivity
– can target particular species in mixed samples (e.g.,
Staphylococcus)
– common in wastewater and other water analyses

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4.4 Viable Counting of Microbial Cell
Numbers (4 of 4)
• “The great plate count anomaly”: Direct microscopic counts
of natural samples reveal far more organisms than those
recoverable on plates.
• Why is this?
– Different organisms may have vastly different
requirements for growth.
– May underestimate actual by one to several orders of
magnitude

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4.5 Turbidimetric Measures of Microbial
Cell Numbers (1 of 3)
• Cell suspensions are turbid (cloudy) because cells
scatter light.
• More cells, more light scattered, more turbid suspension
• Turbidity measurements are rapid, widely used for
estimates

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4.5 Turbidimetric Measures of Microbial
Cell Numbers (2 of 3)
• Optical Density and Its Relationship to Cell Numbers
– Measured with a spectrophotometer (Figure 4.7)
– Unit is optical density (OD) at specified wavelength
(e.g., OD540 for measurements at 540 nm [green light]).
– For unicellular organisms, OD is proportional to cell
number within limits.
– To relate a direct cell count to a turbidity value, a
standard curve must first be established.

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Figure 4.7 Turbidity Measurements of
Microbial Growth

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4.5 Turbidimetric Measures of Microbial
Cell Numbers (3 of 3)
• Other Issues with Turbidimetric Growth Estimates
– quick and easy to perform
– typically do not require destruction or significant
disturbance of sample
– Same sample can be checked repeatedly.
– sometimes problematic (e.g., microbes that form
clumps or biofilms in liquid medium)

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II. Dynamics of Microbial Growth
Two

4.6 Binary Fission and the Microbial Growth Cycle

4.7 Quantitative Aspects of Microbial Growth
4.8 Continuous Culture
4.9 Biofilm Growth
4.10 Alternatives to Binary Fission

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4.6 Binary Fission and the Microbial
Growth Cycle (1 of 4)
• Growth: increase in the number of cells
• Binary fission: cell division following enlargement of a cell to twice
original size (Figure 4.8)
• Septum: partition between dividing cells, pinches off between two
daughter cells (Figure 4.9)
• Generation (doubling) time: time required for microbial cells to
double in number
– Differs for microbes and varies depending on conditions
– example: Escherichia coli = 20 minutes
• During cell division, each daughter cell receives a chromosome and
sufficient copies of all other cell constituents to exist as an
independent cell.

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Figure 4.8 Binary Fission in a
Rod-Shaped Bacterium

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Figure 4.9 Septa

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Bacterial Growth: Overview

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Binary Fission

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4.6 Binary Fission and the Microbial
Growth Cycle (2 of 4)
• The Microbial Growth Cycle
– Batch culture: a closed-system microbial culture of
fixed volume
– Typical growth curve for population of cells grown in
a closed system is characterized by four phases.
(Figure 4.10)
▪ lag phase

▪ exponential phase

▪ stationary phase

▪ death phase

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Figure 4.10 Typical Growth Curve for a
Bacterial Population

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Bacterial Growth Curve

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4.6 Binary Fission and the Microbial
Growth Cycle (3 of 4)
• Lag and Exponential Phases
– Lag phase: interval between inoculation of a culture and
beginning of growth
▪ new conditions require altering metabolic state
▪ time needed for biosynthesis of new enzymes and to produce
required metabolites before growth can begin
– Exponential phase (balanced growth): doubling at regular
intervals
▪ close to metabolically identical
▪ rates vary greatly, influenced by media, conditions, organism
itself
▪ continues until conditions can no longer sustain growth

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4.6 Binary Fission and the Microbial
Growth Cycle (4 of 4)
• Stationary and Death Phases
– Growth limited by nutrient depletion or waste
accumulation
– Stationary phase: growth rate of population is zero
– Metabolism continues at greatly reduced rate
– Decline phase: total number decreases due to cell
death
– Cryptic growth: subpopulations adapt

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4.7 Quantitative Aspects of Microbial
Growth (1 of 3)
• Plotting Growth Data
– Exponential growth: cell numbers double at regular
intervals (Figure 4.11)
▪ Semilogarithmic relationship

– Generation time (g) (Figure 4.12) of the exponentially

growing population is:
g =t /n
▪ t is the duration of exponential growth
(days/hours/minutes)
▪ n is the number of generations during the period of
exponential growth
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 Figure 4.11 The Rate of Growth of a
Microbial Culture
(a) Data for a cell population that
doubles every 30 min. (b) Data plotted
on arithmetic (left ordinate) and
logarithmic (right ordinate) scales.

The data for a cell population that

doubles every 30 minutes.

Part B. A graph that plots number of

cells on an arithmetic scale versus time
in hours.
• A logarithmic plot increases by a
factor of 10 every hour and a half
• Arithmetic plot starts out slowly but
rises with increasing steepness,
surpassing the logarithmic scale at
about 5 hours.
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4.7 Quantitative Aspects of Microbial
Growth (2 of 3)
• The Mathematics of Bacterial Growth
– A relationship exists between the initial number of cells
present in a culture and the number present after a
period of exponential growth:

Nt = N0 2 n

▪ Nt is cell number at time t

▪ N0 is the initial cell number (time 0)
▪ n is the number of generations during the period of
exponential growth.

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Figure 4.12 Calculating Microbial
Growth Parameters

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4.7 Quantitative Aspects of Microbial
Growth (3 of 3)
• Specific Growth Rate
– Specific growth rate (k) expresses rate of growth at
any instant.
– Specific growth rate (k) is calculated as

k = 0.693 / g

• Consequences of Exponential Growth

– Initial increase is slow but increases, resulting in huge
increase in cell numbers (e.g., lactic acid bacteria
spoiling milk).

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4.8 Continuous Culture
• Continuous culture: an open system
• Chemostat: most common type of continuous culture
device (Figure 4.13); known volume added while spent
medium is removed at same rate
• Steady state: cell density and substrate concentration do
not change over time

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Figure 4.13 Continuous Culture Device
(Chemostat)

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4.8 Continuous Culture
• The Chemostat and the Concept of Steady State
– Controls both specific growth rate (k) and yield (biomass/ml)
(Figures 4.14 and 4.15)
– Both growth rate and population density of culture can be
controlled independently and simultaneously, depending on:
▪ Dilution rate (D): F / V (F is flow rate of adding fresh
medium and removing spent medium, V is culture
volume.)
▪ Growth yield controlled by concentration of a limiting
nutrient.

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Figure 4.14 Steady-State Relationships
in the Chemostat
Dilution Rate (x-axis): This refers to how fast
fresh medium (with nutrients) is added to the
system and how fast the culture volume is
removed. It’s measured in h−1, representing how
many times per hour the culture is "replaced"
with fresh medium.

Bacterial Biomass (green line):

• This is the concentration of bacterial cells (in

grams per liter, g/L) in the culture. • However, as the dilution rate
increases (to the right of the graph),
• It remains constant at lower dilution rates the biomass starts to decrease
because the bacteria can divide and grow sharply after a certain point, leading
effectively given the nutrient supply. to washout (the bacteria cannot
reproduce fast enough to replace
cells being removed).

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4.8 Continuous Culture
Specific Growth Rate (red line):

• This is the rate at which the

bacteria grow and divide.
• It increases with the dilution
rate because as more Limiting Nutrient (blue line):
nutrients enter the system, the
bacteria grow faster. • This represents the concentration of the nutrient
that limits bacterial growth (often something like
• But, after a certain point, even glucose, nitrogen, or another essential factor).
if more fresh medium is • Initially, this nutrient is used up quickly, so its
added, the growth rate concentration stays low.
plateaus. • But as the dilution rate increases, the nutrient
concentration begins to rise because bacteria
can’t consume it fast enough, especially after
they start washing out of the system.

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 4.8 Continuous Culture
Key Phases on the Graph:

• Low Dilution Rate (left side of the graph): The bacteria have enough time to consume
the limiting nutrient, so bacterial biomass stays high, and the specific growth rate
increases gradually.

• Intermediate Dilution Rate: The bacteria are growing at their optimal rate because they
have enough nutrients and time to reproduce. The bacterial biomass remains stable.

• High Dilution Rate (right side of the graph): At high dilution rates, the bacteria cannot
grow fast enough to replace those being washed out of the system. The biomass
declines rapidly, and eventually, the system reaches a point where all the bacteria are
washed out (no biomass remains). This is the washout point.

• Washout (indicated by the red arrow): This occurs when the dilution rate exceeds the
bacteria’s growth rate. The cells are flushed out of the system faster than they can divide
and grow, resulting in no bacterial biomass remaining in the system.

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4.8 Continuous Culture
• The relationship between growth rate (green line), growth
yield (red line), and nutrient concentration in a bacterial
culture.
• This kind of graph is used to illustrate how bacteria
respond to varying amounts of nutrients in their
environment.

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4.8 Continuous Culture
• Nutrient Concentration (x-axis): This represents the
amount of nutrients available to the bacteria, measured in
(mg/ml).
• As nutrient levels increase, we observe changes in both
the growth rate and the growth yield of the bacteria.

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4.8 Continuous Culture
• Growth Rate (green line, left y-axis): The rate at which
bacterial cells are growing and dividing.
• Initially, as nutrient concentration increases, the growth
rate increases quickly because bacteria have more
resources to divide.
• However, at a certain point, the growth rate levels off, and
adding more nutrients doesn’t increase how fast the
bacteria grow.

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4.8 Continuous Culture
• Growth Yield (red line, right y-axis): This refers to the
total amount of biomass (bacterial cells) produced.
• Unlike the growth rate, the yield continues to increase with
higher nutrient concentrations, even after the growth rate
has plateaued.

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4.8 Continuous Culture
• Experimental Uses of the Chemostat
– can maintain exponential growth phase for
weeks/months
– used to study physiology, microbial ecology and
evolution, enrichment and isolation of bacteria from
nature

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4.9 Biofilm Growth (1 of 3)
• Planktonic growth: growth in suspension of free-
floating/free-swimming cells
• Sessile growth: attached to surface
– can develop into biofilms
▪ Important in medical and industrial applications

▪ Different properties than planktonic cells

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4.9 Biofilm Growth (2 of 3)
• Biofilm: cells enmeshed polysaccharide matrix attached
to surface (Figure 4.16)
– Form in stages:
▪ Planktonic cells attach (flagella, fimbriae, pili)

▪ Colonization: growth and extracellular

polysaccharide (EPS) production

▪ Development: metabolic changes

▪ Dispersal: colonize new sites

– Can study in flow chamber

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Figure 4.16 Pseudomonas Aeruginosa
Biofilm Development

Confocal scanning laser micrographs of a developing Pseudomonas

aeruginosa biofilm in a flow cell continuously irrigated with nutrient-rich medium.
P. aeruginosa cells first attach to the glass surface (day 0), then rapidly grow and
move on the surface to cover the entire surface (day 1); by day 4 mushroom-
shaped microcolonies over 0.1 mm high have developed.

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4.9 Biofilm Growth (3 of 3)
• Biofilms and Humans
– Microbial mats: multilayered sheets with different
organisms in each layer (e.g., hot springs, intertidal
regions) (Figure 4.17a)
– Implicated in joint infections, implanted medical
devices
– Responsible for cavities and cause gum disease
– Foul, plug, and corrode pipes
– Form in fuel tanks and on ship hulls

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Figure 4.17 Examples of Biofilms
(a) A microbial mat of the
purple phototrophic
bacterium
Thermochromatium
tepidum that developed in
a small sulfidic hot spring
in Yellowstone National
Park (U S A).

(b)Scanning electron
micrograph of a biofilm of
cells of Staphylococcus
aureus that formed on an
indwelling catheter.

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4.10 Alternatives to Binary Fission (1 of 2)
• Balanced growth: result of binary fission, producing
nearly identical cells
• Budding cell division (Figure 4.18)
– Unequal cell growth forming different daughter cells.
– Some budding bacteria form cytoplasmic extensions
such as stalks (Caulobacter and Hyphomicrobium)
and appendages (Ancalomicrobium).

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Three Environmental Effects on Growth:
III.
Temperature
4.11 Temperature Classes of Microorganisms
4.12 Microbial Life in the Cold
4.13 Microbial Life at High Temperatures

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4.11 Temperature Classes of
Microorganisms (1 of 2)
• Cardinal Temperatures
– Minimum, optimum, and maximum temperatures at
which an organism grows (Figure 4.20)
– Characteristic of any given microorganism
– Differ dramatically between species
– Range is typically < 40°C
– At optimum, all/most cellular components are
functioning at maximum rate

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Figure 4.20 The Cardinal Temperatures:
Minimum, Optimum, and Maximum

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4.11 Temperature Classes of
Microorganisms (2 of 2)
• Temperature Classes of Organisms
– Four broad classes related to growth temperature
optima (Figure 4.21)
▪ psychrophile: low, found in cold environments

▪ mesophile: midrange, most commonly studied

▪ thermophile: high, found in hot environments

▪ hyperthermophile: very high, found in extremely

hot habitats such as hot springs and deep-sea

hydrothermal vents

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Figure 4.21 Temperature and Growth Response in
Different Temperature Classes of Microorganisms

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IV. Environmental Effects on Growth: pH,
Four

Osmolarity, and Oxygen

4.14 Effects of pH on Microbial Growth
4.15 Osmolarity and Microbial Growth
4.16 Oxygen and Microbial Growth

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4.14 Effects of pH on Microbial
Growth
• pH expresses acidity or alkalinity of a solution.
• pH 7 = neutral (Figure 4.26)
• acidic pH < 7, alkaline pH > 7
• Each microbe has a pH range ~2–3 pH units within which
growth is possible.
• Most natural environments are pH 3–9 (Table 4.4)

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Figure 4.26 The pH Scale

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Table 4.4 Relationships of
Microorganisms to pH
Physiological class Approximate pH optimum Example organisma
(optima range) for growth
Neutrophile (pH > 5.5 and
7 Escherichia coli
< 8)

Acidophile (pH < 5.5) 5 Rhodopila globiformis

Acidithiobacillus
Acidophile (pH < 5.5) 3
ferrooxidans

Acidophile (pH < 5.5) 1 Picrophilus oshimae

Alkaliphile (pH ≥ 8) 8 Chloroflexus aurantiacus

Alkaliphile (pH ≥ 8) 9 Bacillus firmus

Alkaliphile (pH ≥ 8) 10 Natronobacterium gregoryi

aPicrophilus and Natronobacterium are Archaea; all others are Bacteria.

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4.15 Osmolarity and Microbial
Growth
• Water availability important; depends on environmental
moisture/dryness and concentration of solutes
• Water activity (aw): water availability (Table 4.5)
– ratio of vapor pressure of air in equilibrium with a
substance or solution to vapor pressure of pure water
– varies from zero (no free water) to one (pure water)
• Osmosis: Water diffuses from high to low concentrations.

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4.15 Osmolarity and Microbial
Growth
• Typically, the cytoplasm has a higher solute
concentration than the surrounding environment; thus,
the tendency is for water to move into the cell (positive
water balance).
• When a cell is in an environment with a higher external
solute concentration, water will flow out unless the cell
has a mechanism to prevent this.

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4.15 Osmolarity and Microbial
Growth (3 of 5)
• Halophiles and Related Organisms
– Seawater contains ~3% NaCl
– Halophiles: grow best at aw = 0.98 (seawater); have
a specific requirement for NaCl (Table 4.6, Figure
4.27)
– Halotolerant: tolerate some dissolved solutes but
generally grow best in the absence of added solute
– Extreme halophiles: require very high levels (15-
30%) of NaCl; often unable to grow at lower
concentrations

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Figure 4.27 Effect of NaCl Concentration on Growth
of Microorganisms of Different Salt Tolerances or
Requirements

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4.15 Osmolarity and Microbial
Growth
• Halophiles and Related Organisms (Table 4.6)
– Osmophiles: live in environments high in sugar
Xerophiles: able to grow in very dry environments
– Lowest aw = 0.61 for life; physiochemical constraints
on obtaining water at lower aw

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4.16 Oxygen and Microbial Growth (1 of 5)
• Oxygen Classes of Microorganisms (Table 4.7)
– Aerobes: grow at full O2 tension (~21%) and respire O2
– Microaerophiles: can use O2 only at levels reduced
from that in air (microxic) due to limited respiration or
oxygen sensitivity
– Facultative organisms: can live with or without oxygen

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Table 4.7 Oxygen Relationships of
Microorganisms
Aerobes

Group Relationship to O 2 Type of metabolism Examplea Habitatb

Obligate Required Aerobic respiration Micrococcus luteus (B) Skin, dust

Facultative Not required, but growth Aerobic respiration, Escherichia coli (B) Mammalian large
better with O2 anaerobic respiration, intestine
fermentation
Microaerophilic Required but at levels Aerobic respiration Spirillum volutans (B) Lake water
lower than atmospheric
Anaerobes
Group Relationship to O 2 Type of metabolism Examplea Habitatb
Aerotolerant Not required, and growth Fermentation Streptococcus mutans Oral cavity
no better when O2 (B)
present
Obligate Harmful or lethal Fermentation or anaerobic Methanobacterium Sewage sludge,
respiration formicicum (A) anoxic lake
sediments

aLetters in parentheses indicate phylogenetic status (B, Bacteria; A, Archaea). Representatives of either domain of
prokaryotic cells are known in each category. Most eukaryotes are obligate aerobes, but facultative aerobes (for example,
yeast) and obligate anaerobes (for example, certain protozoa and fungi) are known.
bListed are typical habitats of the example organism; many others could be listed.

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Figure 4.28 Growth Versus O 2
Concentration
• From left to right,
aerobic, anaerobic,
facultative,
microaerophilic, and
aerotolerant anaerobe
growth, as revealed by
the position of microbial
colonies (depicted in
this drawing as black
dots) within tubes of
thioglycolate broth
culture medium.

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Figure 4.28 Growth Versus O 2
Concentration
• A small amount of agar
has been added to keep
the liquid from becoming
disturbed.

• The redox dye resazurin,

which is pink when
oxidized and colorless
when reduced, has been
added as a redox
indicator.

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Figure 4.28 Growth Versus O 2
Concentration
a. O2 penetrates only a
short distance into the
tube, so obligate aerobes
grow only close to the
surface.
b. Anaerobes, being
sensitive to O2, grow only
away from the surface.

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Figure 4.28 Growth Versus O 2
Concentration
c. Facultative aerobes
are able to grow in
either the presence or
the absence of O2 and
thus grow throughout
the tube. However,
growth is better near
the surface because
these organisms can
respire O2.

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Figure 4.28 Growth Versus O 2
Concentration
d. Microaerophiles grow
away from the most oxic
zone.
e. Aerotolerant anaerobes
grow throughout the
tube. Growth is not
better near the surface
because these
organisms can only
ferment.

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Figure 4.28 Growth Versus O 2
Concentration

• In nature, many different habitats exist for each of these

oxygen classes.
• In addition, a single habitat, such as a soil particle, may
support growth of both aerobes and anaerobes.

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Figure 4.29 Incubation Under Anoxic
Conditions

(a) Anoxic jar. A chemical reaction in the envelope in the jar generates H 2 + CO2
The H2 reacts with O2 in the jar on the surface of a palladium catalyst to yield H 2 O; the
final atmosphere contains N 2, H2, and CO2
(b) Anoxic glove bag for manipulating and incubating cultures under anoxic conditions.
The airlock on the right, which can be evacuated and filled with O 2-free gas, serves as a
port for adding and removing materials to and from the glove bag.

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4.16 Oxygen and Microbial Growth (4 of 5)
• Why is oxygen toxic?
– Molecular oxygen (O2) is not toxic.
– Exposure to oxygen yields toxic byproducts. (Figure
4.30)
▪ superoxide anion (O2 )
−

▪ hydrogen peroxide (H2O2)

▪ hydroxyl radical (OH·)

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Figure 4.30 Four-Electron Reduction of O2 to
H2O by Stepwise Addition of Electrons

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4.16 Oxygen and Microbial Growth

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4.16 Oxygen and Microbial Growth
• Superoxide reductase in some strict anaerobes converts
O2− to H2O2 without producing O2.

Figure 4.32 Method for Testing a Microbial Culture for

the Presence of Catalase
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V.
Five Controlling Microbial Growth

4.17 General Principles and Microbial Growth Control by

Heat
4.18 Other Physical Control Methods: Radiation and
Filtration
4.19 Chemical Control of Microbial Growth

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4.17 General Principles and Growth
Control by Heat (1 of 3)
• Decontamination
– the treatment of an object to make it safe to handle
• Disinfection
– directly targets pathogens, not necessarily all
microorganisms
– kills or severely inhibits growth

• Heat Sterilization

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4.17 General Principles and Growth
Control by Heat (3 of 3)
• The Autoclave and Pasteurization
– Autoclave: sealed heating device that uses steam under
pressure. (Figure 4.34)
▪ steam under pressure yields temperature of 121°C elsius

▪ kills endospores

▪ not the pressure but the high temperature that kills the
microbes
– Pasteurization: uses heat to significantly reduce the
microbial load in heat-sensitive liquids.
▪ does not kill all organisms, so it is different from

sterilization
▪ all known pathogenic bacteria are killed

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Figure 4.34 The Autoclave and Moist
Heat Sterilization
A graph of the autoclave cycle
• The first line is the autoclave
itself which has flowing steam to
keep it at 100 degrees when off.
• When turned on the pressure
begins and the temperature
goes from 100 to 121 in about
10 minutes.
• The autoclave stays on for 15
minutes and stays hot for one
minute after the steam is turned
off then decreases over 24
minutes back to 100 degrees.

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 Figure 4.34 The Autoclave and Moist
Heat Sterilization

• The steam and pressure

combine for the autoclave time.
• The object in the autoclave
placed at 0 minutes when the
machine is turned on.
• Temperature increases over 15
minutes to 120 degrees and
stays there for 15 minutes which
is the sterilization time.
• The temperature then decreases
to 100 degrees over 30 minutes.

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4.18 Other Physical Control Methods:
Radiation and Filtration (1 of 4)
• Ultraviolet and Ionizing Radiation
– Ultraviolet (UV) radiation (between 220 and 300 nm)
affects DNA leading to death
▪ useful for decontaminating surfaces (Figure 4.35)

▪ poor penetration

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Figure 4.35 A Laminar Flow Hood

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4.18 Other Physical Control Methods:
Radiation and Filtration (3 of 4)
• Filter Sterilization
– Filtration used on heat-sensitive liquids and gases.
▪ pores of filter (0.45 and 0.2 μm) are too small for

living organisms to pass through but do not trap

most viruses
▪ pores allow liquid or gas to pass through

– Depth filters: fibrous sheet made of overlapping paper

or glass fibers that traps particles (Figure 4.37a)
▪ HEPA (High efficiency particulate air) filters remove

0.3 μm+ particles from air

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4.18 Other Physical Control Methods:
Radiation and Filtration (4 of 4)
• Filter Sterilization
– Membrane filters (Figures 4.37b and 4.38)
▪ Most common for liquid sterilization

▪ High-strength polymers with many tiny pores

▪ Syringe or pump forces liquid into sterile vessel

– Nucleopore filters
▪ Thin irradiated film etched to make holes (Figure

4.37c)
▪ Used for scanning electron microscopy

▪ Microorganisms observed directly on filter

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Figure 4.38 Membrane Filters

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4.19 Chemical Control of Microbial
Growth (1 of 4)
• Antimicrobial agent: chemical that kills or inhibits growth.
• -cidal kills microorganisms (e.g., bactericidal, fungicidal,
viricidal)
• -static inhibits growth (e.g., bacteriostatic, fungistatic,
viristatic)

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4.19 Chemical Control of Microbial
Growth (2 of 4)
• Effect of Antimicrobial Agents on Growth
– Antibacterial agents can be classified as bacteriostatic,
bacteriocidal, and bacteriolytic by observing effects on
cultures (Figure 4.39)
▪ Bacteriostatic agents inhibit important biochemical

processes and bind weakly

▪ Bactericidal agents bind tightly and kill the cell

without lysis
▪ Bacteriolytic agents kill by lysis (e.g., detergents).

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Figure 4.39 Different Types of
Antimicrobial Agents

(a) Bacteriostatic agents inhibit but do not kill.

(b) Bactericidal agents kill.
(c) Bacteriolytic agents lyse cells. In each graph, the arrow indicates the
time at which a growth-inhibitory concentration of an antimicrobial
agent was added to an exponentially growing culture.

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Figure 4.39 Different Types of
Antimicrobial Agents

Bacteriostatic cell count and viable cell count go up steadily and stay
the same for about the same amount of time before going up equally
again when the agent is removed.
Bactericidal cell count and viable cell count go up equally until the agent
is added, then total cell count stays the same and viable cell count
goes down steadily.
Bacteriolytic cell count and viable cell count go up steadily, once the
agent is added both go down steadily and equally.
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4.19 Chemical Control of Microbial
Growth (3 of 4)
• Assaying Antimicrobial Activity
– Minimum inhibitory concentration (MIC): smallest
amount of an agent needed to inhibit growth of a
microorganism.
▪ Can be determined with liquid medium and agent

dilutions (Figure 4.40)

▪ Disk diffusion assay on solid media. (Figure 4.41)

– Antimicrobial agent added to filter paper disk,

diffuses into agar.
– MIC is reached at some distance.
• Zone of growth inhibition: area of no growth
around disk

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Figure 4.40 Antimicrobial Agent
Susceptibility Assay Using Dilution Methods

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Figure 4.41 Antimicrobial Agent Susceptibility
Assay Using Diffusion Methods

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4.19 Chemical Control of Microbial
Growth (4 of 4)
• Chemical Antimicrobial Agents
– Antimicrobial agents (Table 4.9) used to prevent pathogen
growth on inanimate surfaces and external body surfaces.
▪ Sterilants destroy all microorganisms, including

endospores.
▪ Disinfectants kill microorganisms but not necessarily
endospores, used on surfaces.
▪ Sanitizers are less harsh, reduce microbial numbers, do
not sterilize.
▪ Antiseptics (germicides) kill or inhibit microbial growth
and are nontoxic enough to be applied to living tissues.

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Table 4.9 Antiseptics, Sterilants,
Disinfectants, and Sanitizers a (1 of 3)
Agent Mode of action Use
Antiseptics (germicides) blank blank

Alcohol (60–85% ethanol or Lipid solvent and protein denaturant Topical antiseptic
isopropanol in water)
Phenol-containing compounds Disrupts cytoplasmic membrane Soaps, lotions, cosmetics, deodorants,
(hexachlorophene, triclosan, topical disinfectants; paper, leather,
chloroxylenol, chlorhexidine) and textile industries
Cationic detergents, especially Disrupts cytoplasmic membrane Soaps, lotions, topical disinfectants;
quaternary ammonium compounds metal and petroleum Industries
(benzalkonium chloride)
Hydrogen peroxide (3% solution) Oxidizing agent Topical antiseptic
Iodophors (Betadine®) Iodinates proteins, rendering them Topical antiseptic
nonfunctional; oxidizing agent
Octenidine Cationic surfactant, disrupts Topical antiseptic
cytoplasmic membrane

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Table 4.9 Antiseptics, Sterilants,
Disinfectants, and Sanitizers a (2 of 3)
Agent Mode of action Use
Sterilants, disinfectants, and blank blank
sanitizers
Alcohol (60–85% ethanol or Lipid solvent and protein denaturant General-purpose disinfectant for
isopropanol in water) virtually any surface
Cationic detergents (quaternary Interacts with phospholipids Disinfectant/sanitizer for medical
ammonium compounds, Lysol® and instruments, food and dairy equipment
many related disinfectants)
Chlorine gas Oxidizing agent Disinfectant for drinking water and
electrical/nuclear cooling towers
Chlorine compounds (chloramines, Oxidizing agent Disinfectant/sanitizer for medical
sodium hypochlorite, sodium instruments, food/dairy equipment, and
chlorite, chlorine dioxide) in water purification
Copper sulfate Protein precipitant Algicide in swimming pools

Ethylene oxide (gas) Alkylating agent Sterilant for temperature-sensitive

materials such as plastics
Formaldehyde Alkylating agent Diluted (3% solution) as surface
disinfectant/sterilant; Concentrated
(37% solution) as sterilant
Glutaraldehyde Alkylating agent Disinfectant or sterilant as 2% solution

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Table 4.9 Antiseptics, Sterilants,
Disinfectants, and Sanitizers a (3 of 3)
Agent Mode of action Use
Hydrogen peroxide Oxidizing agent Vapor used as sterilant
Iodophors (Wescodyne®) Iodinates proteins; oxidizing agent General disinfectant

OPA (ortho-phthalaldehyde) Alkylating agent Powerful disinfectant used for

sterilizing medical instruments
Ozone Strong oxidizing agent Disinfectant for drinking water

Peroxyacetic acid Strong oxidizing agent Disinfectant/sterilant

Phenolic compounds Protein denaturant General-purpose disinfectant

Pine oils (Pine-Sol®) (contains Protein denaturant General-purpose disinfectant for

phenolics and detergents) household surfaces

aAlcohols,hydrogen peroxide, and iodophors can be antiseptics, disinfectants, sanitizers, or sterilants depending on
concentration, length of exposure, and form of delivery.

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