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Chapter 6

Microbial Growth

© 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case

Copyright © 2013 Pearson Education, Inc.


Lectures prepared by Christine L. Case
© 2013 Pearson Education, Inc.
Microbial Growth

 Increase in number of cells, not cell size


 Populations
 Colonies

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The Requirements for Growth

 Physical requirements
 Temperature
 pH
 Osmotic pressure
 Chemical requirements
 Carbon
 Nitrogen, sulfur, and phosphorous
 Trace elements
 Oxygen
 Organic growth factor

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Physical Requirements

 Temperature
 Psychrophiles (cold-loving microbes)
 Mesophiles (moderate temperature loving microbes)
 Thermophiles (heat-loving microbes)
 Most bacteria grow only within a limited range of
temperatures, 30 celsius
 Minimum growth temperature- lowest temperature at
which the species will grow
 Optimum growth temperature- temperature at which
the species grows best
 Maximum growth temperature- highest temperature at
which growth is possible
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Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature.

Thermophiles

Hyperthermophiles
Mesophiles
Psychrotrophs
Psychrophiles

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Applications of Microbiology 6.1 A white microbial biofilm is visible on this deep-sea hydrothermal vent.
Water is being emitted through the ocean floor at temperatures above 100°C.

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Psychrotrophs

 Grow between 0°C and 20–30°C


 Cannot grow about 40 celsius
 Much common than psychrophiles and the most
likely to be encountered in low temperature food
spoilage because they grow fairly well at refrigerator
temp
 Cause food spoilage

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Refrigeration

 Most common method of preserving household food


supplies
 Microbial reproductive rates decrease at low
temperature
 Mesophiles- 25 to 40 celsius, most common type of
microbe, most of the common spoilage and disease
organisms, optimum for many pathogenic bacteria is
about 37 celsius
 Thermophiles- microorganisms capable of growth at
high temp, optimum growth of 50-60 celsius

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Figure 6.2 Food preservation temperatures.

Temperatures in this range destroy most microbes,


although lower temperatures take more time.

Very slow bacterial growth.

Danger zone Rapid growth of bacteria; some may produce toxins.

Many bacteria survive; some may grow.


Refrigerator temperatures; may allow slow growth
of spoilage bacteria, very few pathogens.
No significant growth below freezing.

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Figure 6.3 The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage.

15 cm (6′′) deep

5 cm (2′′) deep Approximate temperature


range at which Bacillus cereus
multiplies in rice

Refrigerator air

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pH

 Most bacteria grow between pH 6.5 and 7.5


 Molds and yeasts grow between pH 5 and 6
 Acidophiles grow in acidic environments
 Alkalinity also inhibits microbial growth but is rarely
used to preserve foods

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Osmotic Pressure

 Hypertonic environments, or an increase in salt or


sugar, cause plasmolysis
 Extreme or obligate halophiles require high
osmotic pressure
 Facultative halophiles tolerate high osmotic
pressure

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Figure 6.4 Plasmolysis.

Plasma Plasma
membrane Cell wall membrane
H2O

Cytoplasm Cytoplasm

NaCl 0.85% NaCl 10%


Cell in isotonic solution. Plasmolyzed cell in hypertonic
solution.

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Chemical Requirements

 Carbon
 Structural organic molecules, energy source
 Chemoheterotrophs use organic carbon sources
 Autotrophs use CO2

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Chemical Requirements

 Nitrogen
 In amino acids and proteins
 Most bacteria decompose proteins
 Some bacteria use NH4+ or NO3–
 A few bacteria use N2 in nitrogen fixation

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Chemical Requirements

 Sulfur
 In amino acids, thiamine, and biotin
 Most bacteria decompose proteins
 Some bacteria use SO42– or H2S
 Phosphorus
 In DNA, RNA, ATP, and membranes
 PO43– is a source of phosphorus

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Chemical Requirements

 Trace elements
 Inorganic elements required in small amounts
 Usually as enzyme cofactors

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Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

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Toxic Oxygen

 Singlet oxygen: 1O2− boosted to a higher-energy


state
 Superoxide free radicals: O2
Superoxide dismutase
O2 + O2 + 2 H+ H 2 O2 + O 2

 Peroxide anion: O22–


Catalase
2 H 2 O2 2 H 2 O + O2

Peroxidase
H 2 O2 + 2 H + 2 H2O

 Hydroxyl radical (OH•)


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Organic Growth Factors

 Organic compounds obtained from the environment


 Vitamins, amino acids, purines, and pyrimidines

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Biofilms

 Microbial communities
 Form slime or hydrogels
 Bacteria attracted by chemicals via quorum sensing

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Figure 6.5 Biofilms.

Clumps of bacteria
Migrating
adhering to surface
clump of
bacteria

Surface Water currents

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Biofilms

 Share nutrients
 Sheltered from harmful factors

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Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.

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Biofilms

 Patients with indwelling catheters received


contaminated heparin
 Bacterial numbers in contaminated heparin were too
low to cause infection
 84–421 days after exposure, patients developed
infections

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Culture Media

 Culture medium: nutrients prepared for microbial


growth
 Sterile: no living microbes
 Inoculum: introduction of microbes into medium
 Culture: microbes growing in/on culture medium

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Agar

 Complex polysaccharide
 Used as solidifying agent for culture media in Petri
plates, slants, and deeps
 Generally not metabolized by microbes
 Liquefies at 100°C
 Solidifies at ~40°C

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Culture Media

 Chemically defined media: exact chemical


composition is known
 Complex media: extracts and digests of yeasts,
meat, or plants
 Nutrient broth
 Nutrient agar

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Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia
coli

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Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides

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Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria

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Anaerobic Culture Methods

 Reducing media
 Contain chemicals (thioglycolate or oxyrase) that
combine O2
 Heated to drive off O2

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Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.

Clamp with
Lid with clamp screw
O-ring gasket

Envelope containing
sodium bicarbonate
and sodium
borohydride

Palladium
Anaerobic indicator catalyst pellets
(methylene blue)

Petri plates

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Figure 6.7 An anaerobic chamber.

Air
lock

Arm
ports

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Capnophiles

 Microbes that require high CO2 conditions


 CO2 packet
 Candle jar

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Biosafety Levels

 BSL-1: no special precautions


 BSL-2: lab coat, gloves, eye protection
 BSL-3: biosafety cabinets to prevent airborne
transmission
 BSL-4: sealed, negative pressure
 Exhaust air is filtered twice

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Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.

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Differential Media

 Make it easy to distinguish colonies of different


microbes

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Figure 6.9 Blood agar, a differential medium containing red blood cells.

Bacterial
colonies

Hemolysis

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Selective Media

 Suppress unwanted microbes and encourage


desired microbes

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Figure 6.10 Differential medium.

Uninoculated

Staphylococcus Staphylococcus
epidermis aureus

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Enrichment Culture

 Encourages growth of desired microbe


 Assume a soil sample contains a few
phenol-degrading bacteria and thousands of
other bacteria
 Inoculate phenol-containing culture medium with the soil,
and incubate
 Transfer 1 ml to another flask of the phenol medium, and
incubate
 Transfer 1 ml to another flask of the phenol medium, and
incubate
 Only phenol-metabolizing bacteria will be growing

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Obtaining Pure Cultures

 A pure culture contains only one species or strain


 A colony is a population of cells arising from a
single cell or spore or from a group of attached cells
 A colony is often called a colony-forming unit
(CFU)
 The streak plate method is used to isolate pure
cultures

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Figure 6.11 The streak plate method for isolating pure bacterial cultures.

Colonies

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Preserving Bacterial Cultures

 Deep-freezing: –50° to –95°C, placed in


suspending liquid,quick frozen
 Lyophilization (freeze-drying): frozen
(–54° to –72°C) and dehydrated in a vacuum,
water is removed by high vacuum (sublimation)

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Reproduction in Prokaryotes

 Binary fission
 Budding
 Conidiospores (actinomycetes)
 Fragmentation of filaments

ANIMATION Bacterial Growth: Overview

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Figure 6.12a Binary fission in bacteria.
Cell wall
Cell elongates and
DNA is replicated. Plasma
membrane

Cell wall and plasma DNA


membrane begin to constrict. (nucleoid)

Cross-wall forms,
completely separating
the two DNA copies.

Cells separate.

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(a) A diagram of the sequence of cell division
Figure 6.12b Binary fission in bacteria.
Partially formed cross-wall
DNA (nucleoid) Cell wall

(b) A thin section of a cell of Bacillus licheniformis


starting to divide
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Figure 6.13a Cell division.

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Figure 6.13b Cell division.

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Generation Time
 Tome required for a cell to divide and its population
to double
 If 100 cells growing for 5 hours produced
1,720,320 cells:
Log number of cells (end) − Log number of cells (beginning)
Number of generations =
0.301
60 min × hours
Generation time = = 21 minutes/generation
Number of generations

ANIMATION Binary Fission

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Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed
line) and arithmetically (solid line).

(1,048,576)
Log10 = 6.02

Log10 = 4.52
Log10 of number of cells

Number of cells
(524,288)
Log10 = 3.01

Log10 = 1.51 (262,144)

(131,072)
(65,536)
(32,768)
(32) (1024)

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Figure 6.15 Understanding the Bacterial Growth Curve.

Lag Phase Log Phase Stationary Phase Death Phase


Intense activity Logarithmic, or Period of equilibrium; Population Is
preparing for exponential, microbial deaths decreasing at a
population growth, increase in balance production of logarithmic rate.
but no increase in population. new cells.
population.
The logarithmic growth
in the log phase is due to
reproduction by binary
fission (bacteria) or
mitosis (yeast).

Staphylococcus spp.

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Figure 6.16 Serial dilutions and plate counts.

1 ml 1 ml 1 ml 1 ml 1 ml

Original 9 m broth
inoculum in each tube

Dilutions 1:10 1:100 1:1000 1:10,000 1:100,000

1 ml 1 ml 1 ml 1 ml 1 ml

Plating

1:10 1:100 1:1000 1:10,000 1:100,000


(10-1) (10-2) (10-3) (10-4) (10-5)
Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml
(For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)

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Plate Counts

 After incubation, count colonies on plates that have


25–250 colonies (CFUs)

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Figure 6.17 Methods of preparing plates for plate counts.
The pour plate method The spread plate method

Inoculate 1.0 or 0.1 ml 0.1 ml


empty plate. Inoculate plate
containing
solid medium.

Bacterial
dilution Spread inoculum
over surface
Add melted
evenly.
nutrient
agar.

Swirl to
mix.
Colonies grow
Colonies only on surface
grow on of medium.
and in
solidified
medium.

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Figure 6.18 Counting bacteria by filtration.

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Most Probable Number

 Multiple tube MPN test


 Count positive tubes

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Figure 6.19a The most probable number (MPN) method.

Volume of
Inoculum for
Each Set of
Five Tubes

(a) Most probable number (MPN) dilution series.

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Most Probable Number

 Compare with a statistical table

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Figure 6.19b The most probable number (MPN) method.

(b) MPN table.


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Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.

Grid with 25 large squares

Cover glass

Slide

Bacterial suspension is added here and fills the


shallow volume over the squares by capillary
action.

Bacterial
suspension Microscopic count: All cells in
several large squares are
Cover glass counted, and the numbers are
averaged. The large square shown
Slide here has 14 bacterial cells.
Location of squares The volume of fluid over the
Cross section of a cell counter. large square is 1/1,250,000
The depth under the cover glass and the area of a milliliter. If it contains 14 cells,
of the squares are known, so the volume of the as shown here, then
bacterial suspension over the squares can be there are 14 × 1,250,000 =
calculated (depth × area). 17,500,000 cells in a milliliter.
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Direct Microscopic Count

Number of cells counted


Number of bacteria/ml =
Volume of area counted

14
−7
= 17,500,000
8 × 10

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Figure 6.21 Turbidity estimation of bacterial numbers.

Light source

Spectrophotometer
Light

Light-sensitive
Scattered light Blank
that does not
detector
reach detector

Bacterial suspension
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Measuring Microbial Growth

Direct Methods Indirect Methods


 Plate counts  Turbidity
 Filtration  Metabolic activity
 MPN  Dry weight
 Direct microscopic count

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