PowerPoint ® Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State...

PowerPoint® Lecture Presentations prepared byMindy Miller-Kittrell,North Carolina State University

C H A P T E R

© 2014 Pearson Education, Inc.

6

Microbial Nutrition and Growth


Growth Requirements

• Microbial growth

• Increase in a population of microbes

• Due to reproduction of individual microbes

• Result of microbial growth is discrete colony

• An aggregation of cells arising from single parent cell


Growth Requirements

• Organisms use a variety of nutrients for their

energy needs and to build organic molecules and

cellular structures

• Most common nutrients contain necessary

elements such as carbon, oxygen, nitrogen, and

hydrogen

• Microbes obtain nutrients from variety of sources


Growth Requirements

• Nutrients: Chemical and Energy Requirements

• Sources of carbon, energy, and electrons

• Two groups of organisms based on source of carbon

• Autotrophs

• Heterotrophs

• Two groups of organisms based on source of energy

• Chemotrophs

• Phototrophs


Figure 6.1 Four basic groups of organisms based on their carbon and energy sources.


Growth Requirements


• Sources of carbon, energy, and electrons

• Two groups of organisms based on source of electrons

• Organotrophs

• Lithotrophs


Growth Requirements


• Oxygen requirements

• Oxygen is essential for obligate aerobes

• Oxygen is deadly for obligate anaerobes

• How can this be true?

• Toxic forms of oxygen are highly reactive and excellent

oxidizing agents

• Resulting oxidation causes irreparable damage to cells


Growth Requirements



• Four toxic forms of oxygen

• Singlet oxygen

• Superoxide radicals

• Peroxide anion

• Hydroxyl radical


Figure 6.2 Catalase test.


Growth Requirements



• Aerobes

• Anaerobes

• Facultative anaerobes

• Aerotolerant anaerobes

• Microaerophiles


Loose-fittingcap

OxygenconcentrationHigh

LowObligateaerobes

Obligateanaerobes

Facultativeanaerobes

Aerotolerantanaerobes

Figure 6.3 Using a liquid thioglycollate growth medium to identify the oxygen requirements of organisms.


Growth Requirements


• Nitrogen requirements

• Anabolism often ceases due to insufficient nitrogen

• Nitrogen acquired from organic and inorganic nutrients

• All cells recycle nitrogen from amino acids and nucleotides

• Nitrogen fixation by certain bacteria is essential to life on

Earth


Growth Requirements


• Other chemical requirements

• Phosphorus

• Sulfur

• Trace elements

• Only required in small amounts

• Growth factors

• Necessary organic chemicals that cannot be

synthesized by certain organisms


Growth Requirements

• Physical Requirements

• Temperature

• Temperature affects three-dimensional structure of proteins

• Lipid-containing membranes of cells and organelles are

temperature sensitive

• If too low, membranes become rigid and fragile

• If too high, membranes become too fluid


Maximum

Optimum

Minimum

37ºC30ºC22ºC

Figure 6.4 The effects of temperature on microbial growth.


Figure 6.5 Four categories of microbes based on temperature ranges for growth.


Figure 6.6 An example of a psychrophile.


Growth Requirements


• pH

• Organisms sensitive to changes in acidity

• H+ and OH– interfere with H bonding

• Neutrophiles grow best in a narrow range around neutral pH

• Acidophiles grow best in acidic habitats

• Alkalinophiles live in alkaline soils and water


Growth Requirements


• Physical effects of water

• Microbes require water to dissolve enzymes and nutrients

• Water is important reactant in many metabolic reactions

• Most cells die in absence of water

• Some have cell walls that retain water

• Endospores and cysts cease most metabolic activity

• Two physical effects of water

• Osmotic pressure

• Hydrostatic pressure


Growth Requirements



• Osmotic pressure

• Pressure exerted on a semipermeable membrane by a

solution containing solutes that cannot freely cross

membrane

• Hypotonic solutions have lower solute concentrations

• Cell placed in hypotonic solution swells

• Hypertonic solutions have greater solute concentrations

• Cell placed in hypertonic solution shrivels

• Restricts organisms to certain environments

• Obligate and facultative halophiles


Growth Requirements



• Hydrostatic pressure

• Water exerts pressure in proportion to its depth

• Barophiles live under extreme pressure

• Their membranes and enzymes depend on

pressure to maintain their three-dimensional,

functional shape


Growth Requirements

• Associations and Biofilms

• Organisms live in association with different species

• Antagonistic relationships

• Synergistic relationships

• Symbiotic relationships


Growth Requirements

• Associations and Biofilms

• Biofilms

• Complex relationships among numerous microorganisms

• Form on surfaces, medical devices, mucous membranes

of digestive system

• Form as a result of quorum sensing

• Many microorganisms more harmful as part of a biofilm

• Scientists seeking ways to prevent biofilm formation


Figure 6.7 Biofilm development.

Free-swimming microbes are vulnerable to environmentalstresses.

Bacteria

Some microbes land on a surface, such as a tooth, and attach.

The cells begin producing an intracellular matrix and secrete quorum-sensing molecules.

Quorum sensing triggers cells to change their biochemistry and shape.

New cells arrive, possibly including new species, and water channels form in the biofilm.

Some microbes escape from the biofilm to resume a free-living existence and perhaps, form a new biofilm on another surface.

Chemical structure of one type of quorum- sensing molecule

Matrix

Water flow

Water channel

Escapingmicrobes

1

2 3 4 5 6


Culturing Microorganisms

• Inoculum introduced into medium

• Environmental specimens

• Clinical specimens

• Stored specimens

• Culture

• Act of cultivating microorganisms or the microorganisms

that are cultivated


Shape

Circular Rhizoid Irregular Filamentous Spindle

Margin

Elevation

Size

Texture

Appearance

Pigmenta-tion

Opticalproperty

Entire Undulate Lobate Curled Filiform

Flat Raised Convex Pulvinate Umbonate

Punctiform Small Moderate Large

Glistening (shiny) or dull

Nonpigmented (e.g., cream, tan, white)Pigmented (e.g., purple, red, yellow)

Opaque, translucent, transparent

Smooth or rough

Colony

Figure 6.8 Characteristics of bacterial colonies.



• Obtaining Pure Cultures

• Cultures composed of cells arising from a single

progenitor

• Progenitor is termed a colony-forming unit (CFU)

• Aseptic technique prevents contamination of sterile

substances or objects

• Two common isolation techniques

• Streak plates

• Pour plates


Figure 6.9 The streak-plate method of isolation.


Sequential inoculations

1.0 ml

Initialsample

9 mlbroth

Colonies Fewer colonies

1.0 ml to eachPetri dish, add9 ml warm agar,swirl gently to mix

1.0 ml 1.0 ml

9 mlbroth

9 mlbroth

Figure 6.10 The pour-plate method of isolation.



• Obtaining Pure Cultures

• Other isolation techniques

• Some fungi isolated with streak and pour plates

• Protozoa and motile unicellular algae isolated through

dilution of broth cultures

• Can individually pick single cell of some large

microorganisms and use to establish a culture



• Culture Media

• Majority of prokaryotes have not been grown in culture medium

• Agar is a common addition to many media

• Six types of general culture media

• Defined media

• Complex media

• Selective media

• Differential media

• Anaerobic media

• Transport media


Slant

Butt

Figure 6.11 Slant tubes containing solid media.


Bacterial colonies Fungal colonies

pH 7.3 pH 5.6

Figure 6.12 An example of the use of a selective medium.


Beta-hemolysis

Alpha-hemolysis

No hemolysis(gamma-hemolysis)

Figure 6.13 The use of blood agar as a differential medium.


Durham tube(inverted tubeto trap gas)

Acid fermentationwith gas

No fermentation

Figure 6.14 The use of carbohydrate utilization tubes as differential media.


Escherichia coli Escherichia coli Escherichia coli

Staphylococcusaureus

Staphylococcusaureus (no growth)

Salmonella entericaserotype Choleraesuis

MacConkey agar MacConkey agarNutrient agar

Figure 6.15 The use of MacConkey agar as a selective and differential medium.


Airtight lid

Palladium pelletsto catalyze reactionremoving O2

Methylene blue(anaerobicindicator)

Envelopecontainingchemicals torelease CO2

and H2

Petri plates

Chamber

Clamp

Figure 6.16 An anaerobic culture system.



• Culture Media

• Transport media

• Used by hospital personnel to ensure clinical specimens

are not contaminated and to protect people from infection

• Rapid transport of samples is important



• Special Culture Techniques

• Techniques developed for culturing microorganisms

• Animal and cell culture

• Low-oxygen culture



• Preserving Cultures

• Refrigeration

• Stores for short periods of time

• Deep-freezing

• Stores for years

• Lyophilization

• Stores for decades


Bacterial Growth: Overview

Bacterial Growth: OverviewPLAYPLAY


Binary Fission

Binary FissionPLAYPLAY


Cytoplasmic membraneChromosome

Cell wall

Replicatedchromosome

Septum

Completedseptum

30 minutes

60 minutes

90 minutes

120 minutes

Septum

1

2

3

4

5

Figure 6.17 Binary fission.


Growth of Microbial Populations

• Generation Time

• Time required for a bacterial cell to grow and divide

• Dependent on chemical and physical conditions


Figure 6.18 A comparison of arithmetic and logarithmic growth.


Figure 6.19 Two growth curves of logarithmic growth.


Figure 6.20 A typical microbial growth curve.


Bacterial Growth Curve

Bacterial Growth CurvePLAYPLAY



• Continuous Culture in a Chemostat

• Chemostat used to maintain a microbial population in a

particular phase of growth

• Open system

• Requires addition of fresh medium and removal of old

medium

• Used in several industrial settings


Fresh medium witha limiting amountof a nutrient

Sterile airor othergas

Flow-rateregulator

Culturevessel

Overflowtube

Culture

Figure 6.21 Schematic of chemostat.



• Measuring Microbial Reproduction

• Direct methods not requiring incubation

• Microscopic counts


Cover slip

Location ofgrid

Bacterialsuspension

Overflow troughs

Place underoil immersion

Bacterialsuspension

Pipette

Figure 6.22 The use of a cell counter for estimating microbial numbers.




• Direct methods not requiring incubation

• Electronic counters

• Coulter counters

• Flow cytometry




• Direct methods requiring incubation

• Serial dilution and viable plate counts

• Membrane filtration

• Most probable number


1 ml of originalculture

9 ml of broth +1 ml of originalculture

0.1 ml of eachtransferred toa plate

Incubation period

1.0 ml

1:10dilution(10-1)

Too numerous to count(TNTC)

TNTC 65 colonies 6 colonies 0 colonies

1:100dilution(10-2)

1:1000dilution(10-3)

1:10,000dilution(10-4)

1:100,000dilution(10-5)

1.0 ml 1.0 ml 1.0 ml

0.1 ml0.1 ml0.1 ml0.1 ml

Figure 6.23 A serial dilution and viable plate count for estimating microbial population size.


Membrane transferredto culture medium

Sample to be filtered

Membrane filterretains cells

To vacuum

Colonies

Incubation

Figure 6.24 The use of membrane filtration to estimate microbial population size.


1:1001:10Undiluted

1.0 ml1.0 ml

Inoculate 1.0 ml intoeach of 5 tubes

Phenol red, pHcolor indicator,added

Incubate

Results

4 tubes positive 2 tubes positive 1 tube positive

Figure 6.25 The most probable number (MPN) method for estimating microbial numbers.



• Measuring Microbial Growth

• Indirect methods

• Turbidity


Direct light

Light source Uninoculatedtube

Light-sensitivedetector

Light source Inoculatedbroth culture

Scattered lightthat does notreach reflector

Figure 6.26 Turbidity and the use of spectrophotometry in indirectly measuring population size.



• Measuring Microbial Growth

• Indirect methods

• Metabolic activity

• Dry weight

• Genetic methods

• Isolate DNA sequences of unculturable prokaryotes

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