Microbe Growth

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M I C R O B I O L O G Ya n i n t r o d u c t i o n

ninth edition TORTORA ⏐ FUNKE ⏐ CASE

6

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings

PowerPoint® Lecture Slide Presentation prepared by Christine L. Case

6Microbial

Growth

Microbial growth is the increase in number of cells,

not cell size

Microbial Growth


The Requirements for Growth: Physical Requirements

Temperature

Minimum growth temperature

Optimum growth temperature

Maximum growth temperature


g p

Temperature

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.1

Psychrotrophs

Grow between 0°C and 20-30°C

Cause food spoilage


Psychrotrophs


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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


p g


Osmotic pressure

Hypertonic environments, increase salt or sugar,

cause plasmolysis

Extreme or obligate halophiles require high osmotic


g p q g

pressure

Facultative halophiles tolerate high osmotic pressure



The Requirements for Growth: Chemical Requirements

Carbon

Structural organic molecules, energy source

Chemoheterotrophs use organic carbon sources

Autotrophs use CO2


p 2


Nitrogen

In amino acids and proteins

Most bacteria decompose proteins

Some bacteria use NH4+ or NO3

–

A few bacteria use N2 in nitrogen fixation

S lf


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


Trace elements

Inorganic elements required in small amounts

Usually as enzyme cofactors


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Oxygen (O2)

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Table 6.1

Toxic Forms of Oxygen

Singlet oxygen: O2 boosted to a higher-energy state

Superoxide free radicals: O2–


Peroxide anion: O22–

Hydroxyl radical (OH•)


Organic growth factors

Organic compounds obtained from the environment

Vitamins, amino acids, purines, and pyrimidines


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

Agar

Complex polysaccharide

Used as solidifying agent for culture media in Petri

plates, slants, and deeps

Generally not metabolized by microbes


y y

Liquefies at 100°C

Solidifies ~40°C

Culture Media

Chemically defined media: Exact chemical composition

is known

Complex media: Extracts and digests of yeasts, meat,

or plants


p

Nutrient broth

Nutrient agar

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Culture Media

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Tables 6.2, 6.4

Growth in Continuous Culture

A “continuous culture” is an open system in which

fresh media is continuously added to the culture at

a constant rate, and old broth is removed at the

same rate.


This method is accomplished in a device called a

chemostat.

Typically, the concentration of cells will reach an

equilibrium level that remains constant as long as

the nutrient feed is maintained.

Basic Chemostat System


Our Chemostat System


Anaerobic Culture Methods

Reducing media

Contain chemicals (thioglycollate or oxyrase) that

combine O2

Heated to drive off O2


2


Anaerobic

jar


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Anaerobic

chamber


Capnophiles Require High CO2

Candle jar


CO2-packet

Figure 6.7

Selective Media

Suppress unwanted

microbes and

encourage desired

microbes.

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.9b–c

Selective Media

Inhibits the growth of some bacteria while selecting for

the growth of others

Example:

Brilliant Green Agar


g

dyes inhibit the growth of Gram (+) bacteria

selects for Gram (-) bacteria

Most G.I. Tract infections are caused by Gram (-)

bacteria

Selective Media

EMB (Eosin Methylene Blue)

dyes inhibit Gram (+) bacteria

selects for Gram (-) bacteria

G.I. Tract infections caused by Gram (-)


y ( )

bacteria

Differential Media

Make it easy to distinguish colonies of different

microbes.

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.9a

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Some media are both selective and differential

Mannitol salt agar is both selective and differential

Selective:Staphylococcus aereus can grow on mannitol salt agar that has a high concentration of salt; the growth of other organisms will be inhibited


inhibited

Differential:Staphylococcus aureus ferments mannitol and the medium will change colorOther organisms that grow on high salt will grow on mannitol salt agar but may not ferment mannitol; the media will not change colors

Selective and Differential Media

Mannitol Salt Agar

used to identify Staphylococcus aureus

M i l S l A


Mannitol Salt Agar

High salt conc. (7.5%) inhibits most bacteria

sugar Mannitol

pH Indicator (Turns Yellow when acid)

Selective and Differential Media

MacConkey’s Agar

used to identify Salmonella

MacConkey’s Agar


Bile salts and crystal violet (inhibits Gram (+)

bacteria)

lactose

pH Indicator

Many Gram (-) enteric non-pathogenic bacteria can ferment lactose, Salmonella can not

Enrichment Media

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


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

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).


y y g ( )

Streak Plate

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.10a–b

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Preserving Bacteria Cultures

Deep-freezing: –50°to –95°C

Lyophilization (freeze-drying): Frozen (–54° to –72°C)

and dehydrated in a vacuum


Reproduction in Prokaryotes

Binary fission

Budding

Conidiospores (actinomycetes)

Fragmentation of filaments


g

Binary Fission

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.11 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.12b

If 100 cells growing for 5 hours produced 1,720,320

cells:


PLAY Animation: Bacterial Growth


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1. Lag Phase

Bacteria are first introduced into an environment or

media

Bacteria are “checking out” their surroundings

cells are very active metabolically


y y

# of cells changes very little

1 hour to several days

2. Log Phase

Rapid cell growth (exponential growth)

population doubles every generation

microbes are sensitive to adverse conditions

antibiotics


anti-microbial agents

3. Stationary Phase

Death rate = rate of reproduction

cells begin to encounter environmental stress

lack of nutrients

lack of water


not enough space

metabolic wastes

oxygen

pHEndospores would form now

4. Death Phase

Death rate > rate of reproduction

Due to limiting factors in the environment


Measuring Microbial Growth

Direct methods

Plate counts

Filtration

MPN

Indirect methods

Turbidity

Metabolic activity

Dry weight


Direct microscopic count

Dry weight

y g

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Direct Measurements of Microbial Growth

Plate counts: Perform serial dilutions of a sample

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.15, step 1

Plate Count

Inoculate Petri

plates from serial

dilutions


Plate Count

After incubation, count colonies on plates that have

25-250 colonies (CFUs)



Filtration



Multiple tube

MPN test.

Count positive

tubes and


compare to

statistical

MPN table.

Figure 6.18b


Direct microscopic count


Microbe Growth

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