Toxic Forms of Oxygen

(1)

M I C R O B I O L O G Y

a n i n t r o d u c t i o n

ninth edition TORTORA⏐FUNKE⏐CASE

6

PowerPoint®_Lecture_Slide_Presentation_prepared_by_Christine_L._Case

6

Microbial

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

Psychrotrophs

Grow between 0°C and 20-30°C

Cause food spoilage

Psychrotrophs

(2)

The Requirements for Growth:

Physical Requirements

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

The Requirements for Growth:

Physical Requirements

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:

Physical Requirements

The Requirements for Growth:

Chemical Requirements

Carbon

Structural organic molecules, energy source

Chemoheterotrophs use organic carbon sources

Autotrophs use CO2

p 2

The Requirements for Growth:

Chemical Requirements

Nitrogen

In amino acids and proteins

Most bacteria decompose proteins

Some bacteria use NH4+or NO3–

A few bacteria use N2in nitrogen fixation

S lf

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

The Requirements for Growth:

Chemical Requirements

Trace elements

Inorganic elements required in small amounts

Usually as enzyme cofactors

(3)

The Requirements for Growth:

Chemical Requirements

Oxygen (O2)

Toxic Forms of Oxygen

Singlet oxygen: O2boosted to a higher-energy state

Superoxide free radicals: O2–

Peroxide anion: O22–

Hydroxyl radical (OH•)

The Requirements for Growth:

Chemical Requirements

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

(4)

Culture Media

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 Chem ost at Syst em

Our Ch em ost at Syst em

Anaerobic Culture Methods

Reducing media

Contain chemicals (thioglycollate or oxyrase) that combine O2

Heated to drive off O2

Anaerobic Culture Methods

Anaerobic jar

(5)

Anaerobic Culture Methods

Anaerobic chamber

Capnophiles Require High CO

₂

Candle jar

CO2-packet

Figure 6.7

Selective Media

Suppress unwanted microbes and

encourage desired

microbes.

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.

(6)

Some media are both selective and

differential

Mannitol salt agaris both selective and differential

Selective:

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

Differential:

Staphylococcus aureusferments mannitol and the medium will change color

Other 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

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

(7)

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

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

PLAY Animation: Bacterial Growth

(8)

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 doublesevery 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

pH

Endospores 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

(9)

Direct Measurements of Microbial Growth

Plate counts: Perform serial dilutions of a sample

Plate Count

Inoculate Petri plates from serial

dilutions

Plate Count

After incubation, count colonies on plates that have 25-250 colonies (CFUs)

Direct Measurements of Microbial Growth

Filtration

Direct Measurements of Microbial Growth

Multiple tube MPN test.

Count positive tubes and

compare to

statistical

MPN table.

Figure 6.18b

Direct Measurements of Microbial Growth

Direct microscopic count

(10)

Direct Measurements of Microbial Growth

Estimating Bacterial Numbers

by Indirect Methods

Turbidity