[micro] growth and culturing of bacteria

Growth & Culturing

of Bacteria

Microbiology 130

Chapter 6

Microbial Growth Defined:

Microbial growth is the increase in number of cells,

not cell size

Mother cells

Daughter cells

Cell Division:

Binary fission

Tetrads

Sarcinae

Budding

Microbial Growth and Cell Division

Reproduction in Prokaryotes

Binary fission

Budding

Conidiospores (actinomycetes)

Fragmentation of filaments

Binary Fission

Phases of Growth

4 Phases: 1) Lag phase-

2) Log (logarithmic) phase

3) Stationary phase

4) Decline phase or death phase

Log Phase

Log Phase with Calculations

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

cells:

Log phase

4 Phases of Microbial Growth

Growth in Colonies

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).

Measuring Bacterial Growth Serial Dilutions Direct Measurements of Microbial Growth

Plate counts: Perform serial dilutions of a sample

Standard Plate Count

Inoculate Petri plates from serial dilutions2 methods:Pour PlateSpread Plate

Plate Count

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

Direct Measurements of Microbial Growth

Filtration

Multiple tube

MPN test.

Count positive

tubes and

compare to

statistical

MPN table.

Direct microscopic count

Most Probable Number

Estimate of number

MPN 5 test tubes 10, 1, and 0.1 ml

Growth is displayed by production of gas bubbles or by

becoming cloudy

Estimates are from a known chart ( table 6.1 pg 149)

Other Methods: Estimating Bacterial Numbers by Indirect Methods

Turbidity

Measuring Microbial Growth

Direct methods

Plate counts

Filtration

Direct microscopic count

Dry weight

Indirect methods

Turbidity

Metabolic activity

Dry weight

Factors Affecting Bacterial Growth The Requirements for Growth:

Physical Requirements Temperature

Minimum growth temperature

Optimum growth temperature

Maximum growth temperature

Temperature

Figure 6.1

Psychrotrophs/Psychrophiles

Grow between 0°C and 20-30°C

Cause food spoilage

Psychrotrophs/Psychrophiles

Figure 6.2

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

Mesophiles & Thermophiles

Mesophiles- grow best between 25 degrees C and 40 degrees C

Thermophiles- Heat loving- grow best at 50 - 60 degrees C

Obligate thermophiles

Facultative thermophiles-

The Requirements for Growth: Physical Factors - Chemical Requirements

Oxygen (O2)

Toxic Forms of Oxygen

Singlet oxygen: O2 boosted to a higher-energy state

Superoxide free radicals: O2–

Peroxide anion: O22–

Hydroxyl radical (OH)

The Requirements for Growth: Physical Factors / Requirements

Osmotic pressure

Hypertonic environments, increase salt or sugar,

cause plasmolysis

Extreme or obligate halophiles require high osmotic

pressure

Facultative halophiles tolerate high osmotic pressure

The Requirements for Growth: Physical Requirements

Figure 6.4

The Physical Requirements for Growth

Moisture-

Hydrostatic Pressure-

Radiation-

The Requirements for Growth: Nutritional Factors - Chemical Requirements

Carbon

Structural organic molecules, energy source

Chemoheterotrophs use organic carbon sources

Autotrophs use CO2

The Requirements for Growth: Nutritional Factors - 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

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

The Requirements for Growth: Nutritional Factors - Chemical Requirements

Trace elements

Inorganic elements required in small amounts

Usually as enzyme cofactors

Vitamins- organic substances and growth factors

Organic compounds obtained from the environment

Vitamins, amino acids, purines, and pyrimidines

Nutritional Complexity

Locations of Enzymes

Adaptations to Limited Nutrients

Toxic Forms of Oxygen

Singlet oxygen: O2 boosted to a higher-energy state

Superoxide free radicals: O2–

Peroxide anion: O22–

Hydroxyl radical (OH)

Sporulation / Endospores

Formation of endospores in Bacillus, Clostridium

and G+ genera

Can Survive long periods of drought

Axial nucleoid

Endospore septum grows

Spore coat and Exosporium

Germination- 3 stages:

1) Activation, 2) germination, 3) outgrowth

Culturing Bacteria

Methods of Obtaining Pure Culture 1) The Streak Plate Method – sterile wire loop and

streaked in different patterns on agar

2) Pour Plate Method- serial dilutions using melted

Streak Plate

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

Synthetic media

Defined synthetic media

Complex media

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 ~40°C

Culture Media

Chemically defined media: Exact chemical composition

is known

Complex media: Extracts and digests of yeasts, meat,

or plants

Nutrient broth

Nutrient agar

Culture Media

Tables 6.2, 6.4

Anaerobic Culture Methods

Reducing media

Contain chemicals (thioglycollate or oxyrase) that

combine O2

Heated to drive off O2

Anaerobic

Figure 6.5

Anaerobic

chamber

Figure 6.6

Capnophiles Require High CO2

Candle jar

CO2-packet

Figure 6.7

Selective Media

Suppress unwanted

microbes and

encourage desired

microbes.

Figure 6.9b–c

Differential Media

Make it easy to distinguish colonies of different

microbes.

Figure 6.9a

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

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

Preserving Bacteria Cultures

Deep-freezing: –50°to –95°C

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

and dehydrated in a vacuum

Methods of Performing Multiple Diagnostic Tests

Enterotube Multitest API- simultaneous testing

To identify enteric bacteria- cause food poisoning,

typhoid, shigellosis, gastroenteritis etc

Living, But Non-culturable organisms:

Some microbs can be observed with microscope,

identify their DNA but they can not be cultured

[micro] growth and culturing of bacteria

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