GROWTH AND CULTURING
OF BACTERIA
CHAPTER 6
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Growth and Cell Division

Growth Definition

Binary Fission

Budding
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Bacterial Growth Curve




Lag phase
– Adjustment to nutrients
– Enzyme synthesis
– DNA synthesis
Log phase
– Exponential cell division
Stationary phase
– Nutrient depletion
– Waste build-up
Death phase
– More die than are
produced
– Always a few survivors
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Counting Large Populations


Serial Dilutions/
Standard plate
counts
– Very large
numbers may
be counted
by this
method
Pour plate or
spread plate
techniques are
used
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Direct Counts

Colony Counts
– 300 upper limit

Hemocytometer
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Counting Small Populations

Most Probable Number

Filtration
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Other Measurements

Spectrophotometer

Tube turbidity
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Physical Influences (pH)

pH
– Bacteria
• Prefer pH 5.4 – 8.0
• Most grow at pH 7.0 – 7.4
• Rare acidophiles and alkaliphiles exist
– Lactobacillus
– Vibrio cholerae
– Fungi
• Can tolerate pH 5-6
• Often the only cause of acidic food spoilage
– Fruit and vegetable juices/products
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Physical Influences (Temperature)


Psychrophiles 0oC – 20oC
– Refrigerator spoilage
– Listeria monocytogenes
Mesophiles 10oC – 45oC
– Food / Disease


Thermophiles 40oC – 70oC
– Soil composters
Extreme Thermophiles
70oC – 100oC
– Archaea
9
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Oxygen’s Influence on Growth





OBLIGATE AEROBE (Micrococcus)
– Must use oxygen
OBLIGATE ANAEROBE (Clostridium)
– Must have anaerobic environment
– Why?
• Lack enzymes to degrade oxygen radicals
– Superoxide dismutase (O2-)
– Catalase (H2O2)
MICROAEROPHILE (Campylobacter)
– Low oxygen, high carbon dioxide environment
– Respiration pathway may contain unique electron carriers
FACULTATIVE ANAEROBE (Escherichia coli, Staphylococcus)
– Use oxygen when it is present
– Can also grow anaerobically
AEROTOLERANT ANAEROBE (Lactobacillus)
– Tolerate oxygen
– Do not use oxygen
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Growth positions in Thioglycollate medium
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Physical Influences on Growth




Moisture
– Endospores
– Fungal preferences
Hydrostatic pressure
– Barophiles
– Undersea vents
Osmotic pressure
– High salt/sugar used as
food preservatives
– Halophiles – salt tolerant
Radiation (Xrays, UV, γrays)
– Deinococcus radiodurans
– DNA repair systems
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Nutritional Factors Affecting
Bacterial Growth


C, N, S, P availability
– Organic versus inorganic
Trace elements
– Minerals for enzyme activity

Vitamins
– Organics not synthesized by the cell

Nutritional complexity
– Determined by the enzyme capabilities of the microbe
– Fewer enzymes = more nutritional needs (or complexity)
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Bacterial Interactions

Quorum sensing

Biofilms
– Antibiotic resistances
– Diseases
• Tuberculosis
• Cystic Fibrosis
• Diabetic Foot Ulcers

Toxin production to limit
competitor “groups”

Sociomicrobiology
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Culturing – Plate techniques

Streak Plates
– ¼ “ spacing minimum for
well isolated colonies
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Culturing Plate Techniques

Pour Plates
16
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
?
Culture Media

Types
– Defined
• Precise concentrations
• Research into new microbes
– Complex
• Nutritionally sufficient
• Unknown molecular composition
– ? Beef extract
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Culture Media Purposes

Selective media
– Suppress unwanted

Differential media
– Distinguishes

Enrichment media
– Favors fastidious
microbes

Propagation media
– General growth
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Controlling Oxygen Content

Brewer jar
– Anaerobic
environment
– Chemicals absorb
oxygen

Candle jar
– Burning a candle
creates low O2, high
CO2 environment
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Applications of Culturing

Maintaining cultures

Preserved cultures

Diagnostic tests

Limitations on Culture
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.