Culturing requirements
Culturing Microorganisms Vocab
• Culture Medium: Nutrients prepared for microbial
growth
• Sterile: No living microbes
• Inoculate: Introduction of microbes into medium
• Incubation – keep (eggs, cells, bacteria, embryos,
etc.) at a suitable temperature so that they
develop
Culturing Microorganisms Vocab
• Culture: Microbes growing in/on culture medium
• Pure culture contains only one species or strain
• Contaminated culture: unwanted microbes
evident
• Isolation - to separate one organism
• 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)
Figure 6.8 Characteristics of bacterial colonies-overview
Culturing Microorganisms
• Obtaining Pure Cultures
– Aseptic technique prevents contamination of
sterile substances or objects
– Two common isolation techniques
• Streak plates
• Pour plates
Figure 6.9 Streak plate method of isolation-overview
http://www.sumanasinc.com/webcontent/anis
amples/microbiology/streakplate.html
Streak Plate
Figure 6.10a, b
Figure 6.10 Pour plate method of isolation-overview
Culturing Microorganisms
• Culture Media
– Majority of prok. have not been cultured
– Six types of general culture media
• Defined media
• Complex media
• Selective media - suppress unwanted microbes and
encourage desired microbes
• Differential media - Make it easy to distinguish colonies of
different microbes
• Anaerobic media
• Transport media
Culture Media
• Chemically Defined Media: Exact chemical
composition is known
• Complex Media: Extracts and digests of
yeasts, meat, or plants
– Nutrient broth
– Nutrient agar
Agar
•
•
•
•
Complex polysaccharide
Generally not metabolized by microbes
Liquefies at 100°C
Solidifies ~40°C
Figure 6.11 Slant tube containing solid media
Slant
Butt
Figure 6.12 An example of the use of a selective medium
Bacterial colonies
pH 7.3
Fungal colonies
pH 5.6
Figure 6.13 The use of blood agar as a differential medium
Beta-hemolysis
Alpha-hemolysis
No hemolysis
(gamme-hemolysis)
Figure 6.14 The use of carbohydrate utilization tubes as differential media
Durham tube
(inverted tube
to trap gas)
No fermentation
Acid fermentation
with gas
Figure 6.15 Use of MacConkey agar as a selective and differential medium-overview
Figure 6.16 An anaerobic culture system
Clamp
Airtight lid
Chamber
Envelope
containing
chemicals to
release CO2
and H2
Petri plates
Palladium pellets
to catalyze reaction
removing O2
Methylene blue
(anaerobic
indicator)
Culturing Microorganisms
• Special Culture Techniques
– Techniques developed for culturing
microorganisms
• Animal and cell culture
• Low-oxygen culture
• Enrichment culture
Culturing Microorganisms
• Preserving Cultures
– Refrigeration
• Stores for short periods of time
– Deep-freezing
• Stores for years
– Lyophilization (freeze-drying): Frozen and
dehydrated in a vacuum
• Stores for decades
Binary Division
• 1 to 2 to 4 to 8 to ?
• Generation Time
– Time required for a
bacterial cell to grow
and divide
– Dependent on
chemical and physical
conditions
Chapter 6
Phases of Growth
• Lag
– Adapt to nutrients
• Log
– Active growth
• Stationary
– Death = Growth rate
• Death
– Nutrients consumed
– pH too low (why?)
• Optimize curves in production
Chapter 6
Log Growth
Chapter 6
Figure 6.20 Typical microbial growth curve
Number of live cells (log)
Stationary phase
Death
(decline)
phase
Log
(exponential)
phase
Lag phase
Time
Chapter 6
Figure 6.17 Binary fission events-overview
Figure 6.18 Comparison of arithmetic and logarithmic growth-overview
Growth of Microbial Populations
• Measuring Microbial Reproduction
– Direct methods
•
•
•
•
•
Serial dilution and viable plate counts
Membrane filtration
Most probable number
Microscopic counts
Electronic counters
Figure 6.22 Estimating microbial population size-overview
Figure 6.23 Use of membrane filtration to estimate microbial population-overview
Figure 6.24 The most probable number (MPN) method for estimating microbial numbers
1.0 ml
Undiluted
1.0 ml
1:10
1:100
Inoculate 1.0 ml into
each of 5 tubes
Phenol red, pH
color indicator,
added
Incubate
Results
4 tubes positive
2 tubes positive
1 tube positive
•
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
Figure 6.25 The use of a cell counter for estimating microbial numbers-overview
Growth of Microbial Populations
• Measuring Microbial Growth
– Indirect methods
• Metabolic activity
• Dry weight
• Turbidity
Figure 6.26 Spectrophotometry-overview
Growth of Microbial Populations
• Measuring Microbial Reproduction
– Genetic methods
• Isolate DNA sequences of unculturable
prokaryotes
– Used to estimate the number of these microbes