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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)
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/anisamples/microbiology/streakplate.html
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 6.12 An example of the use of a selective medium
Fungal coloniesBacterial colonies
pH 7.3 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
No fermentation Acid fermentationwith gas
Durham tube(inverted tubeto trap gas)
Figure 6.16 An anaerobic culture system
Clamp
Chamber
Petri plates
Airtight lid
Envelopecontainingchemicals torelease CO2
and H2
Palladium pelletsto catalyze reactionremoving O2
Methylene blue(anaerobicindicator)
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
Chapter 6
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
Figure 6.20 Typical microbial growth curve
Stationary phase
Death(decline)phaseLog
(exponential)phase
Lag phase
Time
Num
ber o
f liv
e ce
lls (l
og)
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 6.24 The most probable number (MPN) method for estimating microbial numbers
1.0 ml 1.0 ml
1:1001:10Undiluted
Inoculate 1.0 ml intoeach of 5 tubes
Phenol red, pHcolor 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
Growth of Microbial Populations
• Measuring Microbial Growth– Indirect methods
• Metabolic activity• Dry weight• Turbidity