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/anisamples/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

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 6.15 Use of MacConkey agar as a selective and differential medium-overview

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

Chapter 6

Log Growth

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)

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 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

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