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 5

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 9

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 Butt

Figure 6.12 An example of the use of a selective medium Bacterial colonies Fungal 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)

Acid fermentation with gas 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 Palladium pellets to catalyze reaction removing O2 Envelope containing chemicals to release CO2 and H2 Methylene blue (anaerobic indicator) Petri plates

Culturing Microorganisms Special Culture Techniques Techniques developed for culturing microorganisms Animal and cell culture Low-oxygen culture Enrichment culture 19

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 20

Binary Division Generation Time 1 to 2 to 4 to 8 to ? Time required for a bacterial cell to grow and divide Dependent on chemical and physical conditions Chapter 6

Phases of Growth Lag Log Stationary Death 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 Stationary phase Death (decline) phase Log (exponential) phase Number of live cells (log) 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 28

Figure 6.22 Estimating microbial population size-overview

Figure 6.23 Use of membrane filtration to estimate microbial population-overview

Inoculate 1.0 ml into each of 5 tubes Figure 6.24 The most probable number (MPN) method for estimating microbial numbers 1.0 ml 1.0 ml Undiluted 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 36

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 38