Microbial Growth and Pure Cultures

Microbial Growth

• Microbial growth is an increase in the number of cells, not cell size.

Requirements for Growth

• Physical Requirements:

  • Temperature:
    • Minimum growth temperature
    • Optimum growth temperature
    • Maximum growth temperature
  • pH: Most bacteria grow between pH 6.5 and 7.5; molds and yeasts grow between pH 5 and 6; acidophiles grow in acidic environments; acidity inhibits most microbial growth; alkalinity inhibits microbial growth but is not commonly used for food preservation.
  • Osmotic pressure: Hypertonic environments (higher solute concentration in the surrounding environment than inside a cell) cause plasmolysis. Halophiles require moderate to large salt concentrations, obligate halophiles need high salt concentrations, and facultative halophiles tolerate salt.

• Chemical Requirements:

  • Carbon: Structural backbone of organic molecules; chemoheterotrophs use organic molecules; autotrophs use CO2
  • Nitrogen: Component of proteins, DNA, and ATP; most bacteria decompose protein materials for nitrogen; some use NH4+ or NO3–; others, use molecular nitrogen
  • Sulfur: Used in amino acids, thiamine, and biotin; most bacteria decompose protein for the sulfur source; some use SO4 2- or H2S.
  • Phosphorus: Used in DNA, RNA, and ATP; found in membranes; PO43– is a source of phosphorus.
  • Oxygen:
    • Obligate aerobes require oxygen to survive. A disadvantage is that oxygen isn't found in all environments and dissolves poorly in water
    • Facultative anaerobes prefer oxygen but can survive without it
    • Obligate anaerobes cannot use oxygen and are harmed by its presence
    • Aerotolerant anaerobes cannot use oxygen but tolerate its presence
    • Microaerophiles require oxygen, but at low concentrations, are sensitive to oxygen.

• Trace Elements: Inorganic elements needed in small amounts, usually as enzyme cofactors, including iron, copper, molybdenum, and zinc.

• Organic Growth Factors: Vitamins, amino acids, purines, and pyrimidines obtained from the environment.

Biofilms

• Microbial communities that grow in slime or hydrogel layers adhering to surfaces.
• Bacteria communicate via quorum sensing and share nutrients.
• Shelters bacteria from harmful environmental factors.
• Found in digestive systems, sewage treatment systems, and can clog pipes.
• Resistant to microbial control methods and are involved in 70% of infections.

Culture Media

• Culture media: Nutrients prepared for microbial growth.
• Sterile: No living microbes
• Inoculum: Introduction of microbes into a medium.
• Culture: Microbes growing in or on a culture medium.
• Agar: A solidifying agent in Petri plates, slants, and deeps that is often generally not metabolized by microbes. It liquefies at 100°C and solidifies around 40°C
• Chemically defined media: Exact chemical composition is known ; fastidious organisms require many growth factors
• Complex media: Extracts and digests of yeasts, meat, or plants; chemical composition varies.
• Nutrient broth: Liquid medium.
• Nutrient agar: Solid medium.
• Reducing media: Used for cultivating anaerobic bacteria; contains chemicals like sodium thioglycolate that deplete oxygen; heated to remove oxygen
• Anaerobic chamber: Used for cultivating anaerobic bacteria. This environment is fully enclosed and has conditions that eliminate oxygen (e.g. CO2).

Special Culture Techniques

• Capnophiles: Microbes requiring high CO2 conditions (CO2 packet, candle jar)
• Biosafety levels (BSL): Standards for lab safety in handling microbes (BSL-1 to BSL-4). BSL-4 labs are fully enclosed, and air is filtered prior to exhaust, are highly dangerous for the organisms housed and personnel.

Selective and Differential Media

• Selective media: Suppress unwanted microbes and encourage a specific microbe (e.g., Sabouraud's Dextrose Agar, pH of 5.6 prevents bacterial growth and used to isolate fungi).
• Differential media: Allow distinguishing colonies of different microbes on the same plate.
• Some media are both selective and differentiated.

Enrichment Culture

• Encourages the growth of a desired microbe, even in low numbers, to detectable levels.
• Unlike selective media, does not suppress the growth of other microbes.
• Often used for fecal and soil samples.

Obtaining Pure Cultures

• A pure culture has only one species or strain.
• A colony is a population of cells arising from a single cell (or spore) or a group of attached cells . A colony is often called a colony-forming unit (CFU).
• The streak plate method isolates pure cultures.

Preserving Bacterial Cultures

• Deep-freezing: -50° to -95°C
• Lyophilization (freeze-drying): Frozen (-54° to -72°C) and dehydrated in a vacuum.

Bacterial Division

• Increase in the number of cells, not cell size.
• Binary fission is the most common method.
• Budding, conidiospores (actinomycetes).
• Fragmentation of filaments.

Generation Time

• Time needed for a cell to divide.
• 20 minutes to 24 hours
• Binary fission doubles the number of cells each generation.
• Growth curves are represented logarithmically.

Phases of Growth

• Lag phase: Adjustment to new conditions; little or no cell division; intense metabolic activity.
• Log (or exponential) phase: Rapid growth—constant minimum division time. The number of cells produced is greater than the number dying.
• Stationary phase: Population stabilizes; number produced equals number dying. Factors that decrease growth include accumulation of toxic waste, acidic pH, limited nutrients, and limited oxygen.
• Death phase: Population decreases; number of cells dying is greater than the number of cells produced.Cells lose their ability to divide; although, some cells may remain alive for long periods

Direct Measurement of Microbial Growth

• Plate counts: Counting colonies (CFUs) on plates with 30-300 colonies. Serial dilutions are used to ensure the right number of colonies are counted. Pour plate method and spread plate method are used.
• Filtration: Solution passed through a filter, filter collected bacteria and transferred to the plate.
• Most probable number (MPN) method: Statistical estimation of bacteria in a sample.
• Direct microscopic count: Volume placed; average bacteria per viewing field; Petroff-Hausser cell counter.

Estimating Bacterial Numbers by Indirect Methods

• Turbidity: Measuring cloudiness of culture with a spectrophotometer
• Metabolic activity: Amount of metabolic products (e.g., glucose uptake) is proportional to the number of bacteria.
• Weight (dry weight): Bacteria filtered, dried, and weighed (used for filamentous organisms).