Enrichment

• Enrichment using media devoid of fixed nitrogen


  Such as ammonia or nitrate
• Enrichment culture technique


  • Selects strongly for nitrogen-fixing bacteria and their close relatives
  • Counterselects non-nitrogen fixing bacteria and anaerobic nitrogen-fixing bacteria
• Enrichment Culture Outcomes


  • Attention to both the culture medium and the incubation conditions is important
  • Resources (nutrients) and conditions (temperature, osmotic considerations, etc.) must mimic those of the habitat to give the best chance of obtaining the organism of interest
• Enrichment cultures can yield a firm positive conclusion but never a firm negative conclusion
• The isolation of the desired organism from an enrichment culture says nothing about the ecological importance or abundance of the organism in its habitat
• Winogradsky column


  • An artificial microbial ecosystem and a long-term source of various bacteria for enrichment cultures
  • Used to isolate phototrophic purple and green bacteria, sulfate-reducing bacteria, and many other anaerobes
• Preparing a Winogradsky column

  1. Filling a glass cylinder about half full with organically rich, preferably sulfide-containing mud

  2. Mixing in carbon substrates

  3. The substrates determine which organisms are enriched
• Fermentative substrates, such as glucose, can lead to acidic conditions and excessive gas formation which can create gas pockets that disrupt the column
• We now begin a new unit devoted to microorganisms in their natural habitats
• Microbial communities consist of cell populations living in association with other populations in nature
• Microbial ecology


  The science focused on how microbial populations assemble to form communities and how these communities interact with each other and their environments
• Major components of microbial ecology

  • Biodiversity
  • Microbial activity
• Studying biodiversity


  1. Identify and quantify microorganisms in their habitats

  2. Isolate organisms of interest
• Studying microbial activity


  Measure the metabolic processes that microorganisms carry out in their habitats
• Chapter 19 will outline the basic principles of microbial ecology and examine the types of environments that microorganisms inhabit
• Chapters 20, 21, and 22 will complete the coverage of microbial ecology with a consideration of nutrient cycles, applied microbiology, and the role that microorganisms play in symbiotic associations with higher life forms
• Culture-dependent analyses


  A collection of tools for dissecting the structure and function of microbial communities in relation to their natural habitats
• The vast majority of microorganisms, more than 99% of all species, cannot be cultured using standard laboratory techniques
• The recognition of this fact has stimulated the development of new methods for separating out particular microbial species to establish pure cultures
• Culturing a microorganism remains the only way to fully characterize its properties and predict its impact on an environment
• Enrichment


  A time-honored and useful method for isolating microorganisms from nature but one with limitations
• Enrichment


  1. Culturing in a selective growth medium

  2. Tools and methods used in this approach are referred to as culture-dependent analyses
• Progress has been made in culturing the more elusive microorganisms in natural populations by using robotics to set up large numbers of enrichment cultures that can be monitored using molecular tools
• Culture-independent analyses


  Techniques that can tell us much about the structure and function of microbial communities in the absence of actual laboratory cultures
• Enrichment culture


  A medium and a set of incubation conditions are established that are selective for the desired organism and counterselective for undesired organisms
• Effective enrichment cultures duplicate as closely as possible the resources and conditions of a particular ecological niche
• Hundreds of different enrichment strategies have been devised
• Inoculum


  The sample from the appropriate habitat that is used to start the enrichment culture
• Successful enrichment requires an appropriate inoculum containing the organism of interest
• Making an enrichment culture

  Placing the inoculum into selective media and incubating under specific conditions
• Many common prokaryotes can be isolated using enrichment cultures
• Enrichment cultures can yield a firm positive conclusion but never a firm negative conclusion
• The isolation of the desired organism from an enrichment culture says nothing about the ecological importance or abundance of the organism in its habitat
• Winogradsky column


  An artificial microbial ecosystem and a long-term source of various bacteria for enrichment cultures
• Preparing a Winogradsky column

  1. Filling a glass cylinder about half full with organically rich, preferably sulfide-containing mud into which carbon substrates have been mixed

  2. Covering the mud with water and placing the column near a window that receives diffuse sunlight for months
• Winogradsky columns have been used to isolate phototrophic purple and green bacteria, sulfate-reducing bacteria, and many other anaerobes
• Winogradsky columns can also be supplemented with a specific compound to enrich an organism in the inoculum that can degrade it
• There exists a bias, and sometimes a very severe bias, in the outcome of enrichments, where the most rapidly growing organism(s) for the chosen set of conditions dominate
• Using molecular techniques, we now know that the most rapidly growing organisms in laboratory cultures are often only minor components of the original microbial community
• Sampling with a pipette for microscopy, isolation, and characterization

  Table 18.1