Marine Microbial Biodiversity: Lecture 15
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What are the five general (first order) questions that should be considered when studying microbial diversity?

Who's there? What are they doing? Who's doing what? How are they doing it? To what extent?

Outline the general workflow for studying microbial diversity.

The general workflow consists of: Definition of the study question and study design, Sample collection, Sample preservation, Sample processing and data acquisition, Data analysis, Interpretation of results.

The comic on page 4 of the document highlights the difference between the actual scientific method and the ideal scientific method.

True

Which of the following is NOT a problem in studying microbial diversity?

<p>Abundant diversity of microbes</p> Signup and view all the answers

What is the most common technique used for separating biomass from a filtered fluid in microbial diversity studies?

<p>Filtration</p> Signup and view all the answers

Select the method that is NOT used for sampling fluids in marine microbial diversity studies.

<p>Direct swabbing</p> Signup and view all the answers

How are biofilms sampled in marine microbial diversity studies?

<p>Biofilms can be sampled through a syringe, a push corer, or by directly swabbing or scraping the surfaces.</p> Signup and view all the answers

What are three types of corers used for collecting sediment samples from the ocean floor?

<p>Box core</p> Signup and view all the answers

What is the key advantage of using drillships for sampling sediments in deep-sea environments?

<p>Drillships are equipped with advanced dynamic positioning systems and allow for the collection of cylindrical samples that preserve the intact stratification of sediments and rocks, along with associated microorganisms.</p> Signup and view all the answers

Dredging is preferred over other methods for sampling sediments due to its minimal environmental impacts.

<p>False</p> Signup and view all the answers

Name four common preservation strategies used for marine microbial samples, and mention factors considered in choosing a preservation method.

<p>Refrigeration at 4°C, Freezing at -20°C, Freezing at -80°C or liquid nitrogen, Preserving solutions like formaldehyde, glutaraldehyde, RNA later, or PBS. The choice of preservation method depends on the type of downstream analysis that needs to be conducted. This includes whether the research focuses on culturing, viral counts, chemistry, DNA, RNA, proteins, or a combination of these factors.</p> Signup and view all the answers

Explain the difference between culture-dependent and culture-independent techniques in marine microbial diversity studies.

<p>Culture-dependent techniques rely on culturing microorganisms in a laboratory setting to study them, while culture-independent techniques bypass the culturing step and analyze microbial communities directly from environmental samples using methods such as microscopy, molecular biology, or biochemical analysis.</p> Signup and view all the answers

What type of information is obtained from qualitative, semi-quantitative, and quantitative techniques used in microbial diversity studies? Select all that apply.

<p>Exact number of individuals of a specific microbe in a sample</p> Signup and view all the answers

Explain why the choice between culture-dependent and culture-independent techniques is crucial in studying marine microbial diversity.

<p>Given the inability to grow a large portion of marine microbes in a lab setting, it is essential to choose between culture-dependent and culture-independent techniques carefully. While culture-dependent techniques allow for detailed investigation of isolated microorganisms, culture-independent techniques offer a broader perspective on the diversity present in the environment, encompassing both culturable and unculturable microbes.</p> Signup and view all the answers

What technique revolutionized our understanding of marine microbial diversity in the 1970s?

<p>The evolution of molecular biology techniques, particularly DNA analysis, revolutionized our understanding of marine microbial diversity in the 1970s and beyond. It enabled researchers to probe the microbial world in new ways, leading to significant discoveries about microbial diversity and the establishment of a third domain of life: the Archaea.</p> Signup and view all the answers

Describe the significance of fluorescent microscopy in studying marine microbial diversity.

<p>Fluorescent microscopy is a widely used technique in marine microbial diversity research. It enables the counting of microbes in natural samples using DNA staining chemicals that fluoresce under UV light, providing quantitative data on population abundance. Viability staining further differentiates live from dead cells, offering insights into the dynamics of microbial communities. Additionally, FISH (Fluorescence in situ hybridization) uses specific fluorescent nucleic acid probes that detect and localize specific targets, allowing for the visualization of the micron-scale spatial organization of microbes in ecosystems.</p> Signup and view all the answers

What is the key advantage of using confocal laser scanning microscopy (CLSM) in marine microbial diversity studies?

<p>Confocal laser scanning microscopy (CLSM) provides a significant advancement in imaging by enabling the reconstruction of three-dimensional structures within an object. This is achieved by capturing multiple two-dimensional images at different depths within a sample, offering detailed visualization of complex microbial communities and their spatial arrangement within their environment.</p> Signup and view all the answers

Explain the principles behind flow cytometry in marine microbial diversity studies.

<p>Flow cytometry is a technique that uses fluorescent markers to detect and measure physical and chemical characteristics of individual cells in a sample. The cells are passed through a laser beam, and the light scattered is characteristic to each cell's size, shape, and fluorescent properties. Flow cytometry allows for the separation of physically distinct populations of cells, which is especially useful for separating culturable and non-culturable bacteria.</p> Signup and view all the answers

Describe the principle behind activity measurements in marine microbial diversity studies, providing an example.

<p>Activity measurements involve incubating natural samples with specific substrates and monitoring the production of specific compounds or the consumption of substrates over time. This allows researchers to assess the metabolic activities of the microbial community in the sample. For example, the oxidation of lactate by sulfate-reducing bacteria in sediment samples can be tracked by measuring the production of hydrogen sulfide (H₂S) over time.</p> Signup and view all the answers

What is the significance of the 'unculturable dogma' in the context of marine microbial diversity research?

<p>The 'unculturable dogma' refers to the long-held belief that a significant portion of marine microbes could not be cultivated in the laboratory. This presented a significant challenge in studying these microorganisms, as traditional culture-based methods were not effective.</p> Signup and view all the answers

Pure cultures of bacteria are the only officially accepted approach for describing a new microbial species.

<p>True</p> Signup and view all the answers

Where are new species names officially published?

<p>New species names are officially published in the International Journal of Systematic and Evolutionary Microbiology (IJSEM), specifically in the journal of the International Committee on the Systematic of Prokaryotes (ICSP).</p> Signup and view all the answers

Officially described species are readily available to the scientific community without any restrictions.

<p>False</p> Signup and view all the answers

What is the primary advantage of cultivating pure cultures of microorganisms in the laboratory?

<p>Cultivating pure cultures of microorganisms provides a model organism for manipulative experiments, physiological, biochemical, and genetic studies, enabling researchers to study and understand specific functions of these microorganisms.</p> Signup and view all the answers

The term 'microbial dark matter' has been universally accepted by scientists.

<p>False</p> Signup and view all the answers

What are some of the early approaches to studying microbial diversity that relied on microscopy?

<p>Early approaches to microbial diversity studies relied on various microscopy techniques, including light microscopy (which is a general term encompassing techniques like bright-field, dark-field, phase contrast microscopy) transmission electron microscopy (TEM), and scanning electron microscopy (SEM).</p> Signup and view all the answers

Culture-independent techniques have completely replaced culture-dependent approaches in microbial research.

<p>False</p> Signup and view all the answers

What are some of the key aspects of a successful approach to studying microbial diversity?

<p>A successful approach to studying microbial diversity is to combine culture-dependent and culture-independent techniques. Culture-dependent techniques provide a deeper understanding of isolated organisms, while culture-independent techniques offer a broader perspective on the diversity present in the environment. Combining these approaches allows researchers to gain a more complete understanding of both culturable and unculturable microbes.</p> Signup and view all the answers

Explain the concept of a Winogradsky column and its relevance in microbial diversity studies.

<p>A Winogradsky column is an artificial, simplified ecosystem that mimics the conditions found in a natural sediment-water interface. It consists of a glass container filled with mud, water, and various nutrient sources, and is exposed to sunlight. Over time, gradients of oxygen and sulfur develop, creating a microcosm where diverse microbial communities thrive, from oxygenic photosynthetic algae to anaerobic bacteria involved in sulfate reduction and methanogenesis. Researchers can observe the spatial distribution of these communities along these gradients. This provides valuable insights into the interactions and processes occurring within microbial ecosystems.</p> Signup and view all the answers

What are the key ingredients typically included in a Winogradsky column?

<p>Mud, water, carbon sources, sulfur sources, minerals, and vitamins</p> Signup and view all the answers

Describe the role of enrichment cultures in isolating and characterizing specific microorganisms.

<p>Enrichment cultures are designed to preferentially select for specific microorganisms by providing a medium and incubation conditions that favor their growth. The medium contains nutrients that the target organism can use, while other organisms in the sample are unable to grow effectively. After being incubated, the enriched sample is plated onto an appropriate agar medium, where a pure culture can be obtained by isolating a single colony of the desired organism.</p> Signup and view all the answers

What is the main purpose of using microcosms in microbial diversity research?

<p>Microcosms, which are controlled and simplified versions of natural ecosystems, are used in microbial diversity studies to simulate and predict the behavior of natural ecosystems under controlled settings. They allow researchers to study specific ecological processes, such as the influence of disturbances on microbial communities. This can include studying how changes in environmental parameters like temperature, nutrient availability, or salinity affect the relative abundance of different species and the overall functioning of the microbial community. Microcosms can also be employed to understand the ecological role of key species within the microcosm.</p> Signup and view all the answers

What are the two major categories of culture-independent approaches employed to study microbial diversity?

<p>Culture-independent approaches used to study microbial diversity can be categorized based on the approach (microscopy, molecular, biochemical) and the type of information they provide.</p> Signup and view all the answers

The establishment of the Archaea as a third domain of life is directly related to culture-independent techniques.

<p>True</p> Signup and view all the answers

Provide a brief summary of why the study of marine microbial diversity is important in the context of global ecosystems

<p>Marine microbes play a crucial role in global ecosystems, contributing to key biogeochemical cycles like carbon and nutrient cycling, as well as regulating primary production and shaping the food web. They are a vital component in maintaining the stability and function of marine environments and have a profound impact on climate regulation and global biogeochemical processes.</p> Signup and view all the answers

Study Notes

Marine Microbial Biodiversity: Lecture 15

  • The lecture covers methods for studying marine microbial biodiversity
  • Questions to consider when studying microbial biodiversity:
    • Who is present?
    • What are they doing?
    • Who is doing what specific actions?
    • How are they carrying out these actions?
    • To what extent are these activities present?

Approaches to Microbial Diversity

  • A typical workflow for studying microbial diversity includes:
    • Defining the study question and its design
    • Collecting the sample
    • Preserving the sample
    • Processing the sample and acquiring data
    • Analyzing the data
    • Interpreting the results

Approaches to Microbial Diversity: The Scientific Method

  • Observe natural phenomena.
  • Establish hypotheses.
  • Test hypotheses via experiments.
  • Establish theory by repeatedly validating results.

Approaches to Microbial Diversity: Actual Methods

  • Suggest theory based on what funding/managers want.
  • Design minimal experiments to prove the theory is true.
  • Modify the theory to fit the information acquired from the data.
  • Publish results, renaming theory as hypothesis and following the scientific method.

Problems in Studying Microbial Diversity

  • Spatial heterogeneity: Many methods require hundreds of milliliters or tens of grams of sediment or water to test a specific group.
  • Inability to culture: A significant portion of microbial diversity cannot be cultured, making it hard to understand their actions.
  • Taxonomic ambiguities: Defining "species" can be difficult, with high genomic plasticity and horizontal gene transfer among microbes.
  • Technical bias: Different techniques have their unique biases, which need to be considered when interpreting the results.

Sampling for Marine Microbial Biodiversity

  • Methods involve use of hand-operated sterile bottles or containers for acquiring specific small volumes.
  • Seawater pumps are used for collecting and transferring large volumes of water for various applications.
  • The use of different equipment is important depending on the purpose and the need for sampling at different locations.

Sampling Seawater

  • Niskin bottles (cylindrical plastic tubes) are frequently used for collecting water from deeper ocean areas.
  • These bottles can be equipped with spring-loaded or tensioned caps.
  • The deployment and retrieval use a messenger weight, which trips both caps.

Sampling Seawater

  • Some methods use rosette systems to hold multiple bottles simultaneously.
  • Sample volumes can range from 10 to 20 liters per bottle.
  • The bottles are frequently outfitted with sensors to monitor conductivity, temperature, depth, turbidity, dissolved oxygen, fluorescence, and photosynthetically active radiation

Sampling Biomass

  • Filtration separates biomass and filtered fluid
  • Selective filtration differentiates between particle-associated and free-living microbes; it also separates plankton size classes.

Sampling Fluids

  • Fluids (pores, cold seeps, hydrothermal vents) can be sampled with different methods.
  • Common sampling strategies use centrifugation or suction methods, often including push corers or syringes

Sampling Biofilms

  • Biofilms can be sampled using different methods like syringes, or push corers, as well as by swabbing or scraping the surface.

Sampling Sediments

  • Corers collect sediment from the ocean floor using pushing or grabbing methods.
  • Different types of tubular or box corers are implemented depending on sampling objectives and location.
  • Drillships are employed for offshore drilling, having the capability to handle ultra-deepwater sampling and collect sediment.

Sampling Sediments

  • Coring and drilling methods can yield intact sediment and rock structures and their associated microorganism stratifications, revealing the environmental adaptations of diverse microbial habitats

Dredging of Sediments

  • Dredging collects unconsolidated rocks from the ocean floor, facilitating mapping and surveys.
  • Concerns exist related to potential ecological damage or pollution to wildlife resulting from dredging or disturbance of seabed ecosystems.

Sample Preservation

  • Techniques are specific to intended downstream analysis.
  • Methods often involve temperature modulation and the use of preservation solutions.
  • Temperature-based preservation techniques such as refrigeration (4°C), freezing (-20°C, -80°C, liquid nitrogen), include use of solutions like formaldehyde, glutaraldehyde, RNA later, and PBS (depending on the analysis needs).

Sample Analysis

  • Techniques vary depending on the scientific question and analytical principles.
  • Methods can be categorized as either culture-dependent or culture-independent, reflecting whether culturing is a step in the investigation or not..
  • Analytical approaches fall into qualitative, semi-quantitative, and quantitative categories.
  • Techniques employ various principles such as chemical, biochemical, molecular, isotopic, or combined methods.

Geochemical analysis

  • Ion chromatography (IC) and Inductively coupled plasma mass spectrometry (ICP-MS) are used to determine the major ions and trace metals in the environment.
  • The Piper plot is a useful graphical tool for representing and visualizing major ion data from water samples, making it helpful in the classification of water types and the interpretation of relationships among them.

Geochemical analysis: Trace Metals

  • Diagrams illustrate that different conditions influence behaviors related to the chemical forms that trace metals adopt within the environment.

Microbiological Analysis

  • Culture-independent methods are important for probing the natural diversity of microbial communities without the constraint of culturing.
  • Culture-dependent methods are valuable for creating a new model system enabling the exploration of functional diversity through various biological methods.

Microbial Culturing

  • It involves cultivating microbes in controlled environments.
  • Crucial factors include sterile environment, culture media containing nutrients, inoculation process, and incubation within controlled conditions.
  • A colony represents a group of multiplied microbes.

Enrichment Culture

  • It facilitates cultivation by selecting for particular organisms, mimicking the selective pressures in a specific ecological niche.
  • Different media are used to support microbial growth, often under conditions not favorable to the majority of organisms.

Microcosms

  • Mimicking natural ecosystems, microcosms provide controlled environments for studying ecological processes within a particular habitat to understand how disturbances influence the populations of microbes.

Winogradsky Column

  • A layered system mimicking the environment on the ocean floor is used to study different microbial strategies.
  • The system consists of strata including those with high oxygen levels or sulfur gradients, supporting microbial growth and interactions in a simulated natural environment.

Isolation: Pure Culture

  • The process starts with diluting and spreading samples on solid growth media to produce individual colonies.
  • Well-isolated colonies are chosen via sterile tools.

Isolation: Most Probable Numbers

  • A serial dilution and plating method helps in determining numbers of microbes within samples.
  • This method works under conditions where direct counts are difficult or unreliable.

Isolation: High-Throughput Culture

  • It involves diluting or sorting samples to obtain a single cell per well of a microtiter plate.
  • Culture-based methods are robotically monitored over time.

Cultured VS Uncultured

  • Pure cultures create model organisms for physiological, biochemical, and genetic studies that enable researchers to characterize new microbial functions.
  • Isolating organisms from previously uncultured groups can provide valuable insights into microbial ecology and the roles microbes play within ecosystems.

Cultured VS Uncultured

  • "Uncultured" is an operational term, not a definitive characteristic relating to the inherent attribute of an organism.
  • Scientists often refer to uncultured microbes as "microbial dark matter" within their studies.

Culture-Independent Approaches

  • Early methods of study leveraged microscopy, later evolving through molecular biology.
  • The development of DNA-based analysis provided significant advances for investigating microbial diversity.
  • DNA sequencing revolutionized the study of microbial diversity, particularly in the establishment of the archaea domain and the discovery of numerous new microbial species.

Fluorescent Microscopy

  • DNA chemicals that fluoresce under UV light are used to count and quantify microbes in varied samples.
  • This approach allows for population abundance measurement, while viability staining distinguishes living and dead cells.
  • FISH (fluorescence in situ hybridization) uses specific fluorescent probes localized at specific targets, and this is crucial for studying microbial spatial organization within ecosystems.

Confocal Laser Scanning Microscopy

  • Improves image formation contrast and resolution related to micrographs.
  • Optical sectioning yields 3D images of an object's structure and arrangement.

Flow Cytometry

  • Measures physical and chemical traits of cells and particles, utilizing fluorescent markers, light scattering, and sorting.
  • Allows for analyzing population abundance, viability, and separating cell subpopulations.

Activity Measurements

  • Microbial metabolic activity can be quantified by employing selected substrates and monitoring the resultant rates, however, sometimes this may not provide a completely accurate account of in-situ rates.
  • In some instances, direct chemical analysis can be a sufficient method for evaluating microbial activity in environments, such as looking at the fate of lactate in sediment samples with sulfate-reducing bacteria, using specific assays to measure sulfide levels.

This Week's Reading

  • Studies on cultivating marine bacteria illustrate significant developments and focus on overcoming hurdles with cultured vs. uncultured organisms.

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Marine Microbial Biodiversity Lecture 15 PDF

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This quiz covers the key methods and approaches for studying marine microbial biodiversity. Explore questions about the presence and activities of microorganisms, from workflow design to data analysis. Understand how the scientific method applies to microbial diversity research.

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