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Exploration of microbial diversity.pptx Dr Thirnahari Ugandhar
The document discusses various methods for exploring microbial diversity, focusing on both culture-dependent and culture-independent techniques. Key culture-dependent methods include plate count, enrichment culture, and community-level physiological profiling (CLPP), which help identify specific microorganisms and their metabolic traits. Culture-independent approaches, such as fatty acid methyl ester (FAME) analysis and phospholipid fatty acid (PLFA) profiling, allow for the assessment of microbial diversity without the need for culturing, offering insights into the broader microbial community in various environments.
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Exploration of microbial diversity.pptx Dr Thirnahari Ugandhar
- 1. Exploration of Microbial Diversity by Dr. ThirunahariUgandhar Associate Prof of Botany Department of Botany Kakatiya Govt College (A) Hanamkonda
- 2. Exploration of MicrobialDiversity Culture-Dependent Methods Plate Count Method Community-Level Physiological Profiles (CLPP) Culture-Independent Methods Fatty Acid Methyl Ester (FAME) Phospholipid Fatty Acid (PLFA) Fluorescent Antibody Technique Fluorescence In Situ Hybridization (FISH) Whole Cell In Situ Hybridization Reporter Genes Thymidine Incorporation
- 3. • Exploration ofMicrobial Diversity: Culture-Dependent Methods • Culture-dependent methods involve isolating and cultivating microorganisms from environmental samples to study their diversity and characteristics. Key methods include: 1. Serial Dilution and Plating: • Diluting samples serially and plating them on agar media to isolate individual colonies. 2. Selective Media: Media is designed to promote specific microorganisms while inhibiting others. • Examples: MacConkey agar (Gram- negative bacteria) and Sabouraud agar (fungi). 3. Enrichment Culture: Growing samples in media that enhance specific microbial groups with unique metabolic traits, such as nitrogen fixation.
- 4. 4. Streak PlateMethod: • Streaking samples on agar plates to separate and isolate individual colonies. 5. Spread Plate Method: • Spread liquid samples evenly over agar plates using a sterile spreader to enumerate and isolate colonies. 6. Microbial Identification: • Techniques like microscopy, biochemical tests, PCR, and gene sequencing (e.g., 16S rRNA for bacteria, ITS region for fungi). 7. Pure Culture Maintenance: • Isolated colonies are transferred and maintained as pure cultures for further study. • These methods reveal microorganisms' diversity, abundance, and physiology in different environments. However, as many microorganisms have specific growth requirements, culture- independent methods like metagenomics are used alongside these approaches for a broader understanding of microbial diversity.
- 5. • Exploration ofMicrobial Diversity: Culture-Dependent Methods • Microbial diversity can be explored using various methods, with culture-dependent techniques being one approach. • 1. Definition: • Microbes are isolated and grown in controlled laboratory conditions using specific media and techniques in culture-dependent methods. • 2. Steps in Culture-Dependent Exploration: • Sample Collection: Soil, water, air, or other habitats are sampled. • Preparation of Media: Nutrient-rich media (e.g., agar plates, broth) are prepared to support microbial growth. • Culturing: Samples are inoculated onto the media and incubated under appropriate conditions (temperature, pH, oxygen levels). • Observation: Colonies are observed for size, shape, color, and other characteristics
- 7. • 3. Advantages: •Isolation of specific microbes for further study. • Identification of metabolic traits and antibiotic production. • Enables experimental studies on microbial physiology and genetics. • 4. Limitations: • Many microbes are unculturable under standard laboratory conditions, leading to an underestimation of diversity. • Certain microbes require complex or unknown growth conditions. • 5. Examples of Media Types: • General Media: Nutrient agar for non- selective growth. • Selective Media: Contains specific agents to encourage the growth of certain microbes while inhibiting others. • Differential Media: Helps distinguish between microbial types based on biochemical reactions (e.g., blood agar).
- 8. • Applications ofCulture-Dependent Methods in Microbial Diversity 1. Discovery of Novel Antibiotics and Enzymes: Culturing diverse microbes helps identify new bioactive compounds and enzymes for medical and industrial applications. 2. Industrial Fermentation Processes: Utilized in producing food products (e.g., yogurt, cheese), biofuels, and pharmaceuticals through microbial fermentation. 3. Environmental Monitoring and Bioremediation Studies: Aids in isolating microbes capable of breaking down pollutants or monitoring environmental health. 4. Complementary Role in Microbial Research: Culture-dependent methods remain fundamental in microbial studies, complementing molecular and culture- independent approaches to reveal diversity and ecological roles.
- 9. • Plate CountMethod: Overview • The plate count method, also called the viable count method or colony-forming unit (CFU) method, is a widely used microbiological technique to estimate the number of viable microorganisms in a sample. • Steps in the Plate Count Method: • 1. Sample Collection • A sample is collected from the target source, such as soil, water, food, or clinical specimens, containing microorganisms. • 2. Serial Dilution • The sample is diluted stepwise in sterile diluents (e.g., saline or buffer solution) to reduce microbial density, making colonies easier to count on agar plates.
- 10. • Plating • Dilutedaliquots are plated onto solid agar media using techniques like: • Spread Plating: Evenly spreading the sample over the agar surface. • Pour Plating: Mixing the sample with molten agar before solidification. • Plating is often done in duplicates or triplicates for reliability. • 4. Incubation • Plates are incubated under optimal conditions (e.g., temperature, time) for the growth of the target microorganisms, typically for 24–48 hours. • 5. Counting Colonies • After incubation, visible colonies are counted. Each colony corresponds to a viable microorganism in the original sample.
- 11. • 6. Calculations Applications Widely used in environmental microbiology, food safety testing, pharmaceutical quality control, and clinical microbiology. Limitations Counts only viable microorganisms capable of forming colonies under specific growth conditions. May underestimate the total microbial population if some organisms cannot grow on the selected agar medium or under the incubation conditions used. The plate count method remains a reliable and valuable tool for determining the concentration of viable microorganisms in diverse samples.
- 12. Steps Description Sample CollectionCollect a sample from the source (e.g., soil, water, food, or clinical specimen) containing microorganisms. Serial Dilution Dilute the sample stepwise in sterile diluents to reduce microbial density for accurate colony counting. Plating Plate diluted samples using techniques such as spread plating or pour plating; duplicates/triplicates improve reliability. Incubation Incubate plates under optimal conditions (e.g., temperature and time) for 24–48 hours to allow colony growth. Counting Colonies Count visible colonies; each represents a viable microorganism in the original sample. Calculations Applications Used in environmental microbiology, food testing, pharmaceuticals, and clinical microbiology. Limitations Only counts viable microorganisms that grow under specific conditions; some organisms may not form colonies.
- 14. • Culture-Dependent Methods •Plate Count Method • Community-Level Physiological Profiles (CLPP) • Community-level physiological Profiling (CLPP) is a technique for evaluating how microbial communities utilize different carbon sources. • Based on their metabolic activities, it provides insights into the functional diversity of microbial populations.
- 15. Step Description ExpectedOutcome 1. Sample Preparation Environmental sample is diluted to achieve the required microbial concentration. A standardized microbial suspension ready for inoculation. 2. Inoculation The prepared sample is added to the wells of a Biolog microplate containing various carbon substrates. Even distribution of microbial cells across all wells for substrate utilization. 3. Incubation Microplate is incubated under suitable conditions (e.g., 24-48 hours, optimal temperature). Development of metabolic activity as microbes utilize carbon substrates. 4. Data Collection Color development in each well is measured using a plate reader, based on tetrazolium dye reduction. Quantitative data indicating the extent of substrate utilization by the microbial community. 5. Data Analysis Statistical evaluation of carbon utilization patterns using indices like Richness and Evenness or PCA. Functional diversity indices and graphical representations of microbial metabolic capabilities.
- 16. How CLPP Works •1. Inoculation: • Environmental samples are added to Biolog microtiter plates containing wells pre-filled with various carbon substrates and a tetrazolium dye. • 2. Metabolic Activity Detection: • The tetrazolium dye changes color in response to biochemical oxidation when microbes metabolize the carbon substrates. • 3. Data Collection: • The color intensity in each well indicates the level of substrate utilization. This data is used to assess the microbial community’s carbon source utilization patterns (CSUPs
- 17. • Applications ofCLPP • CLPP is widely used for: • Characterizing microbial communities: Understanding the functional roles of microbes in different environments. • Analyzing spatial and temporal trends: Monitoring changes in microbial activity over time or across locations. • Determining growth phase bias: Identifying how microbial communities differ based on their growth stages. • Assessing stressor impacts: Evaluating the effects of environmental stressors like pollutants or temperature changes. • Comparing microbial communities: Classifying and differentiating microbial populations based on their metabolic capabilities.
- 18. • Historical Background •The BIOLOG system, integral to CLPP, was developed in the late 1980s. • Originally designed to identify clinically important bacteria, it has since been adapted for broader ecological and environmental research. • Key Analytical Measures • CLPP uses specific indices to evaluate microbial metabolic activity, including: • 1. Average Well Color Development (AWCD): • Measures the overall metabolic activity of the microbial community. • 2. Richness: • Indicates the number of carbon sources utilized, reflecting community functional diversity.
- 19. Challenges of CLPP •Laboratory Protocol: • The experimental process is straightforward and user- friendly. • Data Analysis: • Interpreting complex data can be challenging, requiring statistical methods and expertise to draw meaningful conclusions. • This structured explanation makes CLPP easier to understand by focusing on its purpose, mechanism, applications, and challenges.
- 21. Exploration of MicrobialDiversity Culture-Dependent Methods Plate Count Method Community-Level Physiological Profiles (CLPP) Culture-Independent Methods Fatty Acid Methyl Ester (FAME) Phospholipid Fatty Acid (PLFA) Fluorescent Antibody Technique Fluorescence In Situ Hybridization (FISH) Whole Cell In Situ Hybridization Reporter Genes Thymidine Incorporation
- 22. • Culture-independent methods: •These methods include denaturing/temperature gradient gel electrophoresis, single-strand conformation • Polymorphism, restriction fragment length polymorphism, terminal restriction fragment length polymorphism, and quantitative polymerase chain reaction (PCR). • The primary advantage of culture- independent methods is their ability to readily identify a large proportion of the bacterial diversity that can be difficult to observe with culture-based studies. • Culture-independent methods allow us to better understand the diversity of microorganisms on Earth. By omitting the culturing step, we can gain access to species previously out of our reach. To better capture and understand the diversity of microorganisms, we have developed an alternative to 16S amplicon sequencing
- 23. Fatty Acid Methyl Ester(FAME) • Fatty Acid Methyl Ester (FAME) analysis measures the fatty acid content of lipid samples from various sources, including microbial cells, foods, oils, and the environment. Here is how it works: • Sample preparation: Lipids are extracted using appropriate procedures, such as Bligh and Dyer or Folch extraction. The extracted lipids undergo transesterification to become fatty acid methyl esters (FAMEs). • Transesterification involves treating extracted lipids with methanol in the presence of an acid catalyst (e.g., sulfuric or hydrochloric acid). This process produces fatty acid methyl esters (FAMEs) and glycerol.
- 24. After separation, FAMEsare identified with a flame ionization detector (FID) or a mass spectrometer (MS). The detector provides signals proportional to the quantity of FAME compounds, allowing for quantification of the sample's fatty acid composition. Separation and Analysis: FAMEs are separated and evaluated by gas chromatography. GC distinguishes between FAMEs based on their volatility and affinity for the stationary phase. Each FAME elutes from the column with a specific retention duration, enabling identification and measurement.
- 25. Data Analysis: Theresulting chromatogram provides information about the types and relative abundance of fatty acids present in the sample. By comparing retention times and peak areas with known standards or reference libraries, fatty acids can be identified and quantified. FAME analysis is widely used in various fields, including microbiology, food science, environmental science, and lipidomics. In microbiology, it is commonly employed to analyze the fatty acid profiles of bacterial and fungal cells for taxonomic identification, microbial community analysis, and physiological studies. In food science, FAME analysis is used to assess the quality and authenticity of fats and oils. Additionally, in environmental science, it can be used to study lipid biomarkers in sediments, soils, and aquatic systems to understand microbial ecology and biogeochemical processes.
- 26. • Exploration ofMicrobial Diversity Culture-Dependent Methods 1.Plate Count Method 2.Community-Level Physiological Profiles (CLPP) Culture-Independent Methods 3.Fatty Acid Methyl Ester (FAME) 4.Phospholipid Fatty Acid (PLFA) 5.Fluorescent Antibody Technique 6.Fluorescence In Situ Hybridization (FISH) 7.Whole Cell In Situ Hybridization 8.Reporter Genes 9.Thymidine Incorporation
- 27. Phospholipid Fatty Acid(PLFA) • Analysis is a robust method for studying microbial community composition and activity in various ecosystems, including soils, sediments, and aquatic environments. • It provides valuable insights into the living microbial biomass and the structure of microbial communities.
- 28. • 2. PhospholipidIsolation: • The extracted lipids are processed to isolate phospholipids using chromatographic techniques such as: • Solid-Phase Extraction (SPE) • Thin-Layer Chromatography (TLC) • Phospholipids are key components of cell membranes, making them reliable indicators of living microbial biomass. • 3. Fatty Acid Methylation: • Isolated phospholipids undergo methylation, where the fatty acids are converted into fatty acid methyl esters (FAMEs). • This step uses methanol and an acid catalyst, similar to transesterification techniques.
- 29. • 4. GasChromatography (GC) Analysis: • The FAMEs are analyzed using gas chromatography (GC). • GC separates FAMEs based on their volatility and interaction with the stationary phase in the column. Each FAME elutes at a specific retention time. • 5. Detection and Identification: • The separated FAMEs are detected using either: • Flame Ionization Detector (FID) • Mass Spectrometer (MS) • A chromatogram is produced, showing peaks corresponding to different fatty acids. • The fatty acids are identified and quantified by comparing the chromatogram to known standards or reference libraries.
- 30. • 6. DataAnalysis: • The fatty acid profile reveals the composition and diversity of the microbial community. • Different microbial groups have characteristic fatty acid profiles, enabling the differentiation of bacteria, fungi, and other microorganisms. • Variations in specific fatty acids provide insights into: Microbial metabolic activity • Responses to environmental changes • Effects of disturbances on microbial communities • Applications of PLFA Analysis • PLFA analysis is widely used in microbial ecology and environmental microbiology for: Assessing living microbial biomass. • Evaluating microbial community composition and diversity. • Monitoring environmental changes and microbial responses. • Studying the effects of stressors like pollutants or climate variations. • Supporting studies in bioremediation and nutrient cycling.
- 31. Integration with MolecularTechniques • PLFA is often combined with molecular methods like 16S rRNA gene sequencing or metagenomics to gain a more comprehensive understanding of microbial communities and their functions. • Advantages of PLFA Analysis • Provides insights into the living microbial community, unlike DNA- based methods that include both active and inactive microbes. • Enables quantitative assessments of microbial biomass. • Offers information about the functional structure of microbial populations. • Limitations: • Cannot provide direct taxonomic resolution, as fatty acid profiles are group-specific but not species-specific. • Requires specialized equipment and expertise for lipid extraction, chromatographic separation, and data interpretation.
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