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    Microbiology Methods of Monitoring Populations

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    Stephen Moore
    This document discusses various methods for monitoring microbial populations, including direct counting methods using counting chambers, fluorescent dyes, flow cytometry, and Coulter counters. It also discusses viable counting methods like plating and turbidometry, as well as measuring biomass through wet/dry weight and membrane filtration. Culture-dependent and independent methods are described for assessing microbial diversity, including enrichment, isolation, staining, FISH, PCR-based methods, microarrays, metagenomics, and the effect of temperature on growth.

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    Microbiology Methods of Monitoring Populations

    Uploaded by

    Stephen Moore
    92 views17 pages
    This document discusses various methods for monitoring microbial populations, including direct counting methods using counting chambers, fluorescent dyes, flow cytometry, and Coulter counters. It also discusses viable counting methods like plating and turbidometry, as well as measuring biomass through wet/dry weight and membrane filtration. Culture-dependent and independent methods are described for assessing microbial diversity, including enrichment, isolation, staining, FISH, PCR-based methods, microarrays, metagenomics, and the effect of temperature on growth.

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    This document discusses various methods for monitoring microbial populations, including direct counting methods using counting chambers, fluorescent dyes, flow cytometry, and Coulter counters. It also discusses viable counting methods like plating and turbidometry, as well as measuring biomass through wet/dry weight and membrane filtration. Culture-dependent and independent methods are described for assessing microbial diversity, including enrichment, isolation, staining, FISH, PCR-based methods, microarrays, metagenomics, and the effect of temperature on growth.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    92 views17 pages

    Microbiology Methods of Monitoring Populations

    Uploaded by

    Stephen Moore
    This document discusses various methods for monitoring microbial populations, including direct counting methods using counting chambers, fluorescent dyes, flow cytometry, and Coulter counters. It also discusses viable counting methods like plating and turbidometry, as well as measuring biomass through wet/dry weight and membrane filtration. Culture-dependent and independent methods are described for assessing microbial diversity, including enrichment, isolation, staining, FISH, PCR-based methods, microarrays, metagenomics, and the effect of temperature on growth.

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    of 17

    Methods of monitoring populations

    Direct Measurement of Cell Numbers:

    The most widely used using a counting


    chamber (e.g., Petroff-Hausser counting
    chamber and hemacytometers).
    Counting chambers consist of specially
    designed slides and coverslips; the space
    between the slide and coverslip creates a
    chamber of known depth. On the bottom of
    the chamber is an etched grid that
    facilitates counting the cells.
    The number of microorganisms in a sample
    can be calculated by taking into account
    the chamber's volume and any dilutions
    made of the sample before counting.

    Advanced methods:
    Fluorescent dye methods. Useful for environmental
    samples. After filtration of the env. Samples the
    microorganisms can be viewed by staining with specific
    fluorescent dye ( DAPI, Acridine orange)
    Flow cytometry: A flow cytometer creates a stream of
    cells so narrow that one cell at a time passes through a
    beam of laser light. As each cell passes through the
    beam, the light is scattered. Scattered light is detected
    by the flow cytometer.
    Coulter counter: In the Coulter counter, a microbial
    suspension is forced through a small hole. Electrical
    current flows through the hole, and electrodes placed
    on both sides of the hole measure electrical resistance.
    Every time a microbial cell passes through the hole,
    electrical resistance increases (i.e., the conductivity
    drops), and the cell is counted

    Viable Counting Methods


    Plating methods (TVC): Total viable count
    CFU: Colony forming Unit

    Turbidometry :

    Biomass estimation

    Spectrophotometry : Spectrophotometry depends on the


    fact that microbial cells scatter light that strikes them.
    Because microbial cells in a population are of roughly
    constant size, the amount of scattering is directly
    proportional to the biomass of cells present and indirectly
    related to cell number.
    The extent of light scattering (i.e., decrease in transmitted
    light) can be measured by a spectrophotometer and is
    called the absorbance (optical density) of the medium.
    Absorbance is almost linearly related to cell concentration
    at absorbance levels less than about 0.5. If the sample
    exceeds this value, it must first be diluted and then
    absorbance measured.
    Thus population size can be easily measured as long as the
    population is high enough to give detectable turbidity.

    Wet weight & Dry weight :


    Cells growing in liquid medium are collected by
    centrifugation, washed, and weighed before and after
    drying them.
    This is an especially useful technique for measuring the
    growth of filamentous fungi.

    Membrane filtration methods :


    Traps bacteria in aquatic samples on a membrane filter. The filter is
    then placed on an agar medium or on a pad soaked with liquid media
    and incubated until each cell forms a separate colony.
    A colony count gives the number of microorganisms in the filtered
    sample, and selective media can be used to select for specific
    microorganisms. This technique is especially useful in analyzing water
    purity.

    Measurement of microbial diversity


    Culture dependent
    Culture independent
    Measuring microbial activities (products)

    Culture dependent :
    Enrichment : appropriate inoculum,
    selective medium, The Winogradsky
    Column
    Enrichment Bias

    Isolation :
    Agar plate streaking
    Roll tube methods

    Identification
    Biochemical identification
    Molecular identification techniques

    Culture independent
    General Staining Methods
    Fluorescent Staining with Dyes That Bind Nucleic Acids
    Dyes that stain DNA are widely used for the enumeration of
    microorganisms in environmental, food, and clinical samples.
    DAPI (4,6-diamidino-2-phenyl indole)
    DNA staining is nonspecific; all microorganisms in a sample are
    stained.
    DAPI and acridine orange fail to differentiate between living and
    dead cells or between different species of microorganisms

    Viability Staining
    Viability staining differentiates live cells from dead ones.
    The basis of differentiating between live and dead cells lies with
    whether a cells cytoplasmic membrane is intact.
    Two dyes that fluoresce green and red are added to a sample; the
    green fluorescing dye penetrates all cells, viable or not, whereas the
    red dye, which contains the chemical propidium iodide, penetrates
    only those cells whose cytoplasmic membrane is no longer intact
    and that are therefore dead. Thus, when viewed microscopically,

    Fluorescent In Situ
    Hybridization (FISH);
    The fluorescent probes can be used
    to identify organisms that contain a
    nucleic acid sequence
    complementary to the probe.
    Phylogenetic Staining Using FISH
    Phylogenetic FISH stains are fluorescing
    oligonucleotides complementary in base
    sequence to sequences in ribosomal
    RNA.

    PCR Methods of Microbial


    Community Analysis
    Specific genes are used as measures of
    biodiversity and metabolic capacity
    PCR, Denaturing gradient gel
    electrophoresis (DGGE), Terminal
    restriction fragment length
    polymorphism (T-RFLP)
    Phylotype
    Phylogenic tree

    Microarrays and Microbial


    Diversity: Phylochips
    Phylochips are constructed by
    affixing rRNA probes or rRNA gene
    targeted oligonucleotide probes to
    the chip surface in a known pattern.
    Each phylochip can be made as
    specific or general as required for
    the study by adjusting the
    specificity of the probes, and
    several thousand different probes
    can be added to a single phylochip.

    Metagenomics: A more encompassing approach to


    the molecular study of microbial communities is
    environmental genomics, also called
    metagenomics, which uses the sequencing and
    analysis of all microbial genomes in a particular
    environment as a means of characterizing the entire
    genetic content of that environment.
    Metagenomics was made possible by the
    development of
    extremely high-throughput DNA sequencing

    Environmental factors affecting growth


    Effect of Temperature on Growth
    For every microorganism there is a
    minimum temperature below which
    growth is not possible, an optimum
    temperature at which growth is most
    rapid, and a maximum temperature
    above which growth is not possible.
    These three temperatures, called the
    cardinal temperatures, are
    characteristic for any given
    microorganism.

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