Methods in Microbial Ecology - PowerPoint PPT Presentation

Loading...

PPT – Methods in Microbial Ecology PowerPoint presentation | free to download - id: 3c893b-MjY3M



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Methods in Microbial Ecology

Description:

CHAPTER 18 Methods in Microbial Ecology Microbial diversity isolation, identification, and quantification Microbial activity in the habitat – PowerPoint PPT presentation

Number of Views:905
Avg rating:3.0/5.0
Slides: 30
Provided by: www2Oakla5
Learn more at: http://www2.oakland.edu
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Methods in Microbial Ecology


1
  • CHAPTER 18
  • Methods in Microbial Ecology

Microbial diversity isolation, identification,
and quantification Microbial activity in the
habitat Methods Enrichment and isolation Cell
staining Gene isolation and characterization Enric
hment culture technique- medium and culture
conditions favors growth of desired organism and
countersellect for undesired organisms Azotobacter
(N2-fixing bacterium) was first bacterium
isolated by enrichment techniques
2
Culture-Dependent Analyses of Microbial
Communities  Enrichment and Isolation
  • Microbial ecology deals with how microorganisms
    interact with one another and their environment.
  • The enrichment culture technique is a means of
    obtaining microorganisms from natural samples.
    Hundreds of different enrichment strategies have
    been devised (Table 18.1).

3
(No Transcript)
4
(No Transcript)
5
(No Transcript)
6
(No Transcript)
7
(No Transcript)
8
  • A classic enrichment strategy is shown in Figure
    18.1.
  • Azotobacter selection

9
  • The Sergei Winogradsky column is a miniature
    anoxic ecosystem that can be used as a long-term
    source of bacteria for enrichment culture
    purposes (Figure 18.2).

Gradient of H2S
10
Thiosperillum Chromatium
Chlorobium Jenense okenii
limicola
Winogradysky columns used for isolation of
sulfate reducers
11
  • Although the enrichment culture is a powerful
    tool, in most enrichments there exists a bias,
    and sometimes a very severe bias, in the outcome.
    Enrichment bias can be demonstrated by comparing
    the results obtained in dilution cultures with
    classical liquid enrichment.
  • Dilution eliminate rapidly growing but
    quantitatively insignificant organisms

12
Isolation in Pure Culture
  • Once a successful enrichment culture has been
    established, a pure culture can be obtained by
    conventional microbiological procedures,
    including streak plates, agar shakes, and
    dilution methods.

13
  • In the most probable number (MPN) technique
    (Figure 18.4), pure cultures can be obtained from
    repeated serial dilutions.

14
Laser tweezers allow one to "pick" a cell from a
microscope field and literally move it away from
contaminants (Figure 18.5).
15
(No Transcript)
16
Molecular (Culture-Independent) Analyses of
Microbial Communities Viability and
Quantification Using Staining Techniques
17
  • DAPI is a general stain for identifying
    microorganisms in natural samples. It stains DNA.

DAPI stained cells
  • Some stains can differentiate live versus dead
    cells, and fluorescent antibodies that are
    specific for one or a small group of related
    cells can be prepared.

Live green Dead red
P. fluorscens tagged with GFP
  • The green fluorescent protein makes cells
    autofluorescent and is a means for tracking cells
    introduced into the environment. Unlike in pure
    cultures, morphologically similar cells may
    actually be quite different genetically in
    natural samples.

18
Genetic Stains
  • A variety of fluorescent-staining methods employ
    the power of nucleic acid probes and thus are
    highly specific in their staining properties.
    These include phylogenetic staining, chromosome
    painting, and reverse transcription fluorescent
    in situ hybridization (FISH).

Ammonia oxiding bacteria red Nitrite oxiding
bacteria green
Sewage sludge - stained with three probes, red,
green and purple Confocal micrograph
19
Linking Specific Genes to Specific Organisms
Using PCR
20
  • The polymerase chain reaction (PCR) can be used
    to amplify specific target genes such as small
    subunit ribosomal RNA genes or key metabolic
    genes (Figure 18.13).

21
  • The polymerase chain reaction (PCR) can be used
    to amplify specific target genes such as small
    subunit ribosomal RNA genes or key metabolic
    genes.

22
  • Denaturing gradient gel electrophoresis (DGGE)
    can be used to resolve slightly to greatly
    different versions of these genes present in the
    various species inhabiting a natural sample.

First PCR six distinct rRNA sequences Separated
by second PCR And DGGE
23
Environmental Genomics (Metagenomics)
  • Environmental genomics involves shotgun
    sequencing and analysis of the collective genomes
    of the organisms present in a microbial community.
  • In environmental genomics, all genes in the
    microbial communitythe metagenomeare sampled.

24
Measuring Microbial Activities in
Nature Radioisotopes and Microelectrodes
25
  • The activity of microorganisms in natural
    samples can be assessed very sensitively using
    radioisotopes and/or microelectrodes (Figure
    18.18a).

26
  • In most cases, measurements are of the net
    activity of a microbial community rather than of
    a population of a single species (Figure 18.16).

27
  • Radioisotopes can be used as measures of
    microbial activity in a microscopic technique
    called microautoradiography (MAR).

28
Stable Isotopes
  • Isotope fractionation can reveal the biological
    origin of various substances.
  • Fractionation is a result of the activity of
    enzymes that discriminate against the heavier
    form of an element when binding their substrates.

29
12C is preferentially fixed compared to 13C by
the enzyme systems
About PowerShow.com