Microbial ecology techniques

Culturing Culturing The best way to find about physiology capabilities and phylogenetic characteristics of microorganisms Isolate and grow organisms in pure culture Pure culture methods Streak plate Agar shakes MPN

Enrichment Enrichment – provision of favorable specific conditions for the growth of a particular type of microorganism Help to isolate microorganisms that can metabolize a particular substrate or can live under certain conditions which may be present in very small numbers in the original sample

Non-culturable microorganisms Fewer than 1% of bacteria can be cultured in the laboratory using conventional culturing techniques These microorganisms are not capable of growing under the conditions provided May require some factor(s) that was in the environment or may be living as symbionts in plants and animals not provided in the laboratory Non-culturable methods: Lipid profiling Nucleic acids probing Molecular techniques Microscopy

Lipid profiling Each microorganism having a characteristic pattern of lipid composition and proportions Methods: Fatty acid methyl ester (FAME) analysis Phospholipid-linked fatty acid (PLFA) – for composition and abundance of microbial populations

Figure: 07-01 Caption: Synthesis of the three types of informational macromolecules. Note that in any particular region, only one of the two strands of the DNA double helix is transcribed.

Figure: 07-04 Caption: DNA structure. Complementary and antiparallel nature of DNA. Note that one chain ends in a 59-phosphate group, whereas the other ends in a 39-hydroxyl. The red bases represent the pyrimidines cytosine (C) and thymine (T), and the yellow bases represent the purines adenine (A) and guanine (G).

Figure: 07-13 Caption: DNA replication is a semiconservative process in both prokaryotes and eukaryotes. Note that the new double helices each contain one new and one old strand.

Figure: 07-02a Caption: Contrast of information transfer in prokaryotes and eukaryotes. (a) Prokaryote. A single mRNA often contains more than one coding region (such mRNAs are called polycistronic).

Figure: 07-02b Caption: Contrast of information transfer in prokaryotes and eukaryotes. (b) Eukaryote. Noncoding regions (introns) are removed from the primary RNA transcript before translation. The mRNAs of eukaryotes are almost always monocistronic. Please note the two types of cells are not drawn to scale. A typical prokaryotic cell would be 1 to 2 mm in diameter, and a typical eukaryotic (animal) cell about 25 mm in diameter.

Figure: 07-34a-c Caption: Possible reading frames in an mRNA. An interior sequence of an mRNA is shown. The “correct” (or 0) reading frame is determined by the start codon of the mRNA. (a) The amino acids that would be encoded by this region of the mRNA if the ribosome were in the –1 reading frame. (b) The amino acids that would be encoded if the ribosome were in the correct reading frame. (c) The amino acids that would be encoded if the ribosome were in the +1 reading frame.

Figure: 10-03 Caption: Possible effects of base-pair substitution in a gene encoding a protein: three different protein products from changes in the DNA for a single codon.

Figure: 10-35b Caption: Agarose gel electrophoresis of DNA. (b) A photograph of a stained agarose gel. The DNA has been loaded into wells toward the top of the gel as shown, and the positive pole of the electrical field is at the bottom. The sample in lane A is used as a standard where the size of the fragments was known. Using the standards, one can determine the sizes of the fragments in the other lanes. Although each band in a lane will contain the same number of fragmented molecules, the bands stain less intensely at the bottom of the gel because the fragments are smaller and chemically there is less DNA to stain.

Nucleic acid techniques Nucleic acid hybridization Bonding together short complementary nucleic acid strands (probes) to a target sequence Probe is radio-labeled Positive hybridization signal means that complementary base-paring has occurred between the probe and the target sequence

Nucleic acid techniques FISH – Fluorescent In-situ Hybridization uses oligonucleotide probe with flourescent molecule Bind to complementary sequence in the rRNA of the 16S subunit of the ribosomes of the bacterial cells Active cells contain large number of ribosomes Visualize and identify microorganisms in their natural environment Usually combined with confocal laser microscopy

Molecular methods PCR – Polymerase Chain Reaction http://www.dnalc.org/ddnalc/resources/animations.html http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf Methodology Denaturation: separation of a double-stranded DNA Annealing: hybridization of the oligonucleotide primers to the template DNA Extension: elongation of the primer-template hybrid Repeats again 25-30 times PCR is temperature-regulated process Temperature chosen for each step are specific for each protocol; the critical temperature is the annealing – higher temperature means more stringent and specific binding, lower temperature means more “mismatch” can occur Primer selection is specific for the type of microorgansm or community of microorganisms

Electrophoresis Amplified PCR products are visualized by running samples on an agarose gel PCR product is stained with ethidium bromide, SYBR Green I, or another dye that has high affinity to bind DNA The gel is run under an electric current Nucleic acids are negatively charged and will run to the positive pole DNA fragments with smaller size will move through the gel faster A standard molecular weight marker is run along with the sample to allow for identification of the size of the PCR product

T-RFLP T-RFLP – terminal restriction fragment length polymorphism The obtained PCR product is digested with restriction enzymes Labeled terminal restriction fragments are generated The products are detected using electrophoresis

Acknowledgments Svetoslava Todorova contributed to the creation of this PowerPoint presentation