Analysis of

Microbial
Diversity
Prepared By: Fahmina Akhtar
Objectives
• By the end of this topic, you will be able to,

• Describe the fundamental reasons for studying

microbial diversity.

• Describe factors governing microbial diversity.

• Differentiate between traditional and molecular

methods of microbial diversity analysis.

• Describe various molecular approaches for

microbial diversity detection.
 FUNDAMENTAL REASONS FOR STUDYING
MICROBIAL DIVERSITY

 Increase the knowledge of the diversity of genetic

resources and understand the distribution of
organisms.

 Increase the knowledge of the functional role of

diversity.

 Identify differences in diversity associated with

management disturbing.

 Understand the regulation of biodiversity.

 Understand the consequences of biodiversity.

FACTORS GOVERNING
MICROBIAL DIVERSITY
• Abiotic factors include both physical and
chemical factors such as water availability, salinity,
oxic/anoxic conditions, temperature, pH,
pressure, chemical pollution, heavy metals,
pesticides, antibiotics etc.

• Biotic factors include plasmids, phages,

transposons that are types of accessory DNA that
influence the genetic properties and, in most
cases,, the phenotypes of their host and thus have
a great influence on microbial diversity.
Traditional
Approach
Study
to Study Culture
organisms
Isolate pure
culture
metabolism
of culture
Microbial
Diversity
Ceylon Journal of Science (Bio. Sci.) 42(1): 19-33, 2013
The Revolution: Polymerase
Chain Reaction
Ceylon Journal of Science (Bio. Sci.) 42(1): 19-33, 2013
The use of ribosomal RNAs and their
genes as reliable phylogenetic markers
for the assessment of the natural
relatedness between isolated and
uncultured prokaryotes combined with
improved PCR and sequencing
technology.

Ligate PCR amplicon

into a cloning plasmid;
transform host
bacterium; isolate
recombinant plasmids
for sequencing of
inserted 16SrDNA.
Why Is The Small Subunit
rRNA Gene So Useful
• Conserved in parts-highly variable in
other parts. Thus, it a very good
phylogenetic marker.

• Very large database of sequences.

• Cells have many ribosomes which

can be targeted with probes
(FISH, TRFLP) for community
analysis.
 DGGE
FISH SIP

Examples of Implication of
16S PCR
TRFLP
 DGGE (Denaturing Gradient Gel Electrophoresis)

Simple Complex

PCR products of mixed communities are loaded on a gel

with a gradient of denaturant.

Typically, 20-80% formamide.

dsDNA will run down the gel until it melts.

Melting determined by sequence and GC content.

Different sequences migrate different distances.

A barcode of the community will obtain.

DGGE (Denaturing Gradient
Gel Electrophoresis)
• Advantages
• Can cut individual bands and clone or
sequence them.
• Can detect very small differences in DNA
sequences.

• Disadvantages

• High complexity samples give smears

• Requires specialized gel rig.
• Acryl-amide is highly toxic.
TRFLP (Terminal Restriction Length Polymorphism

Mixed population is amplified using a

16S primer with a fluorescent tag.

PCR product is cut with a 4bp cutting

restriction endonucleases.

Different sequences will give different

length fragment.

Sample is injected into a capillary

sequencer to sort fragment by size.
TRFLP (Terminal Restriction
Length Polymorphism
• Advantages

• Vary sensitive
• Fast, easy and cheap.

• Disadvantages

• Cannot cut bands to get sequence data.

• Requires capillary sequencer.
• Hard to distinguish noise from little peaks
sometimes.
 Stable-isotope probing

 It is a technique in microbial ecology for tracing fluxes of

nutrients in biogeochemical cycling by microorganisms.
A substrate is enriched with a heavier stable isotope that
is consumed by the organisms to be studied.

 Sequencing the DNA identifies which organisms were

consuming existing carbohydrates and which were using
carbohydrates more recently produced from
photosynthesis. When DNA is the biomarker, SIP can be
performed using isotopically labeled C, H, O, or N,
though 13C is used most often.
 • Fluorescence in situ hybridization (FISH) is a
molecular cytogenetic technique that uses fluorescent
Fluorescence in probe that bind to only those parts of a nucleic acid
sequence with a high degree of sequence

situ hybridization complementarity. Fluorescence microscopy can be used

to find out where the fluorescent probe is bound to the
chromosomes. It was developed by biomedical
researchers in the early 1980s.