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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.