Microorganisms such as bacteria and archea are important for a plethora of ecological processes such as

nitrogen fixation, organic compound cycling and plant nutrition to name some of them; it is said that all
of the organisms in the biosphere depend on microbial activity (Pace, 1997) hence the importance of
identifying and studying them. There are three important questions that need to be answered when
studying and characterizing microbial entities, the first one is What type of microorganisms is it? the
second one is What is the function of this microorganism and finally What is the role of these
microorganisms in the ecosystem functions? These are the three fundamental questions of microbial
ecology that must be answered by studying microorganisms.
To be able to study a microorganism it is important to have an adequate culture technique. Several
techniques strive to isolate the organism using commercial growth media like nutrient agarose to isolate
a specific microbe by dilution, and then by confirming that the final plate has the required homogeneity
of cells to start the analysis. This can be confirmed by employing a suitable microscopy technique,
indeed the microscopy is a key part of cell study as it allows the localization of fluorescent markers
within the microbial cell (Obara, 2013).
After successful isolation of the microbial cells comes the step of DNA/RNA extraction for further
analysis , the technique used is a combination of chemical and physical methods to achieve lysis, but due
to this process, the lysate contains unwanted contaminants and as such, one extra phase of purification.
One of the fastest, most reliable and inexpensive method to achieve this is by using a silica-gel to absorb
selective nucleic acids; the end product is well suited for applications and further identification such as
polymerase chain reaction (PCR), and TFRLP.
Having genetic material, the next step is to perform a partial community analysis in which PCR is used.
This method uses the DNA/RNA sample as a template to identify the microorganisms. The end product
of PCR reflects genetic signatures for all the organisms that were present in the sample by amplification
of specific genes such as 16S rRNA. Often the 16S rRNA analysis is desirable due to the highly conserved
regions and ample identified data bases of 16S rRNA that make for an easy comparison between the
sample and the database sequences (Rastogi, 2011).
Now that the PCR products are available, a profile of microbial communities in the sample must be
performed, there are several techniques to perform this fingerprinting of the PCR product, one of them
is by electrophoresis in which the sample is elecrophoresed in a gel containing a linear gradient of DNA
denaturing compounds obtaining a series of bands as a product which can be extracted from the gel for

amplification, sequencing and direct comparison from a database (Rastogi, 2011). Another method for
fingerprinting is terminal restriction fragment length polymorphysim (T-RFLP) in which terminal
restriction fragments are labelled with a fluorescent dye in the PCR reaction, this allows for the
detection of only that terminal restriction fragment, simplifying the banding pattern and allowing for
analysis of complex communities (Kirk, 2004). The way to estimate the diversity of the community is by
analyzing the peak heights of the resulting terminal restriction fragments as well as their sizes assuming
that each fragment represents a single operational taxonomic unit (OUT) (Ahmad, 2011).

REFERENCES
Ahmad, I., Ahmad, F., & Pichtel, J. (2011). Microbes and microbial technology: Agricultural and
environmental applications

Kirk, J. L., Beaudette, L. A., Hart, M., Moutoglis, P., Klironomos, J. N., Lee, H., & Trevors, J. T. (2004).
Methods of studying soil microbial diversity. Journal of Microbiological Methods, 58(2), 169-188.
Obara, B., Roberts, M., Armitage, J., & Grau, V. (2013). Bacterial cell identification in differential
interference contrast microscopy images. BMC Bioinformatics, , 134.
Pace, N. R. (1997). A molecular view of microbial diversity and the biosphere. Science, 276(5313), 734740.
Rastogi, G., & Sani, R. K. (2011). Molecular techniques to assess microbial community structure,
function, and dynamics in the environment.