Biofilm Formation by microtiter plate assay as well as metal reduction assay.

1. Submitted to : Maam Saba
Submitted by : M.Phil. I Group: 03
Report : Wastewater Analysis
Department Of Microbiology And Molecular Genetics
University Of The Punjab

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Group Members
Sr.
No.
Members Roll No:
1 Ayesha Siddiqa 01
2 Khaleeqa Ahsan 02
3 Shafiqa Shehzadi 03
4 Nigarish Umer 07
5 Rabiya Ikram 08
6 Asma Kalsoom 09
7 Naima Khan 10
8 Mansoor 13

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Introduction
Biofilms are communities of microbes attached to surfaces, which can be found in medical,
industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode
of growth for microbes in most environments. Mature biofilms have a few distinct
characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that
provides structure and protection to the community. Microbes growing in a biofilm also have a
characteristic architecture generally comprised of macrocolonies (containing thousands of cells)
surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their
resistance to a range of antimicrobial agents including clinically relevant antibiotics.
Figure 1 The formation of a biofilm occurs in several stages, comprising the development, maturation and disassembly of
the bacterial community.
Studies of biofilm development of single-strain microorganisms have focused mainly on
clinically relevant bacteria or strains involved in food spoilage, but areas such as high-grade
chemical production, microorganisms in water distribution systems and bioaugmentation through
natural genetic transformation have also been covered. The development and persistence of
biofilms are affected not only by the surrounding environment but also by the variety of species
present.
Microtiter Plate Assay
The microtiter dish assay is an important tool for the study of the early stages in biofilm
formation, and has been applied primarily for the study of bacterial biofilms. Because this assay
uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms
typically associated with flow cell systems. However, the assay has been effective at identifying
many factors required for initiation of biofilm formation and well as genes involved in
extracellular polysaccharide production. Furthermore, published work indicates that biofilms

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grown in microtiter dishes do develop some properties of mature biofilms, such an antibiotic
tolerance and resistance to immune system effectors. This simple microtiter dish assay allows for
the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput
nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by
multiple strains under various growth conditions.
This assay was used to study biofilm formation by the four different strains isolated from
wastewater. In this assay, the extent of biofilm formation is measured using the dye crystal violet
(CV). However, a number of other colorimetric and metabolic stains have been reported for the
quantification of biofilm formation using the microtiter plate assay. The ease, low cost and
flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.
All of the organisms showed biofilm formation on surfaces of polystyrene within 24-48h. The
biofilm formation was studied under metal stress conditions and showed increased slime
production in metal stress as compared to strains without metal stress. So, under stress conditions
these bacteria showed large biofilm production and their ability to resist metal stress.
Reduction Assay
Industrialization and technological advancements, the hallmarks of civilization have been
increasing heavy metal releases to the environment, that pose a significant threat to environment
and public health because of their toxicity, accumulation in the food chain and persistence in
nature. Heavy metals are toxic because they interfere with the normal biochemical reactions of
the human body. The release of heavy metals into the environment causes an environmental
pollution problem because they are nonbiodegradable and hence accumulate in living organisms.
Some metals (e.g., Cd, Co, Cr, Cu, Fe, Ni, Pb and Zn) are essential metals, which serve as
microelements; they are used for redox reactions to stabilize molecules through electrostatic
interactions, as components of various enzymes, and as regulators of osmotic pressure. However,
the presence of high concentrations of heavy metals in the environment can be detrimental to a
variety of living species. The most important features that distinguish heavy metals from other
toxic pollutants are their nonbiodegradability and propensity in living materials. Metal
processing, finishing and plating are the main sources of metal wastes; however, there are also
many other processes from which heavy metals originate.
Metal uptake by microorganisms is a complicated process that depends on the chemistry of the
metal ions, surface features of the microorganisms, cell physiology and physicochemical
influences from the environment, e.g. pH, temperature and metal concentration.
Heavy metals are released into the environment from a wide range of natural and anthropogenic
sources. The rate of influx of these heavy metals into the environment far exceeds their removal
by natural processes, thus leading to the accumulation of heavy metals in the environment, with
aquatic ecosystems normally at the receiving end. The main problems caused by the release of

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toxic pollutants into receiving waters are toxicity to aquatic organisms and restrictions on the
human use of these waters. As a result of natural and industrial processes heavy metals are
increasingly found in microbial habitats. Thus, microbes have evolved several mechanisms to
tolerate the presence of heavy metals (by efflux, complexation, or reduction of metal ions) and
they also use them as terminal electron acceptors in anaerobic respiration.
The intake and subsequent efflux of heavy metal ions by microbes usually include a redox
reaction involving the metal, and some bacteria can use such reactions for energy and growth.
Therefore, bacteria that are resistant to and grow on metals also play an important role in the
biogeochemical cycling of those metal ions. As a result of these characteristics, microbes play an
important role in cleaning up or remediating metal-contaminated environments.
Chromium and nickel are released into the environment by a large number of processes such as
electroplating, leather tanning, wood preservation, pulp processing, steel manufacturing, etc., and
the concentration levels of chromium and nickel in the environment widely varies. These two
metals are of major concern because of their larger usages in developing countries and their non-
degradability nature. Hexavalent chromium is highly soluble in water and carcinogenic to
human. Ni (II) is more toxic and carcinogenic metal when compared with Ni (IV). Due to their
toxic effects on living systems stringent limits have been stipulated for the discharge of
chromium and nickel into the environment.
The aim of this study was to determine whether the bacteria isolated from the wastewaters have
the ability to produce biofilms in the metal stress and if so, then to what level they are producing
the biofilms. So for that purpose two experiments were performed i.e. microtiter plate and metal
reduction assay.

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Figure 2 Biofilm formation by bacterial isolates on a microtiter plate
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References
http://www.ncbi.nlm.nih.gov/pubmed/21307833
http://link.springer.com/article/10.1007/s00253-007-1108-4
http://femsle.oxfordjournals.org/content/283/1/83.full-text.pdf
http://www.sciencedirect.com/science/article/pii/S0045653507004845
http://www.sciencedirect.com/science/article/pii/S0304389406014737