Biofilm formation by Microtiter plate assay as well as Metal reduction assay

1. BIOFILM FORMATION IN
WASTE WATER TREATMENT
PLANTS
shafiqa shehzadi

2. Biofilms are communities of microbes attached to surfaces,
which can be found in medical, industrial and natural settings.
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.

4. Microtiter Plate Assay
• The microtiter dish assay is an important tool for the study of
the early stages in biofilm formation
• This assay uses static, batch-growth conditions, it does not
allow for the formation of the mature biofilms typically
associated with flow cell systems
• biofilms grown in microtiter dishes do develop some properties
of mature biofilms, such an antibiotic tolerance and resistance
to immune system effectors
• The ease, low cost and flexibility of the microtiter plate assay
has made it a critical tool for the study of biofilms.

5. Protocol of Microtiter plate assay
•It is to determine the biofilm formation ability of bacterial strains.
•In this method bacteria are allowed to grow for 3, 5 and 7 days.
•Before taking the O.D. after respective time a specific procedure was done.
•The media was discarded
•Washed with normal saline twice
•200µl of methanol was added for 15 mins after that it was discarded and air dried
•200µl of 0.1% crystal violet was added for 10 mins
•0.85% NaCl was used to wash it thrice
•It was dried at 30ºC for 30 mins
•200µl of 33% Glacial acetic acid was added and optical density at 578 nm was recorded.

6. Results

7. Metal 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.
• 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.

8. Metal processing, finishing and plating are the main sources of metal
wastes; however, there are also many other processes from which heavy
metals originate.
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.
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.

9. 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.
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.

10. Protocol of Metal reduction assay
•It is to determine the metal utilizing ability of bacterial strains.
•After 24 hours growth cell pellet was made and dried for 24 hours at 100ºC.
•Next day 4mg of pellet was dissolved in 0.5ml of nitric acid and heat shock was
given at 100ºC for 1 hour.
•After that time the eppendofs were cooled at 25ºC and final volume was made
upto 5 ml.
•O.D was taken at 600nm.