2. Introduction
• The ability to form biofilms is a universal attribute of
bacteria.
• Biofilms are multicellular communities held together
by a self produced extracellular matrix.
• The extracellular matrix is the network of proteins
and other molecules that surround, support and impart
structure to cells.
• These biofilms helps the bacteria to grow adhered to
almost every surfaces.
• Biofilms impact humans in many ways as they can
form in natural, medical and industrial settings.
3. Staphylococcus aureus
• It is a Gram positive bacteria.
• The natural habitat of S.aureus in humans is the skin
and nasopharynx.
• It can cause a wide variety of infections involving
skin and soft tissues, endovascular sites and internal
organs.
• S.aureus continues to be an important pathogen in
the community and in hospitals, causing high
morbidity and mortality.
• S.aureus attachment to medical implants and host
tissue, and the establishment of mature biofilm, play
an important role in the persistence of chronic
infections
4. Pseudomonas aeruginosa
• It is a ubiquitous Gram negative environmental bacterium
which causes opportunistic human infections.
• A large number of metabolic pathways and regulatory genes
make this bacterium highly adaptive to various growth
conditions.
• The biofilm formed by the bacteria allows it to adhere to any
surface, living or nonliving and thus Pseudomonal infections
can involve any part of the body.
• Pseudomonas can become resistant to certain antibiotics
which complicates the treatment of its infections. This resistant
arises due to its ability to form biofilm.
6. Standardisation of growth medium and
culture conditions:
• Initial experiments were performed with different culture
media, namely Nutrient broth, LB, TSB, Macconky.
• The suitable media supplemented with 0.5%
Glucose,1%glucose,0.6%yeast
extract,0.5%Glu+0.6%Yeast extract and
0.6%YE+1%Glucose was studied.
• The culture condition such as temperature at 25℃, 30℃,
35℃, 37℃,40℃,45℃ and pH at 4,5,6,7,8,9,10 was
studied.
• Bacterial growth was measured as optical density at
610nm using colorimeter.
• The suitable medium and growth conditions for both the
species was selected for further procedures.
7. Cultivation of biofilms:
• A resuspended preculture of individual species was
diluted with LB+YE+1%Glu.
• In addition, 200µL each of the diluted suspension
were added to 96-well sterile microtitre plates, four
wells with 200µL of medium served as blanks.
• To minimize the evaporative loss, the outermost rows
of each plate were filled with sterile water.
• The plates were sealed with parafilm and incubated
at 37 ℃.
8. • The bacterial growth at different hour was studied.
• After incubation, bacterial growth was determined by
measuring the OD at 610nm.
• For assessing the biofilm strength, the method of Djordjevic
et al.(2002) was used with slight modifications.
• The medium was decanted and unattached cells were removed
by rinsing with PBS.
• Then the biofilms were stained by adding crystal violet
solution to each well and incubated for 30mins at room
temperature.
• The stain was solubilized by adding 95% ethanol and
incubated at 4℃.
• The OD was read at 610nm using colorimeter.
10. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 5 10 15 20 25 30
OD
READAINGS[610nm]
TIME [HOURS]
BLANK LB + 0.5% GLU
LB + 1% GLU LB + 0.6% YE
LB + 0.5% GLU + 0.6% YE LB + 1% GLU + 0.6% YE
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 5 10 15 20 25 30
OD
READAINGS[610nm]
TIME [HOURS]
BLANK LB + 0.5% GLU LB + 1% GLU
LB + 0.6% YE LB + 0.5% GLU + 0.6% YE LB + 1% GLU + 0.6% YE
11. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25 30
OD
READAINGS[610nm]
TIME [HOURS]
Growth curve of P.aeruginosa at different temperature
BLANK 25 ̊ C 30 ̊ C 35 ̊ C 37 ̊ C 40 ̊ C 45 ̊ C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 5 10 15 20 25 30
OD
READAINGS[610nm]
TIME [HOURS]
Growth curve of S. aureus at different temperature
BLANK 25 ̊ C 30 ̊ C 35 ̊ C 37 ̊ C 40 ̊ C 45 ̊ C
