The document discusses biofilms formed by bacteria and focuses on Staphylococcus aureus and Pseudomonas aeruginosa. Some key findings include: - Bacteria can adhere to surfaces and form multicellular communities embedded in a self-produced matrix, known as biofilms. This helps bacteria colonize many environments. - S. aureus is a common pathogen that causes skin and soft tissue infections. It can form biofilms on medical implants which contributes to persistent infections. - P. aeruginosa is an opportunistic pathogen that can infect many parts of the body. Its ability to form antibiotic-resistant biofilms complicates treatment. - Experiments studied the effects of growth media, temperature, and time on

1. BIOFILMS

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.

5. Objectives
•

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.

9. Outcome :

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

12. • pH

13. • Time