1.0 INTRODUCTION:
Biofilms are
complex microbial communities embedded in an extracellular matrix composed of
proteins, extracellular DNA (eDNA), lipids, and exo-polysaccharides. Biofilm
mode of growth of bacteria provides several advantages, one of the most
essential characteristics of microbial biofilms is that the bacteria stay in the microenvironment as long as the conditions are favorable. Biofilm-associated
cells can adhere irreversibly to a wide variety of surfaces, including living tissues and indwelling medical devices such as catheters, valves,
prostheses, and so forth. They are considered an important virulence factor
that causes persistent chronic and recurrent infections; they are highly
resistant to antibiotics and host immune defenses.
2.0 METHODOLOGY:
·
Microorganisms of required
strain will be procured from authorized vendors cultured from suitable media
supplemented with Mueller-Hinton (MH) agar or Luria–Bertani (LB) media or as
applicable.
· The effect of the test sample on
biofilm formation will be evaluated in 96-well polystyrene flat-bottom plates.
·
300µL of inoculated fresh
trypticase soy yeast broth (TSY) (final concentration 106 CFU/mL) will
be aliquoted into each well of the microplate and cultured in the presence of sublethal
concentrations (75, 50, and 25% of MBC).
·
Wells containing medium and
those without extracts and only with methanol will be served as controls.
·
Plates will be incubated at 37˚C
for 48 h and supernatant will be removed and each well will be washed
thoroughly with sterile distilled water to remove free-floating cells.
·
Plates will be air-dried for
30 min and the biofilm formed will be stained for 15 min at room temperature
with a 0.1% aqueous solution of crystal violet. Following incubation, the excess stain will be removed, and wash the plate three times with sterile distilled
water.
·
Finally, the dye bound to the
cells will be solubilized by adding 250µL of 95% ethanol into each well and
after 15 min of incubation, absorbance will be measured using a microplate reader
at a wavelength of 570 nm.
·
Biofilm determination will be
made using the formula
SBF
= (AB − CW)/G,
Where
SBF is the specific biofilm formation, AB is the absorbance of 570nm of the
attached and stained bacteria, CW is the absorbance of 570nm of the stained
control wells containing only bacteria-free medium, and G is the absorbance of 630nm
of cell growth in broth.
3.0 REFERENCE(S):
3.1 Eduardo Sánchez, Catalina Rivas Morales, Sandra
Castillo, Catalina Leos-Rivas, Ledy García-Becerra, and David Mizael Ortiz
Martínez. Antibacterial and Antibiofilm Activity of Methanolic Plant Extracts
against Nosocomial Microorganisms. Evidence-Based Complementary and Alternative
Medicine Volume 2016, Article ID 1572697, 8 pages http://dx.doi.org/10.1155/2016/1572697.
3.2 Famuyide, I.M., Aro, A.O., Fasina, F.O. et
al. Antibacterial and antibiofilm activity of acetone leaf extracts of
nine under-investigated south
African Eugenia and Syzygium (Myrtaceae) species and their
selectivity indices. BMC Complement Altern Med 19, 141 (2019). https://doi.org/10.1186/s12906-019-2547-z.
3.3 Khan Alam, Dunia A. AlFarraj, SyedaMah-e-Fatima, Muhammad ArfatYameen, Mohamed
SolimanElshikh, Roua M.Alkufeidy, Abd El-Zaher
M.A.Mustafa, PramodBhasme, Maryam K.Alshammari, Noorah A.Alkubaisi, Arshad MehmoodAbbasi, Tatheer AlamNaqvi.
Anti-biofilm activity of plant derived extracts against infectious
pathogen-Pseudomonas aeruginosa PAO1.Journal
of Infection and Public Health. Volume 13, Issue 11, November 2020, Pages 1734-1741.
END OF DOCUMENTS
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