Effect of Medicinal Plants on Biofilm-Forming …
191
(24 cm). The filtrates were then concentrated and were evaporated to dryness in water
bath. The obtained crude extract was stored in airtight container in the refrigerator
at −4 °C for further studies.
2.3 Antimicrobial Activity
The antimicrobial activities of the plant extracts were determined against 30 strong
biofilm-forming clinical strains collected from tertiary health care hospital by agar
well diffusion method. Mueller–Hinton Agar (MHA) plates were prepared and the
sample cultures were swabbed uniformly using sterile cotton swabs. Plates were
incubated at 37 °C for 24 h with the plant extracts in the agar wells (Balouiri et al.
2015).
2.4 Study of Anti-biofilm Activity of Plant Extract
One of the strong biofilm strains, Staphylococcus aureus QS1, was further studied
along with reference strain Staphylococcus aureus ATCC 25923. The effect of all
the plant extracts on biofilm inhibition was studied at concentration of 128*MIC,
256*MIC, and 512*MIC of the extracts. Bacterial cultures with 1 × 10
5 CFU/ml
concentration were transferred to a 96-well polystyrene microtiter plate treated with
different concentrations of plant extracts and incubated for 24 h at 37 °C. Control was
prepared in TSB without plant extracts. After 24 h, the culture medium was discarded,
and the wells were washed twice with distilled water to remove the planktonic cells.
Plates were completely dry, and 1% crystal violet solution was added to each well.
The wells were then washed with distilled water and acetic acid was added to each
well. The optical density (OD) was measured at 570 nm (Quave et al. 2008).
2.5 EPS Production
The selected strain of Staphylococcus aureus QS1 culture was added in freshly
prepared sterile nutrient broth and was treated with Curcuma longa (haldi) and
Azadirachta indica (neem) plant extracts of different concentrations, 128*MIC,
256*MIC, and 512*MIC. Vancomycin was used as the standard antibiotic with
control, i.e., without the addition of plant extracts. The samples were incubated
at 37 °C for 24 h at 120 rpm. Samples were centrifuged and the supernatants were
collected to which ice cold isopropanol was added and incubated overnight. The
incubated samples were centrifuged and EPS pellets were collected. The EPS of
samples was air dried and stored for further analysis (Teanpaisan et al. 2016).
191
(24 cm). The filtrates were then concentrated and were evaporated to dryness in water
bath. The obtained crude extract was stored in airtight container in the refrigerator
at −4 °C for further studies.
2.3 Antimicrobial Activity
The antimicrobial activities of the plant extracts were determined against 30 strong
biofilm-forming clinical strains collected from tertiary health care hospital by agar
well diffusion method. Mueller–Hinton Agar (MHA) plates were prepared and the
sample cultures were swabbed uniformly using sterile cotton swabs. Plates were
incubated at 37 °C for 24 h with the plant extracts in the agar wells (Balouiri et al.
2015).
2.4 Study of Anti-biofilm Activity of Plant Extract
One of the strong biofilm strains, Staphylococcus aureus QS1, was further studied
along with reference strain Staphylococcus aureus ATCC 25923. The effect of all
the plant extracts on biofilm inhibition was studied at concentration of 128*MIC,
256*MIC, and 512*MIC of the extracts. Bacterial cultures with 1 × 10
5 CFU/ml
concentration were transferred to a 96-well polystyrene microtiter plate treated with
different concentrations of plant extracts and incubated for 24 h at 37 °C. Control was
prepared in TSB without plant extracts. After 24 h, the culture medium was discarded,
and the wells were washed twice with distilled water to remove the planktonic cells.
Plates were completely dry, and 1% crystal violet solution was added to each well.
The wells were then washed with distilled water and acetic acid was added to each
well. The optical density (OD) was measured at 570 nm (Quave et al. 2008).
2.5 EPS Production
The selected strain of Staphylococcus aureus QS1 culture was added in freshly
prepared sterile nutrient broth and was treated with Curcuma longa (haldi) and
Azadirachta indica (neem) plant extracts of different concentrations, 128*MIC,
256*MIC, and 512*MIC. Vancomycin was used as the standard antibiotic with
control, i.e., without the addition of plant extracts. The samples were incubated
at 37 °C for 24 h at 120 rpm. Samples were centrifuged and the supernatants were
collected to which ice cold isopropanol was added and incubated overnight. The
incubated samples were centrifuged and EPS pellets were collected. The EPS of
samples was air dried and stored for further analysis (Teanpaisan et al. 2016).