Determination of the minimum inhibitory concentration (MIC)
Initially, an overnight culture for every bacterial and fungal strain
was prepared following the Clinical and Laboratory Standards Institute
(CLSI) guidelines. Each microorganism was plated onto Columbia blood
agar plates (CBA) or yeast-cysteine blood agar plates (HCB). Facultative
anaerobic bacteria and Candida albicans were cultivated on CBA
agar plates at 37°C in a 5%-10% CO2 atmosphere for 24
hours. Meanwhile, the anaerobic bacteria were plated on HCB agar plates
and incubated at 37°C for 48 hours within an anaerobic chamber
(Anaerocult, Merck Chemicals GmbH, Darmstadt, Germany). A 0.5 A / 1 A
McFarland standard suspension was generated in 0.9% saline (NaCl) for
facultative anaerobic bacteria and Candida albicans ,
respectively. For the microdilution assay, all facultative anaerobic
strains and Candida albicans were subsequently 1:10 diluted in
BBL™ Mueller Hinton II Broth-Cation-Adjusted (MHB, BD, Heidelberg,
Germany). The anaerobic bacteria were prepared in Wilkins–Chalgren
broth (WCB) at a 0.5 A McFarland standard suspension. As stipulated by
ISO 20776-1:2006, tests involving facultative anaerobic bacteria
required a cell density of approximately 5 x 10^5colony forming units (CFU) per ml, while fungi tests utilized 5 x
10^4 CFU / ml, and tests involving obligate
anaerobic bacteria used 5 x 10^6 CFU / ml.
Subsequently, suitable volumes of the MHB / WCB microbial cultures were
transferred into a 96-well microtiter plate using a multi-channel
pipette. The prepared natural plant extracts were dissolved in dimethyl
sulfoxide (DMSO, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at a
concentration of 100 mg/ml. Concentration series of extract solutions in
DMSO ranged from 10 mg/ml to 0.02 mg/ml, employing dilution levels from
10-fold to 5120-fold. Each well in the 96-well microtiter plate held a
total volume of 100 µl. To rule out any potential antimicrobial effects
of residual DMSO, a parallel dilution series of DMSO was investigated.
Wells containing solely MHB / WCB, as well as a dilution series of 0.1%
chlorhexidine (CHX), served as negative and positive controls for
bacterial growth, respectively. Additionally, wells containing MHB / WCB
and the added microbial strain were designated as growth controls.
Contamination risks were minimized through the use of sterile MHB / WCB.
Subsequently, E. coli , S. aureus , E. faecalis , andC. albicans were incubated at 37°C for 18 hours, while the three
streptococci strains were incubated at 37°C under a 5%-10% CO2
atmosphere for 24 hours. Anaerobic bacteria were maintained at 37°C for
48 hours within an anaerobic jar (Anaerocult, Merck Chemicals GmbH,
Darmstadt, Germany). All assays for each bacterial and fungal strain
were carried out in duplicates, and the highest minimum inhibitory
concentration (MIC) values were considered if MIC values exhibited minor
discrepancies. If differences between two rows exceeded two dilution
levels, the determination involving that specific extract was repeated.
MIC was defined as the lowest concentration of each natural plant
extract that visibly inhibited bacterial growth. The inhibitory impact
of DMSO was factored in if bacterial growth was observed within the
concurrently tested DMSO dilution series.