Page 41 - MDJ 2022 Jan-Jun, Volume 45 Number 1

Antibacterial Effect of Ziziphus mauritiana on Streptococcus mutans

by using a rotary evaporator to remove all the solvent The tested bacteria used in this study was
(Figure 2). The collected crude extract was then stored in Streptococcus mutans ATCC 25175 strain obtained from
a 50 mℓ Falcon tube at -80°C. Then it was lyophilized the Research Lab, Faculty of Dentistry, Universiti
using a freeze dryer for 3 days (Figure 3). The extract was Teknologi MARA, Sungai Buloh Campus.
in crystal form and was stored at 4°C for further use.
Preparation of Media

Brain Heart Infusion Agar (BHI agar)

52 g brain heart infusion (BHI) (Oxoid,
Hampshire, England) agar powder was dissolved in 1 litre
(ℓ) of purified water. It was then heated and agitated
frequently until completely dissolved using a hot plate and
a magnetic stirrer. Then, it was autoclaved for 15 minutes
at 121 °C. The sterilised media (20 mℓ) was dispensed into
15×100 mm petri dishes. The media was allowed to
solidify and undergo condensation to dry. Then, the plates
were sterilised under ultraviolet (UV) for 30 minutes then
stored in a sterile plastic bag at 4 °C.

Brain Heart Infusion Broth (BHI broth)
Figure 1: Z. mauritiana leaf powder was soaked in 500 mℓ of
ethanol for three days on a shaker. 37 g of BHI broth powder (Oxoid, Hampshire,
England) was suspended in 1 ℓ of distilled water. It was
then poured into a 1 ℓ Schott bottle and then autoclaved for
15 minutes at 121 °C. The broth was stored at 4 °C until
further use.

Preparation of Bacterial Suspension

The bacterial stock obtained from the Research
Lab was sub-cultured on BHI agar and incubated for 18–
24 hours at 37 °C. The single colony was sub-cultured into
BHI broth for the preparation of bacterial suspension. The
optical density (OD) reading of the bacterial suspension
was adjusted to 0.08–0.13 using a spectrophotometer
Figure 2: Concentrated ethanolic extract of Z. mauritiana 8
leaves. (OD 625 nm) which is equivalent to 10 cells per mℓ prior to
every antimicrobial assay.
Antimicrobial Assay

The antimicrobial assay of the ethanolic extract
of Z. mauritiana leaves were evaluated by antimicrobial
susceptibly test (AST), minimum inhibitory concentration
(MIC), and minimum bactericidal concentration (MBC).

Antimicrobial Susceptibly Test (AST)
The in vitro antibacterial susceptibility test (AST)
of the ethanolic extract of Z. mauritiana leaves was carried
out by disc diffusion method as described by Upadhyay et
11
al. (2015). The extracts were dissolved in 20 % dimethyl
sulfoxide (DMSO) to a final concentration of 1g/mℓ. The
working solution was prepared by diluting the stock
solution to 200 mg/mℓ and 500 mg/mℓ. Chlorhexidine
(CHX) (chlorhexidine digluconate, 0.12 %) and 20 %
Figure 3: Frozen concentrated ethanolic extract of Z. mauritiana
leaves was inserted into a freeze dryer to obtain a powder form DMSO (final concentration: 10 %) were used as positive
of the crude extract. and negative controls respectively. A blank sterilized disc
was impregnated with 20 µℓ of the 200 mg/mℓ and
Bacterial Strains 500 mg/mℓ of plant extract, chlorhexidine and 20 %

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