Research Methods
times with ethanol solution for the first 24
Place and Time of Research
This research was carried out at the
integrated laboratory of the Faculty of
Science and Technology, Sunan Ampel
State Islamic University Surabaya on
November 29 – December 6, 2022.
Tools and Materials
The tools used for bacterial isolation
research are petri dishes, test tubes,
inoculation needles, incubators, spreaders,
vortex mixers, and incubator shakers. The
materials used for bacterial isolation
research are 70% alcohol, NA, TSB, TSA,
EMB media, cotton label paper, aluminum
foil, and samples
Tools used for extraction and antibacterial
tests include mortal pestle, funnels,
erlenmeyer 500 ml, vacuum, rotary
evaporator, analytical scales, measuring
cups 500 ml, drip pipettes, tweezers , and
micro pipettes. The materials used for
extraction and antibacterial tests in this
study were filter paper, disc paper, plastic
warp, ethanol, alcohol 70%, DMSO 20%,
tetracycline, and yellow tips.
Sample Capture and Setup
The sample used in this study was
Avicennia marina mangrove leaves which
are still fresh and have a good shape and are
not deformed. The leaves are cleaned and
washed with clean water (running water)
then dried using an oven with a temperature
of 100 °C for 24 hours. The dried samples
were then ground using a mortal pestle and
then put into erlenmeyer 500 ml.
Extraction
The mashed bay leaf is then macerated 3
hours, after which it is vacuumed and
filtered using filter paper to produce filtrate
1 and residue 1. Residue 1 is then added
with ethanol solution and allowed to stand
for 17 hours and tightly closed using
aluminum foil and plastic warp. Filtrate 1
is then rotary so that mangrove leaf paste
extract is obtained. Residue 1 that has been
allowed to stand for 17 hours is then
filtered using filter paper to produce filtrate
2 and residue 2 which is allowed to stand
again for 7 hours using ethanol solution.
Filtrate 2 is concentrated using a rotary
evaporator to obtain mangrove leaf paste
extract. Residue 2 is then re-filtered so as
to produce a dirotary filtrate to produce an
additional extract of the paste. Mangrove
leaf paste extract is made into one into a
boto, vial and stored in the freezer.
Antibacterial Activity Test
1. Tool Sterilization
The tools used in the test of
antibacterial activity are sterilized in
advance. Glassware is sterilized in
an autoclave at 121°C for 15
minutes while for ose needles and
insets it is sterilized by burning
using spiritus fire.
2. Media Manufacturing NA
(Eschericia coli) and TSA
(Staphylococcus aureus)
Nutrient agar (NA) of 2.2 gr is
dissolved in 100 ml of aquades using
Erlenmeyer, after which it is heated
to a homogeneous state. The
homogenized media was then
sterilized for 15 minutes in an
autoclave with a temperature of
121°C. The sterilized medium is
 then poured on a petri dish of 15 Diameter Categories
ml, after which it is tightly closed Inhibition
using plastic warp. ≥20 mm Very Strong
10-20 mm Strong
3. Manufacture of Antibacterial Test 5-10 mm Keep
Media ≤5 mm Weak
Avicennia marina mangrove leaf
paste extract was weighed as much
as 0.1 gr and 0.2 gr. The weighed
samples were then homogenized
using a DMSO solution of 10 ml
using a vortex mixer. The
manufacture of antibacterial test
media was carried out using
Eschericia coli and Staphylococcus
aureus bacteria with a concentration
of 1% and 2% in each media.
Avicennia marina samples were then
inserted into the medium using disc
paper. The media is tightly closed
using a plastic warp and marked,
after which it is incubated for 2x24
hours at an incubator with a
temperature of 37 °C.

4. Observation and Measurement of

Antibacterial Activity
Observations were made by
repetition 2 times after 1x24 hours
of incubation at an incubator with a
temperature of 37 °C. The purpose
of this observation is to determine
the presence of antibacterial activity
through the inhibitory zone formed
on the medium. The inhibitory zone
formed on the medium is then
measured using a caliper and
recorded in diameter. The diameter
categories of the inhibition zone are
as follows :