Methodology is a strategy used by researchers to effectively collect data and attain the

predefined goals of their study. In this study, the methodology is shown through the depiction of
a flowchart as follows.

4th picture. Research flow diagram.

1. Process of using crab shells as coagulants and antibacterial agents:

1. Deproteination

In the deproteinization stage, 50 g of pulverized crab shell was mixed with a 3.5% sodium
hydroxide (NaOH) solution in a 1:10 weight ratio. The solution was subjected to controlled
heating at 65°C for 2 h and agitated using a magnetic stirrer.The composite was subjected to
filtration through a filter cloth and meticulously washed with AquaDES until neutral pH
equilibrium. The blend then undergoes a drying process in an oven set at a precise temperature
of 100 degrees Celsius for an extended period of 24 h.

2. Demineralization

The second phase involves demineralization. The outcome of the deproteinization step was
combined with 1 N hydrochloric acid (HCl) in a 1:10 ratio and heated at 70°C for 2 h under
magnetic stirring. Subsequently, the mixture was filtered and rinsed until a neutral pH was
achieved using AquaDES. Subsequently, the mixture was subjected to a 24-h drying process in
an oven set at 100ºC.

3. Deacetylation

In the final step, known as deacetylation, the demineralized mixture is blended with a 50%
sodium hydroxide (NaOH) solution in a 1:20 ratio. This mixture is stirred and heated to 120°C
for 4 h using a magnetic stirrer. After the heating process, the mixture is subjected to filtering
and thorough washing until it reaches neutral pH. The resulting filtrate was subsequently dried
in an oven maintained at 100°C for 24 h. This controlled process yields chitin extracted from
crab exoskeletons, which is characterized by coagulating and antibacterial properties.

B. Examination
The jar test is a method employed to assess the coagulation capacity of a substance and to
determine the optimal operational parameters (dose) in the context of water and wastewater
treatment processes. The Jar-test procedure employed in this research is outlined as follows:

● The formation of 250 ml water samples is introduced into chemical glassware.

● Chitosan was added in varying doses of 0.05, 0.10, 0.15, and 0.20 g, each subjected to
three replicates.
● The samples were then settled for a period of 24 h, during which the turbidity differentials
were monitored. These observations are then compared with those of alum, which
serves as the control variable.

Picture 5. Testing as a Coagulant

Furthermore, to assess the antibacterial activity of chitosan, we employed the disk diffusion
method, which is a common approach for testing the sensitivity of microorganisms to
antimicrobial agents. This involves using filter paper disks containing antimicrobial agents,
which are placed onto an agar medium previously inoculated with microorganisms.Following
incubation, the appearance of a clear zone in the vicinity of the disk signifies the manifestation
of microbial growth inhibition attributable to the presence of the antimicrobial substance. This
technique is characterized by its expeditiousness and simplicity and is of paramount
significance in the assessment of the susceptibility of microorganisms to antimicrobial agents.
As an initial step, all apparatuses were sterilized using an autoclave. In this experiment, filter
paper was immersed in AquaDes solution containing chitosan at four different doses: 50, 100,
150, and 200mg, along with 50 mg of aluminum-sulfate (alum). In addition, water from the river
was applied to the bacterial medium using an oasis needle, which in this case was sodium agar
that had been solidified and placed in a Petri dish. Then, small circular filter papers that had
been soaked in AquaDes with varying doses of chitosan and 50mg of alum were placed inside
the Petri dish on top of the bacterial media.​​