Non-staining Squid Ink as an Effective Antibacterial Agent

Abhitya Krishnaraj

American Heritage High School

 Abstract

Squid ink has been used for writing and cooking. Past research shows that squid ink has

antibacterial properties. Squid ink stains due to melanin. This research compares the efficacy of

melanin-free squid ink with natural squid ink. Squid ink harvested freshly was centrifuged. The

supernatant, melanin-free squid ink, is colorless. Antibacterial testing was done with the streak

plate method on three bacteria strains: Staphylococcus epidermidis, Escherichia coli, and

Chromobacterium violaceum. The results showed that this non-staining squid ink is more

effective as an antibacterial agent than natural squid ink. This is a seminal study on efficacy of

melanin-free squid ink.

 Introduction

Bacteria are single-celled organisms that were the first life on the planet and are present

in most of its habitats. Over millions of years, bacteria have adapted and evolved resulting in the

numerous varieties of bacteria that exist on the planet today. Many bacteria are beneficial and

required for life to flourish and evolve. However, some pathogenic bacteria are major causes of

human diseases and infections such as tetanus, typhoid, leprosy, tuberculosis etc. There are

many antibiotics available now, but with their widespread use, many species of bacteria are

acquiring resistance to them (Antibiotic Inhibition of Bacteria, n.d). Further use of natural

antibacterial agents, as opposed to chemical, are also an emerging trend. Thus research around

new antibacterial drugs is important. Some research shows that “Squid Ink” assists with

increasing immunity and also with several other natural health benefits. Squid ink has been used

in cooking internationally. For example, arroz negro (Black Rice dish) from Spain, fettuccine al

nero di sepia (Black pasta) from Italy, and Ikasumi jiru (Ink Soup) from Japan.

The purpose of this research is to study the antibacterial effect of the natural squid ink

(NSI), and identify if melanin-free squid ink (MFSI) has a higher antibacterial characteristic.

NSI is composed mainly of melanin (the pigment that influences skin color), but it also contains

peptides, proteins, lipids, minerals (especially iron) and the amino acid taurine, as well as

dopamine. Melanin helps our body in several ways – skin against sun, hair against age, eyes

against age, etc. Even though NSI has been researched for many applications, its antibacterial

effect has been a topic of special interest, and research shows that squid ink has anti-bacterial

effect (Girija, Priyadharshini, Suba, Hariprasad & Raghuraman, 2011) (Girija, Priyadharshini,

Suba, Hariprasad & Raghuraman, 2014). Most of the testing has been NSI in its natural form.
NSI stains, whereas MFSI does not stain, and so far no studies have been done on the efficacy of

MFSI compared to NSI.

The goal of this research is to compare NSI and MFSI on three different bacteria strains,

Staphylococcus epidermidis, Escherichia coli, and Chromobacterium violaceum, using the streak

plate method on Nutrient and Blood Agar plates. The primary hypothesis is that melanin free

squid ink (MFSI) will have a greater effect on bacteria than natural squid ink (NSI). Based on

the efficacy of antibacterial of NSI and MFSI on the three strains of bacteria the research can

show if MFSI, which does not stain, can be used for applications in the real world.

Method

Materials

Squid Ink harvested from fresh squids (~30lbs), sterile dissecting scissors, inoculating

loop, sterile brown bottle, liquid bacterial cultures - Staphylococcus epidermidis, Escherichia

coli, Chromobacterium violaceum, pre-poured nutrient agar plates, Bunsen burner, striker, sterile

gloves, protective glasses, blood agar plates, and centrifuge with 17,500 RPM or higher.

Procedure

The procedure for this research project involves collecting squid ink by carefully

dissecting the squid. The independent variables are squid ink and melanin-free squid ink, and

the effects of those were analyzed on three different bacterial strains: Staphylococcus

epidermidis, Escherichia coli, and Chromobacterium violaceum, using Agar plates.

Collection of squid

1 Approximately 30 lbs of fresh squids were purchased from Finster Murphy’s Fresh Fish and

Seafood Market in Florida, USA. There were more than 100 squids of varying sizes
2 The squid were freshly caught in the New Jersey region, and they were flown to the shop

over night

3 Squid is identified as Loligo forbesii. Scientific classification is as follows:

o Kingdom: Animalia

o Phylum: Mollusca

o Class: Cephalopoda

o Order: Teuthida

o Family: Loliginidae

o Genus: Loligo

o Species: L.forbesii

4 The squid were quickly transported to the lab in a cooler

Ink extraction

1 Clean the squid well to remove any external material

2 Assembled materials required were: a dissecting plate, 2 pairs of sterile scissors, paper

towel, and sterile brown bottles for collection of ink and squids

3 Carefully examine the squid to locate the ink sac

4 Place the squid on a plastic plate with its dorsal side (darker side) up

5 Identify the mantle of the squid; the mantle is the main part of the squid’s body that

contains all of its organs

6 With the squid on its ventral side (lighter side), carefully cut the mantle upwards to avoid

puncturing internal organs. Make the cut all the way to the tip of the tail

7 Find the ink sac, usually on top of the liver, and pull it out with the tip of the finger. Cut it

off from the rest of the squid

8 Cut the ink sac with a pair of sterile scissors. Carefully squeeze the ink sac, and collect the

squid ink in sterile brown bottles.

9 Repeat the above procedure for all of the squids

10 Store the ink at 4oC until its use

11 Dispose the rest of the squid

Preparation of melanin-free squid ink

1 Use one bottle of the squid ink collected earlier

2 Add sterile cold water (4oC) in a 1:1 ratio to prepare the liquid for centrifuging (4oC)

3 Transfer the liquid to sample tubes to use in centrifuge

4 Place the sample tube in one of the slots

5 Set the centrifuge with 17500 RPM speed

6 Set the timer to 30 minutes

7 Close the lid of the centrifuge carefully

8 Switch on the centrifuge (centrifuge is a laboratory equipment that can rotate an object

around a fixed axis and potentially apply strong force on the object)

9 Due to the radical acceleration, denser particles settle to the bottom of the sample tube

whereas low-density substances rises to the top

10 The supernatant (liquid remaining after the centrifugal process) is the melanin free ink

11 Keep the samples at a cool temperature (4oC)

Antibacterial testing

There are several ways to conduct antibacterial testing based on material. The streak

plate method of Agar plates was used in this research. The procedure for it is as follows:

1 For each bacterial culture in the experiment, three Agar plates are needed
 a. Control Plate – Agar plate that contains only the bacterial culture streaked

b. Melanin Plate – Agar plate that was streaked with bacterial culture and squid ink

with melanin

c. Melanin Free Plate – Agar plate that was streaked with bacterial culture and

melanin-free squid ink

2 Nutrient Agar plates are used for Escherichia coli and Staphylococcus epidermidis bacterial

cultures, whereas Blood Agar plates are used for Chromobacterium violaceum

3 Wear gloves to protect and also ensure no contamination

4 Take 3 Nutrient Agar plates. Name the Agar plates on the sides individually with type of

the plate (Control, Melanin, Melanin free), and the name of the bacterium (Escherichia coli)

that will be streaked

5 Light the Bunsen burner with a striker

6 Take the bacterial culture test tube and sterilize the top of it in the Bunsen burner flame to

remove any contamination that may have been found

7 Sterilize the inoculating loop in the Bunsen burner by clicking on the loop and dragging it to

the burner

8 Put the loop into the flame until it is red-hot. Allow it to cool but do not let the loop touch

any other surface to avoid contamination

9 Open the test tube with the Escherichia coli culture and lower the cooled loop just below the

surface of the culture

10 Withdraw the loop without touching the sides of the tube

11 Open the Agar plate and streak ¼ of the plate with the culture

12 Carefully close the plate and keep it aside

13 Repeat the steps 7 through 12 for all the three Agar plates

14 Keep the Agar plate marked Control aside

15 Carefully take the bottle with normal squid ink

16 Open the Agar plate marked as Melanin

17 Sterilize the inoculating loop once more by exposing it to Bunsen burner until it turns red

hot and cool the loop

18 Now carefully pick normal squid ink from the brown bottle using the loop and streak it in

the agar plate closer to where the bacterial culture was streaked

19 Carefully close the plate and keep it aside

20 Take the bottle with melanin free ink

21 Open the Agar plate named Melanin free

22 Repeat the steps 17 through 19 for melanin free ink

23 Put all the three plates in incubator in the temperature 30oC

24 Take three more Nutrient agar plates. Name the agar plates on the sides individually with

type of the plate (Control, Melanin, Melanin free) and name of the bacterium

(Staphylococcus epidermidis) that will be streaked

25 Repeat steps 4 through 23 with Staphylococcus epidermidis bacterial samples

26 Take 3 Blood Agar plates. Name the Agar plates on the sides individually with type of the

plate (Control, Melanin, Melanin free) and name of the bacterium (Chromobacterium

violaceum) that will be streaked

27 Repeat steps 4 through 23 with Chromobacterium violaceum bacterial samples

28 Let the bacteria grow in the incubator and watch the growth closely after 72 hours of

incubation
 Measurements

1 For each agar plate (nine in total), growth area was measured to see how much of the plate

was covered for Control, With Melanin and without Melanin

2 Pictures were taken of the final stage at which the growth was recorded, so that the

observations could be verified

3 Careful checks were done to ensure that results for each agar plate was measured for that

particular bacteria strain and for the type (control, with melanin and without melanin)

Results

Observation

Growth of the bacteria in all the three agar plates was observed after 72 hours and every

24 hours after that for a week. Results were noted down. The results were based on the

percentage of bacteria growth on the plate observed.

Agar plate type Approximate growth area of the bacteria on the plate

Staphylococcus Chromobacterium
epidermidis Escherichia coli violaceum
Control 90% 65% 65%
With Melanin 25% 30% 40%
Without Melanin 20% 25% 35%

Staphylococcus epidermidis observations: The hypothesis is that melanin free squid

ink will have a greater effect on Staphylococcus epidermidis bacteria than natural squid ink.
 Staphylococcus epidermidis
100
% of bacteria growth on the plate 90
80
70
60
50
40
30
20
10
0
Control With Melanin Without Melanin
Nutrient Agar Plate

For the Staphylococcus epidermidis control plate, the bacteria grew steadily and the

bacteria grew over most of the plate (approximately 90%). In the plate with natural squid ink,

the growth was slower and the bacteria did not cross the ink. The bacterium was seen to cover

only 25% of the plate. In the plate with melanin free ink, the growth was slightly restricted and

slow. Bacteria did not grow beyond where the melanin free ink was applied and overall growth

covered approximately 20% area of the plate.

The data supports the hypothesis by showing that the melanin free squid ink had a greater

effect on the Staphylococcus epidermidis than the natural squid ink.

Escherichia coli observations: The hypothesis is that melanin free squid ink will have a

greater effect on Escherichia coli bacteria than natural squid ink.

 Escherichia coli
100
% of bacteria growth on the plate 90
80
70
60
50
40
30
20
10
0
Control With Melanin Without Melanin
Nutrient Agar Plate

For the Escherichia coli’s control plate, bacterial growth was steady and the bacteria grew

on approximately 65% of the plate. In the plate with melanin ink, the bacteria grew around

where the ink was streaked covering 30% of the plate. In the plate with melanin free ink, the

growth was slower and limited. Bacteria did not grow beyond where the melanin free ink was

applied and the overall growth was around 25%. The data shows how the Escherichia coli is less

affected by both squid inks than the Staphylococcus epidermidis, but how melanin free squid ink

is still more effective that the natural squid ink.

Chromobacterium violaceum observations: The hypothesis is that melanin free squid

ink will have a greater effect on bacteria than natural squid ink.
 Chromobacterium violaceum
100
% of bacteria growth on the plate 90
80
70
60
50
40
30
20
10
0
Control With Melanin Without Melanin
Blood Agar Plate

For the Chromobacterium violaceum control plate, bacterial growth was steady and the

bacteria grew on almost 60% of the plate. In the plate with melanin ink, the growth was slower

and the bacteria grew around the squid ink streak. The bacteria grew on 40% of the plate. In the

plate with melanin free ink, the growth was slower and very limited, it is clearly seen that the

bacteria grew around where the ink was streaked. The bacteria only grew on 35% of the plate.

This data shows how the Chromobacterium violaceum is very similar to the Staphylococcus

epidermidis because the melanin free squid ink had only a slightly greater effect on the bacteria

than the squid ink with melanin.

Summary observations:

Results show that the hypothesis of melanin free squid ink being a stronger agent

compared to natural form squid ink is true.

 Comparison of effect on bacteria
100

90
% of bacteria gorwth on the palte

80

70

60
Staphylococcus Epidermidis
50
Escherichia Coli
40
Chromobacterium Violaceum
30

20

10

0
Control With Melanin Without Melanin

Discussions

Summary of findings

The efficacy of natural squid ink and melanin-free squid ink was evaluated against three

different bacterial strains. Streak plate method was used to assess the efficacy of the inks on the

bacterial growth. The results clearly show that squid ink affects the bacterial growth at least by

25% (with Chromobacterium violaceum, bacterial growth on control was 65% whereas the

growth on the plate with natural ink was only 40%). Greater efficacies were observed in the agar

plate with melanin-free squid ink for Staphylococcus epidermidis bacterial strain. In this case

the growth was restricted by 70% (Control plate had 90% of growth whereas melanin-free squid

ink plate had only 20% of bacterial growth). For all the bacteria stains, it is clear that the growth

was restricted on the agar plate when the squid ink was streaked on the plate. The bacteria

colonies either never crossed the squid ink streaks or they grew only in surrounding areas of the
ink but never on the area that was covered with the ink. In case of Staphylococcus epidermidis,

bacterial colonies did not cross the ink streaks in both natural and melanin-free squid inks. In

case of Escherichia coli, colonies grew around the natural squid ink streaks and colonies did not

cross the melanin free squid ink. In case of Chromobacterium violaceum, bacteria only grew in

surrounding areas of both natural and melanin-free squid inks.

Between natural squid ink and melanin-free squid ink, the results are not significantly

different but yet noticeable. In case of Staphylococcus epidermidis, the growth of bacteria was

around 25% of area for natural form ink plate, whereas, for the melanin free ink plate, the growth

was around 20% of area. Thus there is approximately 5% improved efficacy in melanin free ink,

when compared to natural form ink, and 70% improved efficacy comparing to the control

without ink. In case of Escherichia coli, the growth of bacteria was around 30% of area for

natural form ink plate, whereas, the growth was around 25% of area for melanin free ink plate.

Thus there is approximately 5% improved efficacy in melanin free ink, when compared to

natural form ink, and 45% improved efficacy comparing to the control without ink. In case of

Chromobacterium violaceum, the growth of bacteria was around 40% of area for natural form

ink plate whereas the growth was around 35% of area for melanin free ink plate. Thus there is

approximately 5% improved efficacy in melanin free ink, when compared to natural form ink,

and 30% improved efficacy comparing to the control without ink. This collectively shows that

the melanin-free squid ink affected the growth of the bacteria slightly more than the natural squid

ink.

GENERAL DISCUSSION

Squid Ink: Marine water covers approximately 70% of earth’s surface, and marine

ecosystems are among the largest of earth’s aquatic ecosystems. They offer a rich niche for
marine flora and fauna. Besides, they possess very distinctive structures, reproductive systems,

sensory and defense mechanisms due to extreme marine environment (warm to cold temperature

and low to very high pressure). Usage of terrestrial plants and organisms for therapeutic

applications has been a topic of interest among researchers for a long time. In contrast, using

marine organisms for therapeutic application is still in an early stage (Greenwood, 1995). With

their vast genetic and physiological diversity, they are becoming more important topics of

interest in areas like pharmaceuticals and aquaculture.

In specific, Coleoid cephalopods—squid, cuttlefish and octopus— are unique marine

organisms due to their inking behavior. When predators frighten these organisms they squirt ink

to create an ink cloud around them to enable them to hide or just escape. Even though the ink is

composed of many compounds – peptides, melanin, mucus, tyrosinase, dopamine and L-DOPA

and amino acids - melanin has received the most attention due to its black color and

characteristics. Some of the recent research shows that squid ink has anti-bacterial property

(Girija et al., 2011) (Girija et al., 2014). Thus this study was undertaken to study the efficacy of

squid ink with and without melanin.

Bacteria cultures: Even though bacteria are inevitable for life on earth, they are also

cause of some of the major human diseases and infections such as tetanus, typhoid, leprosy,

tuberculosis etc. There are many antibiotics available now, but with widespread use of it many

species of bacteria are acquiring resistance to them (Antibiotic Inhibition of Bacteria, n.d). Thus

research around new antibacterial drugs is important. Staphylococcus epidermidis is a bacterium

that is part of our normal flora. It is an opportunistic pathogen that affects populations that are

susceptible for infection (newborns, elderly, drug users, etc.) typically through biofilms that

grow on medical devices (Otto, 2009). Echerichia coli is commonly found in lower intestine of
warm-blooded organisms. They are typically harmless, but sometimes they can cause serious

food poisoning and contamination. Chromobacterium violaceum is a kind of bacteria isolated

from soil and water in tropical and subtropical regions. Infections caused by Chromobacterium

violaceum are rare among mammals, but a few human infections have been reported (Lee et al.,

1999). Overall, studying the anti-bacterial effect in different bacterial strains will provide best

insight on the efficacy of an antibacterial material and thus all three above bacterial strains were

used in the experiment.

Applications

Squid ink has been used as a writing device in the past centuries. Squid ink is proven to

be rich in Antioxidants, high in iron, and contains glutamic acid. Antioxidants in squid ink are

found in the ink even after the melanin is removed. The antioxidants tend to have strong lipid

prevention; these attributes lead to a lower risk of heart disease in humans. The iron in squid ink,

when consumed, leads to higher red blood cell and hemoglobin counts. Squid ink also adds a

rich flavor to food; this has led to squid ink being used in many cuisines all around the world.

Now, Squid ink research has not only been around its antibacterial characteristics, but also there

is some research that shows that it can be candidate for cancer prevention (Staaf, 2011) (Nithya,

Ambikapathy & Panneerselvam 2011). Given MFSI is colorless, staining will no longer be an

issue. So the applications of using MFSI can be manifold, provided it does not have any other

side effects or impact. As part of the preparation of squid as seafood, the squid ink can be used

to kill bacteria during its preparation. Recently in Japan, McDonalds created a “Black Bun” for

its burger using Squid Ink. It was more for marketing a black burger, but now there might be an

opportunity to see other benefits of using squid ink.

 From this research melanin-free squid ink can be used as an antibacterial agent with

bacteria that requires natural agents to keep the area free of bacteria. There are several types of

antibacterial agents and squid ink can be used to ensure that bacteria does not grow where its

used. In cases where food is prepared certain antibacterial agents should not be used since they

cannot be ingested. In the case of squid ink there are no known side effects on ingestion and so

can be used in areas where food is prepared. Hospitals are another area where use of squid ink as

an antibacterial agent could be used, since chemicals could have other side effects based on

respiratory conditions. Without melanin, the smell of squid ink seems to have changed and so

color and smell could make melanin-free squid ink usable in hospitals as well.

Limitations

One of the main limitations is the amount of squid ink that can be harvested from the

squid. To extract few milliliters of ink, approximately 30lbs of squid was needed. If the squid

ink can be used for therapeutic purposes, it may require large amount of squids to be caught to

harvest sufficient ink. It has to be noted that overfishing can cause issues on sustainability of

these sea creatures. Thus, proper steps need to be taken to make sure the population can be

sustained even if they are used for the therapeutic applications in the future.

Another limitation is the stability of this compound for storage, transportation and shelf

life. Most of the testing done was for fresh caught squid ink, and the squid ink was stored under

cold temperature in sterile brown bottles. If squid ink is to be used for commercial or therapeutic

applications, more work would need to be done on how its constitution would be under normal

conditions, storage and temperature.

Most antibacterial agents today are used in the form of a mixture, and so just efficacy of

squid ink alone is not sufficient. Creating a mixture and testing with that mixture would need to
be done. Comparative efficacy will indicate the benefits. This limitation is critical to determine

how well squid ink performs compared to other agents, to determine if it would be successful in

the market.

Squid ink also has a smell about it. This may not make it usable in certain conditions and

this limitation can be addressed with melanin-free squid ink and other olfactory tests on the

resulting supernatant.

Future Research

In the future, further studies can be done to identify if natural form or melanin free squid

ink is more effective in cancer prevention. This research used squid ink obtained from fresh

squid. Research can also be done to study the anti-bacterial effect of squid ink that is

commercially available. Squid Ink is used for cooking today, and one research project could be

to see if the heat applied while cooking affects its antibacterial property. Another area of

research is to combine other natural antibacterial agents to see the combined impact on bacteria..

Some recent papers have shown that the peptide in squid ink serves as an antioxidant, and this

could be an extension. Understanding the chemical composition of squid ink has not been

completed and could be performed to see if some of this can be synthetically created and have

the same benefits.

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