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IIB 5. Antibacterial Agents in Seaweeds

BackgroundResearchers studying marine cyanobacteria in order to find novel natural products are finding they contain

an abundance of potent bioactive compounds with promising anticancer, antibiotic, and anti-inflammatory

activity. Marine algae are among the largest producers of biomass in the marine environment. They also

produce a wide variety of chemically active metabolites, potentially to protect themselves against other

organisms. These active metabolites, also known as biogenic compounds, produced by several species of

marine macro- and microalgae, have antibacterial, antialgal, antimacrofouling, and antifungal properties.

Many of the metabolites are novel structures that represent unique biosynthetic pathways. Some of the

most promising compounds function in target cells as tubulin polymerization inhibitors (compounds that

inhibit the formation of cellular microtubules during the process of mitosis), actin polymerization inhibitors(compounds that inhibit development of actin filaments in cells), and neurotoxins.

Numerous promising compounds have been identified in the cyanobacterium Lyngbya majuscula

(mermaids hair or fireweed). This filamentous cyanobacterium can be found in tropical and subtropical

marine and estuarine environments around the world. Some strains cause swimmers itch (see William

Gerwicks PowerPoint presentation Introduction of Drug Discovery from Marine Organisms, (II.

Resources. Powerpoint.Gerwick) on this CD).

The multistep process of drug discovery from marine algae and cyanobacteria involves years of research.

The discovery process starts with the collection of samples followed by screening for biological activity.

One of the strategies for natural product drug screening is a simple toxicity assay using cells such as cancercell lines or microbial cultures, or indicator species such as brine shrimp. Extracts demonstrating significant

activity in either of the assays are chosen for further purification and analysis.

In this experiment, students will explore the first steps in drug development by screening different seaweed

samples for antimicrobial activity. Students will make a crude extract of seaweed or cyanobacteria and test

for the presence of antibacterial properties against gram-positive and gram-negative bacteria. Students will

compare the antibacterial effectiveness of their extract against known antibiotics and disinfectants.

Focus QuestionHow are the antibacterial properties of algal extracts determined?

Learning ObjectivesStudents will learn to extract compounds from algae and test them for antimicrobial activity.C


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MaterialsVarious species of fresh seaweed or marine cyanobacteria

Luria broth cultures of gram-positive and gram-negative bacterial species. Suggested species appropriate for

high school level:

Gram-positiveBacillus subtilis(2530oC)

Bacillus megaterium(2530oC)

Micrococcus luteus(2530oC)

Staphylococcus epidermis(37oC)

Gram-negativeE. coliMM294 (or other K12 strain37oC)Serratia marcescens(2530oC)

Pseudomonas aeruginosa(37oC)

Luria Broth or nutrient agar plates

Disinfectants in small beakersbleach, Lysol, Pinesol, etc.

Antibiotic disksampicillin, penicillin G, tetracycline, streptomycin, erythromycin, etc.

(available from Difco atwww.Difco.com Search for BBL antibiotic susceptibility test disks)

Extraction solventsethanol, methanol, hexane, acetone, etc.

Cold mortar and pestle

Scissors

1.5 ml tubes and microcentrifuge

Microfuge tube rack

20100 l Micropipettors and sterile tips

Forceps

Sterile Petri dishes

Beaker of 95% ethanol

Spreading rods

Bunsen burners and strikers

37oC and 30oC bacterial incubators

Beaker for waste/used tips

Permanent markers

Ice bucket with ice

Sterile 1/4-inch Schleicher and Schuell high-purity paper disks or Whatman filter paper

(cut with a hole puncher)

Teaching TimeThree class periods:

Day 1Extraction

Day 2Antibacterial testing

Day 3Recording results and class discussion

ProcedureCrude Extraction of Seaweed

1. Obtain 1 gram of fresh seaweed (* see notes below for collecting tips, including suggested

species to collect) and remove all epiphytes and decayed areas. Rinse several times in distilled water

and squeeze/pat dry.

2. Cut seaweed into small pieces with scissors or razor blade.

3. Using a cold mortar and pestle, thoroughly mash the chopped seaweed with 1 ml of solvent.

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If needed, add more solvent, but try to limit the amount of solvent, because this will dilute your

extract. Prepare different extracts using the solvents listed above.

4. Scrape the mashed seaweed/liquid into a labeled 1.5 ml microfuge tube and centrifuge for 5

minutes at maximum speed.

5. Carefully pipette out the supernatant to another labeled 1.5 ml tube.

6. (Optional) Filter sterilize the supernatant with a 0.2 m cellulose acetate filter.

Preparation of Disks

1. Using sterile tips, pipette 20 l of each extract onto two 1/4-inch filter paper disks in sterile Petri

dishes and allow to dry (approximately 2030 minutes). Note: if micropipettors are not available,

use the following method. Place forceps in a beaker of 95% ethanol or isopropanol and tap off the

excess alcohol when removing the forceps (or pass the forceps through a flame to burn off the

alcohol). Pick up a sterile disk with the sterilized forceps and dip the disk into the extract. Be sure

to touch the disk to the side of the extract tube and tap it to remove excess liquid, and then place

the disk in a sterile Petri dish to dry.

2. Prepare negative control disks by pipetting 20 l of the solvent in the extraction on two paper

disks in sterile Petri dishes and allow to dry.

3. Prepare positive control disks of a store-bought disinfectant (Lysol, Pine-Sol, bleach, etc.) in the

same manner.

Antibacterial Testing

1. Obtain overnight Luria Broth (LB) suspension cultures of a gram-positive and gram-negative

species.

2. Obtain 2 LB agar plates. With a permanent marker, label the bottom of the plate with your

initials, bacterial species, seaweed extract, and date. Be sure to write along the edge of the plate in

small letters so that your labels will not obscure your view.

3. Sterilize the spreading rod and spread 100 l of bacterial suspension over the surface of eachplate.

Dip spreader into the ethanol beaker and brieflypass it through a Bunsen burner flame

to ignite the alcohol. Allow alcohol to burn away fromthe flame!

Lift lid of one plate only enough to allow spreading; do not place lid on the table.

Cool spreader by gently rubbing it on the surface of the agar away from the cell suspension.

Touch spreader to cell suspension, and gently drag it back and forth several times across

the surface of the agar. Rotate plate on quarter turn, and repeat spreading motion. Be

careful not to gouge the agar.

Replace plate lid. Return cell spreader to ethanol withoutflaming.

NOTE: if alcohol or spreading rods are not available, use sterile Q-tips to spread bacteria

over the surface of the agar.


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4. Using forceps, carefully pick up one of the extract disks and place it on the agar. See figure

below. Press down on each disk to ensure close contact of the disk to the agar. Be sure to label the

bottom of the plate.

5. Place the agar plates lid side down in the appropriate incubator overnight.

6. After 16 to 18 hours of incubation, measure the diameter of any inhibition zones to the nearest

millimeter. Measure from the edge of the disk to the edge of the inhibition zone. If your zone is

irregular, measure the zone at four different places (e.g., 12 oclock, 3 oclock, 6 oclock, and 9

oclock) and calculate an average.

7. Create two data tables: one to record your observations and another for class data. Make

sketches showing the results of your antibacterial tests.

* Collecting tips:

Note 1: When collecting limu in Hawaii please be aware of the problems associated with invasive

species. Place the limu in a bleach solution to kill the cells before discarding. This will help to prevent

the species youve collected from spreading should any find their way back into the water.

Note 2: Collection suggestions for Hawaii: Limu kohu (Asparagopsis taxiformis) and limu alani

(Dictyota sandvicensis) have produced positive results in this bioassay.

Collecting suggestion for species in Oregon: Try this assay with the brown seaweed rockweed (also

known as bladder wrack, Fucus spp). Brown seaweeds are very common on the Oregon coast, and

rockweed is present throughout the year rather than dying back at the end of summer as many sea-

weeds do. It contains relatively high amounts of iodine, reportedly from 0.03 to 1 percent, and is a

good specimen to explore for antibacterial actions. The assay can also be performed with the greenalga Enteromorpha intestinalis, the alga that looks like green Easter basket grass.

1 seaweed extract

2 extract control3 disinfectant control

4 antibiotic control


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Antibacterial Agents in SeaweedsStudent Work Sheet

Questions

1. What do the clear areas surrounding a disk indicate? What do differences in the width of the clear areas

indicate?

2. What evidence do you have that the inhibition of the bacteria is due to the extracts on the disk and not

the disks themselves?

3. Summarize the class data. Which seaweed extract was most effective against each of the bacterial species?

What might account for the difference in effectiveness?

4. If a seaweed extract did not produce any inhibition zones, does that mean that the seaweed does not

contain any antibacterial agents? Explain.

5. Compare the effectiveness of your seaweed extract to that of the commercially prepared antibiotics anddisinfectants. How could you increase the effectiveness of your seaweed extract? Identify as many variables

as you can that could affect the potency of your extract.

6. Which disinfectant (Lysol, bleach, Pine-Sol, etc) was most effective against the bacteria you tested?

7. Use a reference like the Merck Indexto determine which chemicals are likely to be the active ingredients

in the disinfectant you tested. Conduct an Internet search to see how these active ingredients affect

bacteria. Include the URLs of the Web sites that provided you with information to answer this question.

8. Which antibiotic was most effective against the bacteria you tested? Conduct an Internet search to

explain the mode of action of this antibiotic. Include the proper citations for your reference materials.

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Antibacterial Agents in SeaweedsTeacher Answer Key

Questions1. What do the clear areas surrounding a disk indicate?

Bacteria have been killed by the extract.

What do differences in the width of the clear areas indicate?

Greater antibacterial action by the extracts.

2. What evidence do you have that the inhibition of the bacteria is due to the extracts on the disk and not

the disks themselves?

The control disks with disinfectants and sterile broth eliminate variables in the experiment.

3. Summarize the class data. Which seaweed extract was most effective against each of the bacterial species?

Answers will vary with individual results

What might account for the difference in effectiveness?

Stronger antibacterial action in different species of seaweeds.

4. If a seaweed extract did not produce any inhibition zones, does that mean that the seaweed does not

contain any antibacterial agents? Explain.

No, it may kill species of bacteria not tested here.

5. Compare the effectiveness of your seaweed extract to that of the commercially prepared antibiotics and

disinfectants. How could you increase the effectiveness of your seaweed extract?

Use a more concentrated sample from the seaweed.

Identify as many variables as you can that could affect the potency of your extract.

Any dilution of the extract, season the seaweed was harvested (i.e. at the end of summer the seaweeds would be

declining)

6. Which disinfectant (Lysol, bleach, Pine-Sol, etc) was most effective against the bacteria you tested?


7. Use a reference like the Merck Indexto determine which chemicals are likely to be the active ingredients

in the disinfectant you tested. Conduct an Internet search to see how these active ingredients affect

bacteria. Include the URLs of the Web sites that provided you with information to answer this question.

8. Which antibiotic was most effective against the bacteria you tested?


Conduct an Internet search to explain the mode of action of this antibiotic. Include the proper citations for

your reference materials.


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Extension Activities

1. If antibacterial properties were discovered in one of the species of seaweed tested, test other

species of the same genus to determine if the bioactive compounds are species specific.

2. Obtain the same species of seaweed from various locations and seasons to determine if habitat

and season have any effect on the production of the bioactive compounds.

3. Traditional healers have often used seaweed as medicines. Research the types of seaweeds used

and how the medicines were prepared by meeting with tribal elders, medicine men, kupuna, orKahuna lapaau.

4. Test the effect of traditional methods of preservations (e.g., salting of limu kohu and rolling

the seaweed into balls) on the maintenance of antibacterial properties. Compare the antibacterial

properties of preserved (salted or dried) seaweeds and fresh seaweeds.

References and Further Reading

Bruckner A.W. 2002. Life Saving Products from Coral Reefs. Issues in Science and Technology online.

http://bob.nap.edu/issues/18.3/p_bruckner.html. Accessed November 2005.

Gerwick, William, Department of Pharmacy, Oregon State University, July 27, 2004. Introduction to

Drug Discovery from Marine Organisms (II.Resources.Gerwick) PowerPoint presentation at Marine

Biotechnology Curriculum Workshop, Hawaii Institute of Marine Biology, Kaneohe, HI

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