Background

IIB 5. Antibacterial Agents in Seaweeds

Researchers studying marine cyanobacteria in order to nd novel natural products are nding they contain an abundance of potent bioactive compounds with promising anticancer, antibiotic, and anti-inammatory 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 laments in cells), and neurotoxins. Numerous promising compounds have been identied in the cyanobacterium Lyngbya majuscula (mermaids hair or reweed). This lamentous 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 cancer cell lines or microbial cultures, or indicator species such as brine shrimp. Extracts demonstrating signicant activity in either of the assays are chosen for further purication and analysis. In this experiment, students will explore the rst 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 Question

How are the antibacterial properties of algal extracts determined?

Learning Objectives

Students will learn to extract compounds from algae and test them for antimicrobial activity.

Materials

Various 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. coli MM294 (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 at www.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 lter paper (cut with a hole puncher)

Teaching Time

Three class periods: Day 1Extraction Day 2Antibacterial testing Day 3Recording results and class discussion

Procedure
Crude 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.

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 lter. Preparation of Disks 1. Using sterile tips, pipette 20 l of each extract onto two 1/4-inch lter 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 ame 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 each plate. Dip spreader into the ethanol beaker and briey pass it through a Bunsen burner ame to ignite the alcohol. Allow alcohol to burn away from the ame! 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 without aming.

NOTE: if alcohol or spreading rods are not available, use sterile Q-tips to spread bacteria over the surface of the agar.

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

1 2 3 4

seaweed extract extract control disinfectant control antibiotic control

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 nd 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 seaweeds 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 green alga Enteromorpha intestinalis, the alga that looks like green Easter basket grass.

Questions

Antibacterial Agents in Seaweeds Student Work Sheet

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 and disinfectants. 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 Index to 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.

Questions

Antibacterial Agents in Seaweeds Teacher Answer Key

1. 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? Answers will vary with individual results 7. Use a reference like the Merck Index to 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? Answers will vary with individual results Conduct an Internet search to explain the mode of action of this antibiotic. Include the proper citations for your reference materials.

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 specic. 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, or Kahuna 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