Student Experiments Using the Disk Diffusion Method:
Testing Plant Extracts for Antibacterial Properties
The disk diffusion method is used to determine the effectiveness of various antibacterial materials. Small
filter-paper disks are soaked in the material to be tested, and placed on a Petri dish that has been seeded with
the bacteria of interest. In the next lab, you will be testing antibiotics, and several other common
antimicrobial compounds available on the market. While many of these are very effective, they have only
been available for use within the last 75 years, or so. Before antibiotics and germicidal chemicals were
available, humans treated bacterial disease using plants and plant products. For thousands of years there was
nothing else available to treat human disease and to preserve foods. For a very long time, the study of botany
was an essential part of medical training.
As our treatment focus has dwelled on drugs, we have “forgotten” many of the old methods that were used
before modern drug therapy was available. Many of us have heard of folk remedies, though, and in recent
years, there has been a renewed interest in natural approaches to treating infectious disease. This renewed
interest in plants and plant products to treat disease has also spread to the scientific community, and across
the world, many plants and their products are being tested for effectiveness using modern scientific
approaches. Some are old remedies being re-evaluated using new techniques. Others are plants that have only
recently come to our attention. Many plants and plant products have been used for treating diseases that are
not bacterial in origin, and are have not been known for their bacteriostatic properties, whereas others appear
to have promise for their ability to inhibit bacterial growth.
In this class you have the opportunity to use your scientific skills to test the effectiveness of several plants or
plant extracts for their ability to inhibit the growth of select bacterial species. You will develop hypotheses,
and plan and carry out experiments to test your hypotheses. You will use the disk-diffusion method
(modified Kirby-Bauer) to test the inhibitory effects of the products you have chosen.
The Report – a scientific paper:
The report for this experiment will be in formal scientific paper format. Although is will not be a long paper,
it requires all the parts of a formal scientific paper:
Abstract
Introduction
Materials and Methods
Results (text and table, figure optional)
Conclusions
References (at least three, two may be online)
Resources to help you with this project:
This is where you will use Writing in the Biological Sciences, required for this class. Use this book to help
you with samples of all the parts of a scientific paper.
How to start:
Think about what you already know about this topic. Most of us know someone who has used a tea or some
other plant extract to treat a medical problem. However, don’t lose sight of the fact that not all diseases are
bacterial. The scope of this assignment is bacterial only! Try going to the online sources on the next page,
and browse. Look for ideas on the Herbalgram site that sound interesting. Do you see a study that seems
interesting? It is considered good science to repeat someone else’s research to confirm it, so it’s ok to think
about re-doing a part of a study that someone else did. It is also good science to take someone else’s research
and apply it to different species, different plant parts, or different extract components. When you write this
up, you will give the original authors credit for their part in your ideas, of course!
The American Botanical Council (the Herbalgram site) has a lot of scientifically sound information
on the medicinal use of plants. The American Society for Microbiology sponsors several scientific journals,
some of which have published articles on the effectiveness of various plants and plant extracts. You can
search all of their journals, or only a select few.
Student Experiments Page 1
Useful online sources:
The American Botanical Council (ABC)
http://www.herbalgram.org
Restricted portions of the site will require use of a login and password, which was provided in class.
The links page may also prove useful.
The American Society for Microbiology
http://www.asm.org
Go to Publications, and then to Journals.
From this part of the site, you can search for journal articles on topics of interest. Type in a keyword
and see what comes up. If the article is very new (published in the last couple of months), you may
not be able to get the full text. Look for older articles to access full-text online. We do not have a
login and password that students are authorized to use for accessing the more recent articles. These
are scientific journals, and the articles may be difficult to read. Sometimes it is worth it to work your
way through it. As a rule of thumb for the purposes of this class, use only articles that you can
understand!
Other Web sites:
If you are interested in using other online resources, you must clear them with me. Many web sites are not
reliable sources of scientific information. You may compromise the quality of your research and/or the
quality of your paper by using resources that are not credible.
Choosing the bacteria:
Choose 1-5 bacterial species to test. You will be given a table of all the bacteria we have stock cultures of.
One approach is to choose a disease you’re interested in. Or look for some other factor that groups the
bacteria you choose together: target organ that they affect (e.g. small intestine), source of inoculum (e.g.
environment), etc. This will help you develop a coherent hypothesis that covers all your bacteria.
Choosing potential inhibitors:
Choose 1-5 plant extracts to use as potential inhibitors. Each inhibitor will be tested on each of the bacteria
you have chosen. You will be given a table of plant extracts that we have on hand to test. You may bring in
other plants or extracts if I approve their use in your experimental design.
Asking a central question, proposing a hypothesis:
Asking a clear central question is the basis of a good experiment. An example of a good question might be:
Are bacteria that inhabit the back of the throat inhibited by extracts of basil, chamomile, or ginger?
Your hypothesis is a proposed answer to your question. For example:
My hypothesis is that Streptococcus pyogenes is inhibited by water extracts of basil, chamomile, and ginger,
but not by essential oils or tinctures of these plants.
Size of the experiment and replication:
The size of your experiment will be determined by your central question and hypothesis. However, I expect
your experiments to be sufficiently complex that you will have a minimum of 3 petri dishes before
replication. That means you will have a minimum of 6 petri dishes altogether. If the experiment you want to
conduct requires less than 6 petri dishes after replication, you must clear that part of your plan with me. All
experimental plans must be approved before you do them. All experiments must be replicated.
Materials and technical details:
Each student will have Petri dishes of Mueller-Hinton agar. Divide each dish into 3-4 quadrants, based on the
experiment you are conducting. Label each Petri dish and each quadrant. For each bacterial species, you will
inoculate one Petri dish using the spread-plate method. Then you will place filter-paper disks impregnated
with the products you choose to test into the quadrants. Incubate, inverted at 37°C – if this is the optimum
temperature for the bacterial species you have chosen! Be sure your disks are pressed onto the agar so that
they don’t fall off!
Student Experiments Page 2
Controls:
At least one quadrant of EVERY Petri dish will be a control quadrant. What will you use for your control?
This will vary with the treatments you are testing. If all your treatments are essential oils, then you will need
to use an oil as a control. If all your treatments are tinctures, you will need to use ethanol as your control. If
your treatments are all water-based, then your control must be water-based. If you have a mixture of
treatment types, then you will need to accommodate several controls in your experimental plan.
Here is an example: I might choose to test garlic tincture and basil essential oil. I divide my Petri dish into
quadrants, place an alcohol control disk into quadrant #1, and place a oil impregnated control disk in
quadrant #2. In quadrant #3, I place a disk soaked with garlic tincture, and in quadrant #4, I place a disk
impregnated with basil essential oil. Your setup will look different based on the choices you make.
Designing your experiment and research proposals:
We will work on your experimental design/research proposal on Tuesday, March 1. On Thursday, March 3,
it must be completed and turned in. We have allocated a short amount of time to review these designs, so that
you have the opportunity to change it needs adjustment. Don’t forget to include the concentrations of your
treatments and the method(s) of extraction.
Your research proposal includes your central question and hypothesis, the general layout of your experiment,
materials, species, number of Petri dishes, layout of each Petri dish, concentrations of each extract, etc.
Ideally, your research proposals should be typed (word-processed), and must have pictures of your petri
dishes and the layout of every treatment. Significant portions of a good proposal can be used in the
Introduction of your scientific paper, and some parts will become part of the Materials and Methods section
of your paper.
Please note that Wikipedia is not an acceptable source for any reference for this class.
See the next page for a list of bacteria we have available for your use.
Student Experiments Page 3
Choose from the following bacteria available for your use. If you look up the bacteria with empty cells, you
can finish filling in this table. Finishing the table will help you become more familiar with the bacteria
available for this experiment.
Gram
Bacterial species
reaction
Disease
Target Organ
Source of Infection
Staphylococcus
Opportunistic
Skin, open wounds, Normal flora on skin,
epidermidis
+
infections
urinary tract
environment
Unwashed hands, poor
+
Food intoxication
Small intestine
hygiene, etc.
Staphylococcus
aureus
Skin and other
Nasal/oral contact, close
+
infections
Skin, open wounds human-to-human contact
Aerobacter
aerogenes
Bacillus cereus
+
Food poisoning
Small intestine
Unrefrigerated foods
Bacillus subtilis
+
Food poisoning
Small intestine
Unrefrigerated foods
Pseudomonas
Opportunistic
aeruginosa
infections
Skin, open wounds Environmental
Enterococcus
faecalis
+
Intestine
Opportunistic
Micrococcus luteus
+
infections
Skin, open wounds Environmental
Opportunistic
Micrococcus roseus
+
infections
Skin, open wounds Environmental
Opportunistic
Poor hygiene
infections
Skin, open wounds (hand washing)
Urinary tract
Bladder, urethra,
Escherichia coli
infections
kidney
Poor hygiene
Food, water, rare/raw beef,
Traveler’s diarrhea Small intestine
poor sanitation/hygiene
Enterobacter
aerogenes
Intestine
Mycobacterium
Respiratory
smegmatis
Acid-fast
(related)
Cattle, poultry, rodents,
Salmonella
fecal contamination of
enteritidis
Food poisoning
Small intestine
foods, unwashed hands
Klebsiella
pneumoniae
Respiratory
Strep throat,
Streptococcus
rheumatic fever,
Upper respiratory
Droplets from human
pyogenes
+
other
tissue
contamination
Streptococcus lactis
+
Opportunistic
Wounds
Sarcina aurantiaca
+
Skin, open wounds
Sarcina lutea
+
Skin, open wounds
Serratia marcescens
Skin, open wounds
Branhamella
catarrhalis
Lower respiratory
Vibrio fischeri
Food-water borne
Gastrointestinal
Food-water-borne
Corynebacterium
Respiratory
xerose
+
(related)
Droplets
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