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.
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!
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.
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.
Staphylococcus epidermidis / + / Opportunistic infections / Skin, open wounds,
urinary tract / Normal flora on skin, environment
Staphylococcus aureus / + / Food intoxication / Small intestine / Unwashed hands, poor hygiene, etc.
+ / Skin and other infections / Skin, open wounds / Nasal/oral contact, close human-to-human contact
Aerobacter aerogenes / -
Bacillus cereus / + / Food poisoning / Small intestine / Unrefrigerated foods
Bacillus subtilis / + / Food poisoning / Small intestine / Unrefrigerated foods
Pseudomonas aeruginosa / - / Opportunistic infections / Skin, open wounds / Environmental
Enterococcus faecalis / + / Intestine
Micrococcus luteus / + / Opportunistic infections / Skin, open wounds / Environmental
Micrococcus roseus / + / Opportunistic infections / Skin, open wounds / Environmental
Escherichia coli / - / Opportunistic infections / Skin, open wounds / Poor hygiene
(hand washing)
- / Urinary tract infections / Bladder, urethra, kidney / Poor hygiene
- / Traveler’s diarrhea / Small intestine / Food, water, rare/raw beef, poor sanitation/hygiene
Enterobacter aerogenes / - / Intestine
Mycobacterium smegmatis / Acid-fast / Respiratory (related)
Salmonella enteritidis / - / Food poisoning / Small intestine / Cattle, poultry, rodents, fecal contamination of foods, unwashed hands
Klebsiella pneumoniae / - / Respiratory
Streptococcus pyogenes / + / Strep throat, rheumatic fever, other / Upper respiratory tissue / Droplets from human 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 xerose / + / Respiratory (related) / Droplets
Student Experiments Page 3