Honors Laboratory Projects in Microbiology

Thomas M. Terry, University of Connecticut, Feb. 2000

General Guidelines

Honors students in MCB 229, "Fundamentals of Microbiology", will work in teams to carry out an investigative laboratory project

Since this is likely to be one of your first experiences in designing and carrying out experiments that are not simply "cook-book" exercises, I have compiled a list of suggestions and guidelines that should help you to carry out this project successfully. Please read the following guidelines carefully, and spend some time planning your exercises accordingly.

1. The more focused your investigation is, the more likely you are to be successful in devising clear-cut experiments and obtaining useful and relevant data. For example, if you focus your experiments on answering the question "Do bacteria survive longer on cutting boards made of wood as opposed to plastic?", you should be able to devise simple experiments that can clearly answer this question "yes" or "no". On the other hand, if you focus on a more general question such as "How do bacteria survive?", you will have a more difficult time deciding what experiments to do and how to carry them out.

2. Realize that the time available for your research topic is limited to roughly 8 weeks of actual research time, and that you must sandwich this project into an already busy schedule. Choose a "bite-sized" project that is manageable within this time frame.

3. In choosing a project, a good beginning is to formulate a hypothesis and set out to confirm it. For example, you might hypothesize that the bacterial content of ground beef is higher than the bacterial content of ground chicken (or vice versa). It's not so important that you guess correctly as that you imagine what you think might be true. The alternative, sometimes called a "fishing expedition", of gathering data without any idea of what might be important is rarely productive.

4. Your project may consist of a single experiment, carried out over several weeks (for example, if you were going to assay the antibiotic resistance of 50 different isolates of E. coli, you'd probably want to break this up into about 3 weeks worth of work). Or, you may do several experiments, starting with one experiment the first week. This is often the way science works -- you set out to prove something, then look at your first set of results and come up with another experiment that might answer questions raised in your initial data.

5. Often the success or failure of a project depends on how well you master the methodology needed. I recommend that you build on methods you will encounter in the first 4-5 weeks of this course (e.g., serial dilutions, viable counts, antibiotic inhibition assays, gram staining, etc.) rather than trying to master entirely new procedures before you can do an experiment. You'd be surprised how many interesting questions you can answer with a fairly small tool kit of microbiological techniques.

6. Once you have carried out an experiment, spend some time trying to critique your results. What unambiguous results (if any) did you obtain? Which results are open to question, and why? It is very typical in science that one experiment will suggest several new experiments to try to answer "loose ends" that turn up in the first experiment. You

7. If you obtain a result that is unexpected, you should repeat the experiment to verify that your result was real, not just a fluke. Often, a first result will suggest a way of not only repeating but extending the experiment that allows you to deepen your investigation.

Media Limitations and Planning

It is challenging for us to be able to supply media for your experiments in a timely fashion. In order to make sure that we can provide what you need, we ask that you observe the following procedures:

1. Before you begin any experiments, you should submit an experimental protocol in writing to your lab instructors. This protocol should list exactly what media you need, and what date you need it on. For Example:

needed on Tues. March 31:
5 petri plates with nutrient medium
5 test tubes with sterile water
5 1-ml pipets
access to a water bath at 45° C.

Sometimes you may need to modify the design of an experiment at the last minute. This is especially likely to happen if you have not thought through how you will carry out the experiment. You should try to visualize exactly how your experiment will be carried out, as if you were standing there watching yourself inoculating plates, tubes, etc. Get in the habit of listing everything you see yourself using in this simulation -- then review the list and see if you have forgotten anything.

2. We can provide both "standard" and "non-standard" media. Examples of "standard media" include: nutrient broth, nutrient agar, TSA-glucose agar, staph broth, and other plate or tube media used so far this semester. Examples of non-standard media: plates with different agar concentrations (e.g., for use in studying motility). Also, we can supply antibiotic assay disks, reagents for enzyme tests, and more.

4. You should consult with Dhaval Nanavati, the Honors lab supervisor, each week during the project. At each consultation, you should show him what you have done, suggest what you think needs to be done next, and request media you will need for the following week. Dhaval will help you design appropriate experiments within the constraints of this project.

5. At the end of the semester, each research team will prepare a poster presentation and give a short oral report using this poster to the honors group.

Ideas for Research Projects

The following list is deliberately vague. I have suggested a number of possible projects, each of which can be varied considerably depending on your interests and ideas. This is by no means a definitive list; you may have other ideas for projects that do not appear here. Dhaval will help you choose and refine ideas for an appropriate project.

Once you have decided on a project, Dhaval or I can help you to choose the most appropriate media to use, and help you formulate an initial protocol.

1. Quantitative Experiments. How many bacteria are in ________ (a kitchen sponge? a thoroughly washed hand? road sand? a gram of ground beef? etc.) This is one of the most straightforward, and yet surprisingly interesting, of experimental questions in microbiology. The basic procedures are simple: dilution series and viable count assays. One can modify these assays to even simpler, "semi-quantitative" assays for an initial screening.

Here are some possible applications:

* comparing the number of bacteria in a variety of water samples. This can be extended by looking for the presence of indicator organisms for fecal contamination: E. coli and Enterococcus faecalis .

* comparing the number of bacteria in eggs, meat, poultry, fish, or other foods. Samples are first weighed, then mixed with sufficient water to produce 1% solutions (1 gm/100 ml), and mixed thoroughly in a blender. Dilutions are assayed for total count. It is also possible to screen for the presence of certain pathogens (e.g. Salmonella) or fecal indicator organisms.

* comparing the survival of a pathogenic indicator organism (such as E. coli, an enteric bacterium that has similar survival abilities to pathogenic enterics such as Salmonella) on wood vs. plastic cutting boards. The FDA requires plastic cutting boards in food preparation, arguing that they are easier to clean. However, recent reports suggest that bacteria disappear from wood surfaces much faster.

* examining "microbiology in the home". Where are bacteria found in greatest abundance? How contaminated is a sponge that is used several times a day to clean food?

* examining the variation of numbers of bacteria in different habitats. For example, last year some students in MCB 229 were trying to isolate Staph. aureus from fellow students, with lower than expected results. They decided to investigate the distribution of S. aureus on different parts of the same person. Surprisingly, they discovered that S. aureus is found more frequently between the toes than anywhere else!

* examining variation over time. For example, we know that bacterial numbers on hands goes down after hand washing. But how soon does it come back up, and to what extent? A careful study, in which the same skin area was swabbed at different times and then assayed for total count, might be very revealing.

2. Population Experiments.

We tend to assume that all bacteria are alike. In fact, however, the genetic variation between a number of independent isolates of a bacterium like E. coli can be enormous, substantially larger than the genetic variation between people.

One important application of variation is to learn how much variation there is in antibiotic resistance in a population. For example, if you were to study 20 or 50 independent isolates of Staph aureus, or E. coli, and find that a substantial % were resistant to commonly used antibiotics, this would be an important finding. You might investigate 20-50 independent isolates of E. coli for antibiotic resistance, using the Kirby-Bauer disc diffusion assay that you will learn in lab. Will all these strains show the same pattern of antibiotic sensitivity, or will there be detectable differences? We don't know, and you could find an answer as you project.

You could apply the same type of antibiotic resistant survey to a population of Staph isolates. Other population experiments might focus on different types of variety; for example, the class isolated many different thermophiles. You could investigate what kind of variety is represented in some of these isolates: how many are endospore formers? How many are gram-positive vs. gram-negative? How do their cardinal temperatures compare: do they all show the same minimum and maximum temperatures, or are there significant variations? How many are aerobes as opposed to facultative?

3. Enrichment - Isolation - Characterization experiments. There are many microbes in nature with interesting properties, whose isolation poses interesting challenges. Here are several types of organisms you might choose to "hunt down" and study:

* Pseudomonas putida: a plant pathogen that secretes an ice-nucleating protein, responsible for the formation of ice crystals. Strains of this organism are commercially used at ski resorts to create finer crystals in snow-making machines.
* Archaea in either the extreme thermophile or halophile groups. (For technical reasons, isolating the fastidiously anaerobic methanogens is beyond our resources).
* Actinomycetes that produce antibiotics. Who knows -- you might discover some organisms that secrete antibiotics not yet known to science! Soil is loaded with actinomycetes; you could isolate a variety and test them to see if they inhibit other bacteria, both gram-positive and gram-negative.
* Lactic acid bacteria. This is a large group, including the Streptococci and the Lactobacilli. You could find out what microbes are present in some soured milk products (only unpasteurized products should be used).
* Bioluminescent bacteria. These organisms glow, like fireflies, in the dark. They are found at low concentrations in seawater, marine sediments, surfaces and GI tracts of marine animals such as squid, fish, or shrimp, and especially on the surfaces of decomposing fishes. Not easy to isolate, but very rewarding to work with.

4. Other types of experiments. You may well have ideas that don't fit into any of the categories suggested above. That's great. We'll work with you to help you design experiments that can be reasonably done in the time available.