The most fundamental rule in this lab will be, do not contaminate any culture or medium except with known inocula from pure cultures. This includes other people's media as well as your own. Appropriate techniques to assure asepsis will be introduced as needed.
USING THE AUTOCLAVE
Assuring asepsis in prepared media usually means sterilizing them under pressure at 121o C. The autoclave uses a steam generator and a pressure chamber to accomplish this. Please follow these instructions for use, and consult the lab instructor if any question arises.
1. To use the autoclave, the steam generator must have an adequate supply of water. There is a dipstick in the reservoir which is accessible on top of the machine. Only distilled water should be added to this reservoir.
2. Make sure the appropriate cycle is selected. This will usually be the liquid cycle, which is indicated by a flask icon. Make sure the appropriate time is indicated on the panel. For liquid volumes less than 500 mL, 20 min is adequate. Up to 1 L, 25 min is adequate.
3. Place media, glassware and other materials into the pressure chamber. Liquid media are most safely sterilized in containers whose volume is approximately twice that of the medium. Arrange them so that glass vessels are not touching each other. Close the door and secure it by turning the lock clockwise. It is not necessary to use excessive force in tightening the door. A light on the panel indicates when the door is closed. It will be necessary to turn the handle about 3 times more after that light comes on.
4. Press the START button. The autoclave will automatically cycle through the sterilization process. If the reservoir is at room temperature when you start, this will take 90-100 min. If the reservoir is already hot, it will be a little shorter.
5. If something spills in the chamber, please be sure to clean it up immediately.
A BRIEF OVERVIEW
Because this lab involves the growth of living organisms, there will always be some unpredictability. You are in charge of your time and your material. You are responsible for making sure that cultures and media you need are prepared in advance. However, because there are some media and cultures that you will all be using, cooperation and division of labor will result in greater efficiency for all concerned. We will use a lot of media and glassware. Please carry your share of the burden of preparation and cleanup. This will count toward your lab grade. When the course is over, please dispose of any remaining cultures or media. It will be tremendously appreciated. This also will have an effect on your grade.
You will have a few projects that will be ongoing for the duration of the course. Others will take one to five days. In order to complete everything, you will often need to work on more than one project on a given day. Again, you will decide how best to schedule your time. There will be plenty of time to get everything done.
The ongoing projects will be the identification of unknown bacterial cultures, the development and observation of a microbial ecosystem, and a project of your own choosing. You will be involved practically on a daily basis with these projects. Please keep working on these so that they are finished in a timely fashion. Make frequent notations in your notebook. Other, shorter projects are designed to give you a sample of the kinds of things that microbiologists do routinely. We have a number of lab manuals that will be available. I encourage you to look through them and pick out one or two other exercises that interest you and work on them.
You will keep a bound lab notebook. Your lab notebook will be organized by project, not as a daily journal of activities. Each project should include all relevant observations, measurements and calculations, as well as one or more summaries of hypotheses and conclusions. The first object of a lab notebook is to allow someone coming after you to exactly repeat your work, given the same materials. Procedures need to be explicit, not vague. Observations should be complete; use drawings freely. Also, please remember that lab counts for 25% of your grade, and so should consume about 25% of your effort in the course.
There are two ongoing projects for the block for the entire class (plus one individual project, below): the Winogradsky column and the identification of bacterial unknowns. You will be working with several unknowns. We will give you a mixed culture of three organisms, from which you will need to prepare pure cultures for identification. You will culture and isolate an organism from among your personal flora (that is, bugs growing on or in you). And you will culture and isolate an organism from the environment - swab a surface indoors, or take a sample from out of doors, or from any other environmental source.
Besides these ongoing projects, you must do a spore staining exercise, the bacterial virus isolation, the phylogenetic tree and the biofilm exercise, as described in the links above.
WINOGRADSKY COLUMN
The Winogradsky column is a method for establishing a microbial ecosystem in which both succession and enrichment can be observed. A sample of sediment from a lake, pond or stream is enriched with some nutrient material(s) and incubated with water from the same environment. The column may have a natural 'life' of months; we, obviously, can observe it for only 3 weeks. Bacteria, algae, protists and fungi (and occasionally some invertebrates) that are naturally found in the water or sediment provide the initial inoculum. The water and mud provide a gradient of nutrient and oxygen concentrations that set up selection pressures on these initial flora and fauna.
You will work in pairs. Bring in some mud and water (about
2 cups of each) from some standing body of water nearby. Try to pick a spot
that is not too sandy or clayey. This is one experiment that won't require
aseptic technique.
Reserve a small amount of mud (about 50 mL). You will want to have 200-300 mL of water above the mud in your column. The added nutrients will be 1 g CaCO3, 1 g CaSO4, 1 g cellulose powder, 0.1 g Na2S per 100 mL mud, plus any added nutrient. Mix these and a handful of dried grass or weeds with your mud (that you haven't reserved). Add enough water to the mud to make it pourable, and pour it into a graduated cylinder, a little at a time. Tamp it with a glass rod or pipet to allow trapped air to escape. Add enough reserved mud to cover. Then add water to about 2 cm from the top of the cylinder. Cover with foil or plastic. Place the column in sunlight or in the light of an incandescent bulb.
Observe the column at least twice a week. You will be comparing your observations sequentially to follow any succession, so be thorough: organism counts, and drawings of different forms should be included. You should draw a representative field from each observation. Does the color of the column change? Is there any noticeable smell, or gas evolution? Include both macroscopic and microscopic observations. Smears and stains will help with contrast. You should take samples from at least three depths of the liquid: top, middle and near the mud. Helps to identifying organisms.
This will be your other ongoing project. Where will you get your unknowns? I will give you one. You may try to get the environmental unknown from your column (above). And you will need one from your own body. In the latter cases, you will have to enrich and isolate a pure culture first. It does no good to try to identify an unknown culture if there is a mixture of organisms in it. The first step, then, is to begin to culture organisms from a variety of sources. Personal samples might be taken from various locations on the skin (hands, scalp, toes, face), the mucus membranes (nose, mouth, throat, genitalia, etc.) or even from urine or stool. Samples may be taken from soil, from animals (insects, especially), from plants, from water, from food. Use anything you think may harbor microbial growth.
What will these samples be grown on? This can be extremely variable. There are a wide variety of media, and not all bacteria will grow on all of them. Most bacteria, however, will grow on a complex medium known as tryptic soy. This is a proteolytic digest of soy meal. More fastidious organisms require a more complete medium, like brain heart infusion or some kind of streptococcus medium.
There are a number of things that you might choose to do. The first thing to consider is an extension of one of the exercises or projects that we are already doing. For instance, you could try to identify the bacteriophage that you obtain from environment. You could try to isolate phages for a variety of organisms. You could obtain a growth curve for the yogurt culture under different conditions of temperature or type of milk. You could isolate the DNA from the phage and do a restriction digest to get its size and position of restriction sites.
You could also try something that interests you from the text or the lecture material. You could try to select for and isolate an organism with unusual metabolic features like a lithotroph, an anoxygenic phototroph, a methanogen, a nitrogen fixer. You could isolate an antibiotic producer and try to characterize the antibiotic. You could isolate an organism with antibiotic resistance and try to determine whether the resistance gene is located on the chromosome or a plasmid. Your book describes some experiments that might be done with chemotaxis (or you might do phototaxis, or even magnetotaxis, maybe). You could do some kind of mutagenesis and screening of mutants. There are a number of motility experiments I would be happy to have you try. There are some simple studies with biofilms that could be done, as well.
As you can see, the possibilities are various. I would be happy to talk to you about your interests and what kind of project you might do that would correspond to those interests.