To supplement this manual an illustrated manual describing our most commonly used assays can be found in Resources on Owl-Space (Assays_illlustrated.pdf).
[Media requirements] [Sterilization of media] [Preparing agar plates] [Preparing broth and agar tubes] [Aseptic technique]
Bacteria live in our soil, streams, food, in us, and in virtually all habitable (and some seemingly inhabitable) locations on earth. They can make us wine, yogurt, and garden compost, and without them we couldn't even digest our food. All nitrogen would eventually be lost to the atmosphere without them. Bacteria are increasingly used as research tools and in biotechnology, supplying us with recombinant DNA, enzymes, and designer drugs. We are even increasingly using them to rid ourselves of toxic wastes.
Bacteria also can make your breath stink, rot your teeth, clog your lungs, give you Montezuma's revenge, and kill you if you (or your physician) are not careful. You have undoubtedly heard of pathogenic Escherichia coli, and "flesh eating bacteria." Perhaps you have also heard of an increasing number of cases of antibiotic-resistant tuberculosis and other diseases of bacterial origin. Microbiology has some exciting (perhaps even scary) years ahead of it. The field encompasses the study of viruses, bacteria, fungi, and protists, however there is plenty to do just studying bacteria. Bacteria are ubiquitous, and are of major importance to biological scientists, physicians, environmentalists, food preparers, and brewmasters, let alone the rest of us who have suffered through bacterial infections at one time or another.
Many of the techniques and strategies that you learn in this laboratory will be useful if you conduct any type of biological laboratory investigation in the future. Even more important is the opportunity to test your ability to use your common sense and exercise self-reliance. You will need to relate reference material and other literature to activity in the laboratory without a set of "cookbook" instructions. Your success will be directly related to your ability to learn hands-on technique, the degree of care you take in working with your cultures and assays, and your conscientiousness in keeping up with your responsibilities. Of course there will be some sets of instructions, in fact there will be training sessions in the first few meetings to acquaint you with the care, feeding, examination, and identification of bacteria.
General and specialized media are required for bacterial growth and for characterization. The media you prepare are, in fact, research tools. Peruse this section and use it as a reference as needed. The basic procedures can be applied to almost any type of assay or culture requirement for propagation of obligate aerobes or faculatative anaerobes. Obligate anaerobes are poisoned by oxygen, and specialized procedures are needed for their maintenance.
Bacteria display a wide range of nutritional and physical requirements for growth including
Microorganisms may be grown in liquid, solid or semisolid media. Liquid media are utilized for growth of large numbers of organisms or for physiological or biochemical studies and assays. Some species, such as Streptococcus or Staphylococcus, often demonstrate typical morphologies only when grown in liquid media. Solid media are useful for observations of characteristic colonies, for isolation of pure cultures and for short-term maintenance of cultures. Usually, the preparation of a solid medium for growth simply includes the addition of 1 to 2% agar to a solution of appropriate nutrients. Agar is a complex carbohydrate extracted from marine algae that solidifies below temperatures of 45šC. It is not a nutritional component.
Usually, bacteria are grown in complex media, because we simply do not know enough about the organism or organisms to define all of their requirements for growth and maintenance. Neither the chemical composition nor the concentration of substrates are defined. Media frequently contain nutrients in the form of extracts or enzymatic digests of meat, milk, plants or yeast. For fastidious organisms we must often use delicious-sounding concoctions such as tomato juice agar or chocolate agar, or something less appetizing (but nutrient-rich) such as brain-heart infusion broth or blood agar.
There is no single medium or set of physical conditions that permits the cultivation of all bacteria, and many species are quite fastidious, requiring specific ranges of pH, osmotic strength, temperature and presence or absence of oxygen. The requirements for growth of bacteria under laboratory conditions are determined by trial and error. You will culture bacteria using a rich, complex medium, namely tryptic soy agar or broth, so that a wide variety of possible unknowns can be mixed into the same culture and grown on the same plates. Agar plates will be used for isolation and some assays, and for short term maintenance of cultures. Agar slant tubes will be used for long term maintenance of isolates. Broths (liquid media) will be used to grow isolates for some assays or for the assays themselves.
About dehydrated media
We purchase pre-mixed dehydrated media in theh form of granules or powder, and rehydrate the media by mixing a measured amount of medium per measured volume of deionized water. Instructions for rehydration are usually printed on the container (40 gms/liter for tryptic soy agar, 18.2 gms/liter for R2A agar). When complex media are required, look first for the pre-mixed powder. Prepare from scratch only if necessary. Some media such as phenol red broth or decarboxylase media require that you add a nutrient component and/or adjust pH before sterilization. Some antibiotics and other heat-labile components must be filter-sterilized and then added to cooled liquid agar. Watch for special instructions on bottles. For example, some analytical media are to be heated to dissolve components, but not steam sterilized.
Tryptic soy agar consists of a pancreatic digest of casein (milk sugar) and a papaic digest of soybean meal, with sodium chloride and agar. It is a general purpose medium for the culture of fastidious and nonfastidious microorganisms. Most isolates should grow on tryptic soy agar provided that you inoculate the plate with living material and culture it at an appropriate temperature. Some isolates, though, may struggle on medium that is too rich. You may also need specialized agar for producing spores or reaction products to reveal properties of individual species.
Reasoner's 2A (R2A) agar consists of proteose peptone, caseamino acids, yeast extract, dextrose, starch, and inorganic salts. It is specifically formulated to allow the culturiing of bacteria that would be crowded out by species that grow much faster on richer, more complex media.
When fungal spores or bacteria-laden microscopic particles make contact with your plates, broths, and tubes colonies happily reproduce and your precious media eventually resemble something out of an abandoned full refrigerator. One can't recognize individual colonies when the plates are covered with fuzz! No untreated surface in the lab is sterile, and nearly all dust and other particles have spores or active cells on their surfaces. Obviously, then, all labware and all media must be sterilized before use. We sterilize most media and supplies using a steam autoclave to produce moist heat. Other methods, including filtration, ethylene oxide, radiation, or ultraviolet light, may be necessary if components are heat-labile or materials are not heat-resistant.
An autoclave is designed to deliver steam into a pressure chamber, generating high heat and pressure at the same time. Heating media to above 121 degrees C for a minimum of 20 min. should destroy all living cells and spores. High pressure (typically 20 lbs/sq. in) allows the temperature to exceed 100 degrees without boiling off the water from the solutions being sterilized. We use an autoclave that starts timing when the temperature reaches 121 degrees, and exhausts the steam slowly after the prescribed time above 121 degrees (to prevent exploding bottles!). The autoclave is effectively a giant pressure cooker.
To properly use an autoclave
Traditional plates were reusable glass petri dishes with lids. Today we use clear plastic disposable petri dishes, typically 95 or 100 mm in diameter, 20 per sleeve. When prepared for inoculation, a plate contains solid agar to provide a surface for growth, mixed with nutrient materials. We prepare agar media either by mixing 1 to 2% agar with individual components or by using a pre-mixed powder. Either way, the dry components must be heated to melt the agar and sterilized in a flask or bottle, then poured into the plates using aseptic technique, preferably in a sterile cabinet (laminar flow hood).
Preparing agar plates
The following procedure was specifically written for our laboratory in the Anderson Biological Laboratory building, Rice University campus.
The only difference between broth and agar media is that broths do not contain an agar component. We use broth tubes primarily for specific assays, or (rarely) for bacteria that will not form colonies on a solid surface. In broth a species may display motility and/or a characteristic pattern of association among individual cells, such as chains or clusters, that is not as obvious in agar cultures. To prepare broth a dry medium is layered onto the surface of a measured volume of water as with agar media, mixed, and distributed into individual loosely capped or vented capped tubes in racks. Heating to dissolve components is sometimes required, but not always. Racks are steam sterilized and then allowed to cool, and caps tightened to prevent evaporation. Unlike preparation of agar plates, tubes are prepared with media already in the incubation vessel. A large volume syringe can facilitate distribution of media into individual tubes.
Agar tubes and agar slant tubes
Preparre agar for a tube as you would agar for pouring plates, but use an open vessel, not a bottle. Beakers are most appropriate. Medium must be uniformly distributed after melting the agar. As with broth tubes, it is easiest to use a syringe or some other repeating dispenser to deliver media to individual tubes.
Some applications call for a tube that is partially filled with agar to give a level surface. For maintaining stocks of isolates or to prepare material for assays, slant tubes are helpful. A slant is simply a tube placed at an angle during cooling to give a large slanted surface for inoculation. The tube can be tightly capped for relatively long term storage of an isolate with low risk of contamination or drying out of the culture. A large "butt," that is, the depth of agar below the start of the surface area, helps prevent drying out. Some liquid near the bottom of the surface also helps serve that purpose.
To prepare an agar slant each tube should be filled sufficiently to allow the agar to flow to just below the neck when the neck is laid over a horizontal 10 ml glass pipet. The tubes are sterilized with caps loose as with all media, then laid on their sides using a pipet to keep them tilted up just enough to create a long slanted surface. After cooling, the caps are tightened and the tubes are ready for use.
The media on which you culture desirable microorganisms will readily grow undesirable contaminants, especially molds and other types of fungus, and bacteria from your skin and hair. It is therefore essential that you protect your cultures from contamination from airborne spores and living microorganisms, surface contaminants that may be on your instruments, and from skin contact.
Bacteria and other contaminants cannot fly. Nearly all forms of contamination are carried on microscopic dust particles that make their way onto sterile surfaces when they are carelessly handled. One exception is insect contamination, such as by ants for fruit flies. Fruit flies are a particular nuisance because they can crawl under the lids of agar plates and lay eggs. You would think that people doing gentics research would have developed a model by now that can't fly into other peoples' experiements!
A contaminated culture can often be rescued, however there is always the risk that you will re-isolate the wrong microorganism. Besides, you don't have that kind of time to waste. Exercise extreme care to keep your cultures pure.
Using a sterile cabinet
Unlike a fume hood, which is designed to keep airborne substances from escaping into the laboratory environment, a sterile cabinet keeps airborne contaminants from getting into the hood. A simple laminar flow hood protects exposed sterile surfaces that are placed inside. A containment hood does both jobs, keeping airborne particulate matter from going in or out. To use a hood properly, remember these points.
Accumulated waste materials can pose a contamination hazard. A microbiology laboratory can become inundated with old cultures unless a well organized system for disposal of is in place. Even a few people can produce so much contaminated material, that if teams don't take care of their own materials someone will spend at least a week just cleaning up the place. All cultures must be sterilized before disposal or cleaning of lab ware. To make disposal as efficient as possible, please get rid of materials you no longer need as soon as possible, as described in the special rules.