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Methods Manual – Applied Microbiology

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]

Introduction

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.

Media

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.

Media requirements

Bacteria display a wide range of nutritional and physical requirements for growth including

  • Water
  • A source of energy
  • Sources of carbon, nitrogen, sulfur, phosphorus
  • Minerals, e.g., Ca2+, Mg2+, Na+
  • Vitamins and growth factors

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.

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Media sterilization

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 4 to 20 min. destroys nearly all living cells and spores. High pressure (typically 20 lbs/sq. in) allows the temperature to exceed 100 degrees, which can't be accomplished with steam at one atmosphere. 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

  • Know the instrument - some are fully automatic, some are fully manual
  • Prepare supplies properly - the more layers or greater the volume, the longer it will take for the interior to heat up
  • Check the steam pressure and ensure that the instrument is set for slow exhaust if liquids are to be sterilized
  • Ensure that the door is closed properly and securely
  • Check that the time and/or automatic cycle are set properly
  • Ensure that the temperature is well below 100 degrees before attempting to open the door
  • Crack the door to allow steam to vent, keeping face and hands well away from the opening
  • ***CAUTION*** Exposing tightly stoppered bottles to variable pressures invites explosion and injury. When heating any liquids using any method, take care disturbing the flask or bottle. Material near the bottom may be superheated and boil over when moved. Stoppers, caps, covers, must be vented - never make them fit tightly.

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Agar plates

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).

Containers used for media must have vented tops and should be capable of holding at least 20% more than the intended volume of medium, to allow for expansion during sterilization. We find 1L capped bottles to be very convenient for preparing large quantities. To facilitate cooling, handling, and pouring, we typically prepare them with 500 ml medium. Beware of vessels with narrow necks - surface area of the liquid should be large enough to prevent superheating. Agar does not distribute uniformly when melted. A safe way to ensure a uniform distribution for pouring plates or tubes is to drop a magnetic stir bar in the flask or bottle, then gently stir the medium after sterilization, while it cools. Stirring distributes the agar evenly. If screw cap bottles are used, the cap must be loosened prior to sterilization.

Here is an outline of the general procedure.

  • Ensure that you have time to prepare the medium (30 min or so), sterilize it (1 hour minimum), cool it with stirring (30 min usually) and pour the plates
  • Measure appropriate volume of deionized water into a flask or bottle
  • Drop in a stir bar
  • Layer the powder on the water surface, allow to soak in
  • Do not use mechanical mixing for most complex media; lumps will form that will not go into solution
  • Stir or swirl to mix then heat in a microwave oven to melt the agar (uncapped)
  • Place cap or foil on opening (do not tighten caps - leave loose to allow venting)
  • Steam sterilize for minimum of 15 min in appropriate autoclave tray (check media instructions for sterilization time)
  • After safely removing the materials, use stir plate to mix sterilized agar and allow it to cool enough to be handled
  • Pour recommended volume (usually 15-20 ml) into each plate in hood (recommended) or with very conscientious aseptic technique, at a bench
  • Allow plates to cool and lose some moisture; best practice is to leave closed in a hood for a day or so
  • Store plates inverted in a closed container
  • Aside to contamination from dust particles due to careless handling, condensation and insect contamination are our worst enemies; usually we do not refrigerate plates; watch for fruit fly larvae

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.

Media are purchased as dehydrated granules or powder, and are rehydrated 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). 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.

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Broth tubes

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.

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Aseptic technique

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!

  • Never leave a culture dish open, even for a short time when viewing colonies of organisms, unless you intend to destroy it.
  • When it is necessary to open a dish, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the lid between your face (and your germs!) and the agar surface.
  • For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an inoculum.
  • Flame a loop or needle to red-hot just prior to use, burning off any organic material
  • Cool the instrument by touching the sterile agar or liquid surface prior to touching a culture (or else you will kill it)
  • Re-sterilize the instrument after performing the procedure, putting down safely without burning the bench, you, or another student.
  • Pass the neck of a culture tube or any container with a culture or sterile contents through a flame before taking off the cap. Hold the cap with opening down, and the tube horizontal or nearly so. Convection from the heated neck will prevent dust from falling into the opening. Flame again before putting the cap back [see 'preparing a bacterial smear' in the staining section]
  • Use sterile disposable pipets to remove samples from a broth culture that must be kept uncontaminated.
  • Always be aware of where your hands are, where your face is, and whether or not your culture is in a position to be contaminated. If you have long hair, make sure it does not hang into your plate. Hair is full of potential contaminants, and is one of the principle sources of contaminating microorganisms.
  • If you have an open flame, long hair that is not tied back or loose clothing can be hazardous to your health.
  • Keep flammables away from the flames, including alcohol used for sterilizing instruments; do not place a heated loop or glass rod into an alcohol dish

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.

  • Keep all surfaces clean and dry
  • Frequently use the UV light to sterilize the interior surfaces; do not stare at the light, which can cause retinal damage
  • The opening must not exceed the recommended sash height
  • Surfaces kept to back of the hood are more likely to remain sterile, as are objects kept close to the table surface
  • Keep non-sterile objects closer to the front, sterile objects to the back
  • Keep the hood fairly uncluttered
  • Never reach over a sterile surface - you WILL contaminate it; reach around sterile surfaces if necessary
  • Watch for long hair hanging over sterile surfaces
  • Place lids with sterile side DOWN; don't turn lids upside down; nothing will jump up and contaminate the lid
  • Use slow, deliberate movements to avoid inadvertant contamination

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.


Created by David R. Caprette (caprette@rice.edu), Rice University 12 Oct 02
Updated 25 Dec 11
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