Culture of bacteria in the laboratory
The microbiologist has many choices to make if wishing to grow microorganisms in the laboratory. The biomass introduced into the growth medium is known as the inoculant, and one of the first questions that need to be addressed is whether to inoculate onto solid or into liquid media. The medium will be chosen to reflect the origin of the inoculant, which might be a mixed culture of many different species of microorganism, or a pure culture of only one species. Solid media are normally held in circular sterile plastic containers with lids (Petri dishes), the solidity being provided by agar. The culture is streaked or plated onto the surface of the medium using a sterile wire or plastic loop, or a sterile glass spreader, respectively.
Microorganisms have an enormous metabolic diversity, so require a medium made up of components to suit. Some organisms can synthesize all their cellular components from simple carbon and energy sources, such as glucose, and so will grow on a minimal (synthetic/defined) medium. Such a medium will contain sources of nitrogen, phosphate, sulfur, calcium, magnesium, potassium, and iron as inorganic salts, and may be supplemented with trace elements, such as zinc, manganese, boron, cobalt, copper, nickel, chromium, and molybdenum.
These elements are required in minute quantities by the organism, and are used as prosthetic groups in some enzymes (e.g. alcohol dehydrogenase contains zinc). A truly minimal medium will not contain any vitamins, but organisms will often grow more quickly if provided with riboflavin, thiamine, nicotinic acid, pyridoxine HCl, calcium pantothenate, biotin, folic acid, and vitamin B12. Again, these are provided in minute quantities, enough to be used during enzyme synthesis but not in sufficient amounts to act as a carbon and/or energy source.
When growing some auxotrophs or bacteria with a requirement for amino acids, it may be necessary to supplement the minimal medium with some or all of the 20 possible acids. This is common in pathogens, where a protected lifestyle in an animal host has led to the loss of one or all the enzymes involved in amino acid synthesis pathways. Identifying amino acid requirements is often time-consuming and costly, in which case a complex medium can be used.
Complex media are defined in the sense that absolute quantities of buffering ions are added to a solution, as well as known amounts of plant, animal, or yeast extracts. The common complex medium Lauria Bertoni broth (LB) contains 5 g l–1 NaCl, but carbon, energy, trace elements, and other growth factors are provided by 5 g l–1 ‘yeast extract’ and 10 g l–1 ‘Tryptone’. Tryptone is casein (milk solids) digested with pancreatic enzymes, so the exact composition in terms of molar concentrations of amino acids, short peptides, and so on is unknown and will vary between manufacturers. Similarly, yeast extract, a hydrolysate of baker’s yeast (Saccharomyces cerevisiae) is of undefined composition.
Pathogenic bacteria associated with bacteremia are frequently grown on a complex medium containing whole or partially hydrolyzed blood, which not only provides the essential growth factors but can also give an indication of the presence of hemolytic organisms by clear haloes in the blood red medium around colonies. Liquid media can be placed in a variety of containers appropriate to the oxygen requirements of the organism to be grown. Facultative anaerobes and anaerobes can be grown in bottles, with gentle shaking to mix the culture, while the more commonly used aerophiles are generally grown in batch culture in Erlenmeyer flasks. These flasks are adapted chemistry apparatus, conical flasks between 5 ml and 5 l in volume. They are filled to 10% of the total volume so that the liquid medium provides sufficient surfacearea for oxygen transfer to the culture. The inoculation of liquid and solid media and the transfer of cultures from one container to another without the ingress of contaminating organisms have become known as aseptic technique.
Media have been developed over the last 100 years in both composition and utility. Selective or differential media are of a composition that only allows the growth of one type or group of organisms. For example, a minimal medium containing methanol as a carbon and energy source will select for methylotrophs, those organisms able to use reduced C1 compounds. The medium can be enhanced further to be diagnostic. For example, solid Baird Parker medium will allow the growth of only a handful of genera, including Micrococcus and Staphylococcus, but only Staphylococcus aureus will grow as gray-black shiny colonies with a narrow white entire margin surrounded by a zone of clearing 2–5 mm. This ‘egg yolk’ colony form is used as a first indication of the presence of potential pathogens before more detailed tests are carried out.
Most microbiological media are adapted to the study of aerobes, but the true anaerobes, particularly those that are damaged by exposure to oxygen (some Clostridia and members of the Archaea), need special culture conditions. The total exclusion of oxygen is difficult, but a series of methods named after their inventor (the Hungate techniques) achieve this.
▶Storage and revival of microorganisms
The Bacteria and Archaea are remarkably resistant to extreme conditions and many can survive freezing or desiccation without ill effect, even in their vegetative states. This means that many, but by no means all, prokaryotes can be stored as pelleted biomass for decades. In the laboratory, long-term storage of biomass frozen at -70°C in a 50% glycerol solution is often used, although this can sometimes cause cell Death. A Freezing in dimethyl sulfoxide (DMSO) can be an alternative, but prokaryotes rarely require cryopreservation in liquid nitrogen as eukaryotic cells can do. Larger laboratories may purchase a freeze-drying apparatus to lyophilize cultures. This is by far the most efficient method of long-term storage. Short-term laboratory storage (for up to a week) is normally done by streaking the biomass out onto solid media held in a suitable container (Petri dish or 30 ml bottle). After storage, the cells are in a starved state and must be revived with a complex medium so as little stress as possible is placed on them.
Once a microbiological experiment has been completed, the live organisms should be safely destroyed. Similarly, before an experiment starts all living cells present should be inactivated so that only the inoculum desired is present. This poses problems for the microbiologist, particularly one working with pathogens, thermophiles, or sporulating bacteria. The process of pasteurization (71.7°C for 15 seconds) will kill many common human bacterial pathogens without affecting the medium a great deal and so is therefore used in food preparation. Moist heat in the form of steam or boiling will kill most vegetative cells as well as some viruses, but thermophiles and endospores will survive.
▶Autoclaving contaminated equipment (121°C for 15 minutes, 15 psi (100 kPa) above atmospheric pressure) kills all cells as well as endospores, but is not suitable for use with polycarbonate (a common material for making plastic containers) and will cause many carbohydrate medium components to caramelize. Several methods have been used to deal with heat-sensitive components:
- Tyndallization – repetitive heating to 90–100°C for 10 minutes, followed by cooling for 1–2 days. Allows endospores to germinate in the medium, which are then killed by the heating.
- Ultraviolet radiation – kills living cells but does not penetrate opaque containers or large volumes of solution well.
- γ (Gamma)radiation – kills living cells but causes brittleness in polycarbonate and polypropylene.
- Filtration of media – 0.22 mm filters can be suitable for aqueous solutions of heat-labile chemical constituents, but it is difficult to filter large (> 500 ml) quantities effectively while maintaining sterility.