Chapter 5: Cultivation of Microorganisms

Cultivation is the process of propagating organisms by providing the proper environmental conditions. Growing microorganisms are making replicas of themselves, and they require the elements present in their chemical composition. Nutrients must provide these elements in metabolically accessible form. In addition, organisms require metabolic energy to synthesize macromolecules and maintain essential chemical gradients across their membranes. Factors that must be controlled during growth include the nutrients, pH, temperature, aeration, salt concentration, and ionic strength of the medium.

Most of the dry weight of microorganisms is organic matter containing the elements carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. In addition, inorganic ions such as potassium, sodium, iron, magnesium, calcium, and chloride are required to facilitate enzymatic catalysis and to maintain chemical gradients across the cell membrane.

For the most part, the organic matter is in macromolecules formed by the introduction of anhydride bonds between building blocks. Synthesis of the anhydride bonds requires chemical energy, which is provided by the two phosphodiester bonds in adenosine triphosphate (ATP; see Chapter 6). Additional energy required to maintain a relatively constant cytoplasmic composition during growth in a range of extracellular chemical environments is derived from the proton motive force. The proton motive force is the potential energy that can be derived by passage of a proton across a membrane. In eukaryotes, the membrane may be part of the mitochondrion or the chloroplast. In prokaryotes, the membrane is the cytoplasmic membrane of the cell.

The proton motive force is an electrochemical gradient with two components, a difference in pH (hydrogen ion concentration) and a difference in ionic charge. The charge on the outside of the bacterial membrane is more positive than the charge on the inside, and the difference in charge contributes to the free energy released when a proton enters the cytoplasm from outside the membrane. Metabolic processes that generate the proton motive force are discussed in Chapter 6. The free energy may be used to move the cell, to maintain ionic or molecular gradients across the membrane, to synthesize anhydride bonds in ATP, or for a combination of these purposes. Alternatively, cells given a source of ATP may use its anhydride bond energy to create a proton motive force that in turn may be used to move the cell and to maintain chemical gradients.

To grow, an organism requires all of the elements in its organic matter and the full complement of ions required for energetics and catalysis. In addition, there must be a source of energy to establish the proton motive force and to allow macromolecular synthesis. Microorganisms vary widely in their nutritional demands and their sources of metabolic energy.

The three major mechanisms for generating metabolic energy are fermentation, respiration, and photosynthesis. At least one of these mechanisms must be used if an organism is to grow.

Fermentation

The formation of ATP in fermentation is not coupled to the transfer of electrons. Fermentation is characterized by substrate phosphorylation, an enzymatic process in which a pyrophosphate bond is donated directly to adenosine diphosphate (ADP) by a phosphorylated metabolic intermediate. The phosphorylated intermediates are formed by metabolic rearrangement of a fermentable substrate such as glucose, lactose, or arginine. Because fermentations are not accompanied by a change in the overall oxidation-reduction state of the fermentable substrate, the elemental composition of the products of fermentation must be identical to those of the substrates. For example, fermentation of a molecule of glucose (C₆H₁₂O₆) by the Embden-Meyerhof pathway (see Chapter 6) yields a net gain of two pyrophosphate bonds in ATP and produces two molecules of lactic acid (C₃H₆O₃).

Respiration

Respiration is analogous to the coupling of an energy-dependent process to the discharge of a battery. Chemical reduction of an oxidant (electron acceptor) through a specific series of electron carriers in the membrane establishes the proton motive force across the bacterial membrane. The reductant (electron donor) may be organic or inorganic (eg, lactic acid serves as a reductant for some organisms, and hydrogen gas is a reductant for other organisms). Gaseous oxygen (O₂) often is used as an oxidant, but alternative oxidants that are used by some organisms include carbon dioxide (CO₂), sulfate (SO₄²⁻), and nitrate (NO₃⁻).

Photosynthesis

Photosynthesis is similar to respiration in that the reduction of an oxidant via a specific series of electron carriers establishes the proton motive force. The difference in the two processes is that in photosynthesis, the reductant and oxidant are created photochemically by light energy absorbed by pigments in the membrane; thus, photosynthesis can continue only as long as there is a source of light energy. Plants and some bacteria are able to invest a substantial amount of light energy in making water a reductant for carbon dioxide. Oxygen is evolved in this process, and organic matter is produced. Respiration, the energetically favorable oxidation of organic matter by an electron acceptor such as oxygen, can provide photosynthetic organisms with energy in the absence of light.

Nutrients in growth media must contain all the elements necessary for the biologic synthesis of new organisms. In the following discussion, nutrients are classified according to the elements they supply.

Carbon Source

As already mentioned, plants and some bacteria are able to use photosynthetic energy to reduce carbon dioxide at the expense of water. These organisms are referred to as autotrophs, creatures that do not require organic nutrients for growth. Other autotrophic microorganisms are the chemolithotrophs, organisms that use an inorganic substrate such as hydrogen or thiosulfate as a reductant and carbon dioxide as a carbon source.

Heterotrophs require organic carbon for growth, and the organic carbon must be in a form that can be assimilated. Naphthalene, for example, can provide all of the carbon and energy required for respiratory heterotrophic growth, but very few organisms possess the metabolic pathway necessary for naphthalene assimilation. Glucose, on the other hand, can support the fermentative or respiratory growth of many organisms. It is important that growth substrates be supplied at levels appropriate for the microbial strain that is being grown: Levels that will support the growth of one organism may inhibit the growth of another organism.

Carbon dioxide is required for a number of biosynthetic reactions. Many respiratory organisms produce more than enough carbon dioxide to meet this requirement, but others require a source of carbon dioxide in their growth medium.

Nitrogen Source

Nitrogen is a major component of proteins, nucleic acids, and other compounds, accounting for approximately 5% of the dry weight of a typical bacterial cell. Inorganic dinitrogen (N₂) is very prevalent, comprising 80% of the earth’s atmosphere. It is also a very stable compound, primarily because of the high activation energy required to break the nitrogen–nitrogen triple bond. However, nitrogen may be supplied in a number of different forms, and microorganisms vary in their abilities to assimilate nitrogen (Table 5-1). The end product of all pathways for nitrogen assimilation is the most reduced form of the element, ammonia (NH₃). When NH₃ is available, it diffuses into most bacteria through transmembrane channels as dissolved gaseous NH₃ rather than ionic ammonium ion (NH₄⁺).

The ability to assimilate N₂ reductively via NH₃, which is called nitrogen fixation, is a property unique to prokaryotes, and relatively few bacteria are capable of breaking the nitrogen–nitrogen triple bond. This process (see Chapter 6) requires a large amount of metabolic energy and is readily inactivated by oxygen. The capacity for nitrogen fixation is found in widely divergent bacteria that have evolved quite different biochemical strategies to protect their nitrogen-fixing enzymes from oxygen.

Most microorganisms can use NH₃ as a sole nitrogen source, and many organisms possess the ability to produce NH₃ from amines (R—NH₂) or from amino acids (RCHNH₂COOH), generally intracellularly. Production of NH₃ from the deamination of amino acids is called ammonification. Ammonia is introduced into organic matter by biochemical pathways involving glutamate and glutamine. These pathways are discussed in Chapter 6.

Many microorganisms possess the ability to assimilate nitrate (NO₃⁻) and nitrite (NO₂⁻) reductively by conversion of these ions into NH₃. These processes are termed assimilatory nitrate reduction and assimilatory nitrite reduction, respectively. These pathways for assimilation differ from pathways used for dissimilation of nitrate and nitrite. The dissimilatory pathways are used by organisms that use these ions as terminal electron acceptors in respiration. Some autotrophic bacteria (eg, Nitrosomonas, Nitrobacter spp.) are able to convert NH₃ to gaseous N₂ under anaerobic conditions; this process is known as denitrification. Our understanding of the nitrogen cycle continues to evolve. In the mid-1990s, the anammox reaction was discovered. The reaction

in which ammonia is oxidized by nitrite is a microbial process that occurs in anoxic waters of the ocean and is a major pathway by which nitrogen is returned to the atmosphere.

Sulfur Source

Similar to nitrogen, sulfur is a component of many organic cell substances. It forms part of the structure of several coenzymes and is found in the cysteinyl and methionyl side chains of proteins. Sulfur in its elemental form cannot be used by plants or animals. However, some autotrophic bacteria can oxidize it to sulfate (SO₄²⁻). Most microorganisms can use sulfate as a sulfur source, reducing the sulfate to the level of hydrogen sulfide (H₂S). Some microorganisms can assimilate H₂S directly from the growth medium, but this compound can be toxic to many organisms.

Phosphorus Source

Phosphate (PO₄³⁻) is required as a component of ATP; nucleic acids, and such coenzymes as NAD, NADP, and flavins. In addition, many metabolites, lipids (phospholipids, lipid A), cell wall components (teichoic acid), some capsular polysaccharides, and some proteins are phosphorylated. Phosphate is always assimilated as free inorganic phosphate (P_i).

Mineral Sources

Numerous minerals are required for enzyme function. Magnesium ion (Mg²⁺) and ferrous ion (Fe²⁺) are also found in porphyrin derivatives: magnesium in the chlorophyll molecule, and iron as part of the coenzymes of the cytochromes and peroxidases. Mg²⁺ and K⁺ are both essential for the function and integrity of ribosomes. Ca²⁺ is required as a constituent of gram-positive cell walls, although it is dispensable for gram-negative bacteria. Many marine organisms require Na⁺ for growth. In formulating a medium for the cultivation of most microorganisms, it is necessary to provide sources of potassium, magnesium, calcium, and iron, usually as their ions (K⁺, Mg²⁺, Ca²⁺, and Fe²⁺). Many other minerals (eg, Mn²⁺, Mo²⁺, Co²⁺, Cu²⁺, and Zn²⁺) are required; these frequently can be provided in tap water or as contaminants of other medium ingredients.

The uptake of iron, which forms insoluble hydroxides at neutral pH, is facilitated in many bacteria and fungi by their production of siderophores—compounds that chelate iron and promote its transport as a soluble complex. These include hydroxamates (-CONH₂OH) called sideramines, and derivatives of catechol (eg, 2,3-dihydroxy-benzoylserine). Plasmid-determined siderophores play a major role in the invasiveness of some bacterial pathogens (see Chapter 7). Siderophore- and nonsiderophore-dependent mechanisms of iron uptake by bacteria are discussed in Chapter 9.

Growth Factors

A growth factor is an organic compound that a cell must have to grow but that it is unable to synthesize. Many microorganisms, when provided with the nutrients listed above, are able to synthesize all of the building blocks for macromolecules (Figure 5-1), which are amino acids; purines, pyrimidines, and pentoses (the metabolic precursors of nucleic acids); additional carbohydrates (precursors of polysaccharides); and fatty acids and isoprenoid compounds. In addition, free-living organisms must be able to synthesize the complex vitamins that serve as precursors of coenzymes.

Each of these essential compounds is synthesized by a discrete sequence of enzymatic reactions; each enzyme is produced under the control of a specific gene. When an organism undergoes a gene mutation resulting in failure of one of these enzymes to function, the chain is broken, and the end product is no longer produced. The organism must then obtain that compound from the environment: The compound has become a growth factor for the organism. This type of mutation can be readily induced in the laboratory.

Different microbial species vary widely in their growth factor requirements. The compounds involved are found in and are essential to all organisms; the differences in requirements reflect differences in synthetic abilities. Some species require no growth factors, but others—such as some of the lactobacilli—have lost, during evolution, the ability to synthesize as many as 30–40 essential compounds and hence require them in the medium.

A suitable growth medium must contain all the nutrients required by the organism to be cultivated, and such factors as pH, temperature, and aeration must be carefully controlled. A liquid medium is used; the medium can be gelled for special purposes by adding agar or silica gel. Agar, a polysaccharide extract of a marine alga, is uniquely suitable for microbial cultivation because it is resistant to microbial action and because it dissolves at 100°C but does not gel until cooled below 45°C; cells can be suspended in the medium at 45°C and the medium quickly cooled to a gel without harming them.

Nutrients

On the previous pages, the function of each type of nutrient is described, and a list of suitable substances presented. In general, the following must be provided: (1) hydrogen donors and acceptors, about 2 g/L; (2) carbon source, about 1 g/L; (3) nitrogen source, about 1 g/L; (4) minerals: sulfur and phosphorus, about 50 mg/L of each, and trace elements, 0.1–1 mg/L of each; (5) growth factors: amino acids, purines, and pyrimidines, about 50 mg/L of each, and vitamins, 0.1–1 mg/L of each.

For studies of microbial metabolism, it is usually necessary to prepare a completely synthetic medium in which the exact characteristics and concentration of every ingredient are known. Otherwise, it is much cheaper and simpler to use natural materials such as yeast extract, protein digest, or similar substances. Most free-living microbes grow well on yeast extract; parasitic forms may require special substances found only in blood or in extracts of animal tissues. Nevertheless, some parasitic microbes (eg, Treponema pallidum) cannot be grown in vitro or grow inside eukaryotic cells (eg, Chlamydia trachomatis).

For many organisms, a single compound (eg, an amino acid) may serve as energy source, carbon source, and nitrogen source; others require a separate compound for each. If natural materials for nonsynthetic media are deficient in any particular nutrient, they must be supplemented.

Hydrogen Ion Concentration (pH)

Most organisms have a fairly narrow optimal pH range. The optimal pH must be empirically determined for each species. Most organisms (neutralophiles) grow best at a pH of 6.0–8.0, although some forms (acidophiles) have optima as low as pH 3.0, and others (alkaliphiles) have optima as high as pH 10.5.

Microorganisms regulate their internal pH over a wide range of external pH values by pumping protons in or out of their cells. Acidophiles maintain an internal pH of about 6.5 over an external range of 1.0–5.0, neutralophiles maintain an internal pH of about 7.5 over an external range of 5.5–8.5, and alkaliphiles maintain an internal pH of about 9.5 over an external range of 9.0–11.0. Internal pH is regulated by a set of proton transport systems in the cytoplasmic membrane, including a primary, ATP-driven proton pump and an Na⁺/H⁺ exchanger. A K⁺/H⁺ exchange system has also been proposed to contribute to internal pH regulation in neutralophiles.

Temperature

Different microbial species vary widely in their optimal temperature ranges for growth (Figure 5-2): Psychrophilic forms grow best at low temperatures (–5 to 15°C) and are usually found in such environments as the Arctic and Antarctic regions; psychrotrophs have a temperature optimum between 20°C and 30°C but grow well at lower temperatures. They are an important cause of food spoilage. Mesophilic forms grow best at 30–37°C, and most thermophilic forms grow best at 50–60°C. Some organisms are hyperthermophilic and can grow at well above the temperature of boiling water, which exists under high pressure in the depths of the ocean. Most organisms are mesophilic; 30°C is optimal for many free-living forms, and the body temperature of the host is optimal for symbionts of warm-blooded animals.

The upper end of the temperature range tolerated by any given species correlates well with the general thermal stability of that species’ proteins as measured in cell extracts. Microorganisms share with plants and animals the heat-shock response, a transient synthesis of a set of “heat-shock proteins,” when exposed to a sudden rise in temperature above the growth optimum. These proteins appear to be unusually heat resistant and to stabilize the heat-sensitive proteins of the cell.

The relationship of growth rate to temperature for any given microorganism is seen in a typical Arrhenius plot (Figure 5-3). Arrhenius showed that the logarithm of the velocity of any chemical reaction (log k) is a linear function of the reciprocal of the temperature (1/T); because cell growth is the result of a set of chemical reactions, it might be expected to show this relationship. Figure 5-3 shows this to be the case over the normal range of temperatures for a given species; log k decreases linearly with 1/T. Above and below the normal range, however, log k drops rapidly, so that maximum and minimum temperature values are defined.

Beyond their effects on growth rate, extremes of temperature kill microorganisms. Extreme heat is used to sterilize preparations (see Chapter 4); extreme cold also kills microbial cells, although it cannot be used safely for sterilization. Bacteria also exhibit a phenomenon called cold shock, which is the killing of cells by rapid—as opposed to slow—cooling. For example, the rapid cooling of Escherichia coli from 37°C to 5°C can kill 90% of the cells. A number of compounds protect cells from either freezing or cold shock; glycerol and dimethyl sulfoxide are most commonly used.

Aeration

The role of oxygen as hydrogen acceptor is discussed in Chapter 6. Many organisms are obligate aerobes, specifically requiring oxygen as hydrogen acceptor; some are facultative anaerobes, able to live aerobically or anaerobically; some are obligate anaerobes requiring a substance other than oxygen as hydrogen acceptor and are sensitive to oxygen inhibition; some are microaerophiles, which require small amounts of oxygen (2–10%) for aerobic respiration (higher concentrations are inhibitory); and others are aerotolerant anaerobes, which are indifferent to oxygen. They can grow in its presence, but they do not use it as a hydrogen acceptor (Figure 5-4).

The natural by-products of aerobic metabolism are the reactive compounds hydrogen peroxide (H₂O₂) and superoxide (O₂⁻). In the presence of iron, these two species can generate hydroxyl radicals (•OH), which can damage any biologic macromolecule:

Many aerobes and aerotolerant anaerobes are protected from these products by the presence of superoxide dismutase, an enzyme that catalyzes the reaction

and by the presence of catalase, an enzyme that catalyzes the reaction

Some fermentative organisms (eg, Lactobacillus plantarum) are aerotolerant but do not contain catalase or superoxide dismutase. Oxygen is not reduced, and therefore H₂O₂ and O₂⁻ are not produced. All strict anaerobes lack both superoxide dismutase and catalase. Some anaerobic organisms (eg, Peptococcus anaerobius) have considerable tolerance to oxygen as a result of their ability to produce high levels of an enzyme (NADH oxidase) that reduces oxygen to water according to the reaction

Hydrogen peroxide owes much of its toxicity to the damage it causes to DNA. DNA repair-deficient mutants are exceptionally sensitive to hydrogen peroxide; the recA gene product, which functions in both genetic recombination and repair, has been shown to be more important than either catalase or superoxide dismutase in protecting E coli cells against hydrogen peroxide toxicity.

The supply of air to cultures of aerobes is a major technical problem. Vessels are usually shaken mechanically to introduce oxygen into the medium or air is forced through the medium by pressure. The diffusion of oxygen often becomes the limiting factor in growing aerobic bacteria; when a cell concentration of 4–5 × 10⁹/mL is reached, the rate of diffusion of oxygen to the cells sharply limits the rate of further growth.

Obligate anaerobes, on the other hand, present the problem of oxygen exclusion. Many methods are available for this: Reducing agents such as sodium thioglycolate can be added to liquid cultures, tubes of agar can be sealed with a layer of petrolatum and paraffin, the culture vessel can be placed in a container from which the oxygen is removed by evacuation or by chemical means, or the organism can be handled within an anaerobic glove box.

Ionic Strength and Osmotic Pressure

To a lesser extent, such factors as osmotic pressure and salt concentration may have to be controlled. For most organisms, the properties of ordinary media are satisfactory; however, for marine forms and organisms adapted to growth in strong sugar solutions, for example, these factors must be considered. Organisms requiring high salt concentrations are called halophilic; those requiring high osmotic pressures are called osmophilic.

Most bacteria are able to tolerate a wide range of external osmotic pressures and ionic strengths because of their ability to regulate internal osmolality and ion concentration. Osmolality is regulated by the active transport of K⁺ ions into the cell; internal ionic strength is kept constant by a compensating excretion of the positively charged organic polyamine putrescine. Because putrescine carries several positive charges per molecule, a large drop in ionic strength is effected at only a small cost in osmotic strength.

Two problems will be considered: the choice of a suitable medium and the isolation of a bacterial organism in pure culture.

The Medium

The technique used and the type of medium selected depend on the nature of the investigation. In general, three situations may be encountered: (1) One may need to raise a crop of cells of a particular species that is on hand, (2) one may need to determine the numbers and types of organisms present in a given material, or (3) one may wish to isolate a particular type of microorganism from a natural source.

A. Growing Cells of a Given Species

Microorganisms observed microscopically to be growing in a natural environment may prove exceedingly difficult to grow in pure culture in an artificial medium. Certain parasitic forms have never been cultivated outside the host. In general, however, a suitable medium can be devised by carefully reproducing the conditions found in the organism’s natural environment. The pH, temperature, and aeration are easy to duplicate; the nutrients present the major problem. The contribution made by the living environment is important and difficult to analyze; a parasite may require an extract of the host tissue, and a free-living form may require a substance excreted by a microorganism with which it is associated in nature. Considerable experimentation may be necessary to determine the requirements of the organism, and success depends on providing a suitable source of each category of nutrient listed at the beginning of this chapter. The cultivation of obligate parasites such as chlamydiae is discussed in Chapter 27.

B. Microbiologic Examination of Natural Materials

A given natural material may contain many different microenvironments, each providing a niche for a different species. Plating a sample of the material under one set of conditions will allow a selected group of forms to produce colonies but will cause many other types to be overlooked. For this reason, it is customary to plate out samples of the material using as many different media and conditions of incubation as practicable. Six to eight different culture conditions are not an unreasonable number if most of the forms present are to be discovered.

Because every type of organism present must have a chance to grow, solid media are used, and crowding of colonies is avoided. Otherwise, competition will prevent some types from forming colonies.

C. Isolation of a Particular Type of Microorganism

A small sample of soil, if handled properly, will yield a different type of organism for every microenvironment present. For fertile soil (moist, aerated, rich in minerals and organic matter), this means that hundreds or even thousands of types can be isolated. This is done by selecting for the desired type. One gram of soil, for example, is inoculated into a flask of liquid medium that has been made up for the purpose of favoring one type of organism, such as aerobic nitrogen fixers (Azotobacter). In this case, the medium contains no combined nitrogen and is incubated aerobically. If cells of Azotobacter are present in the soil, they will grow well in this medium; forms unable to fix nitrogen will grow only to the extent that the soil has introduced contaminating fixed nitrogen into the medium. When the culture is fully grown, therefore, the percentage of Azotobacter in the total population will have increased greatly; the method is thus called enrichment culture. Transfer of a sample of this culture to fresh medium will result in further enrichment of Azotobacter; after several serial transfers, the culture can be plated out on a solidified enrichment medium and colonies of Azotobacter isolated.

Liquid medium is used to permit competition and hence optimal selection even when the desired type is represented in the soil as only a few cells in a population of millions. Advantage can be taken of “natural enrichment.” For example, in looking for kerosene oxidizers, oil-laden soil is chosen because it is already an enrichment environment for such forms.

Enrichment culture, then, is a procedure whereby the medium is prepared so as to duplicate the natural environment (“niche”) of the desired microorganism, thereby selecting for it (Figure 5-5). An important principle involved in such selection is the following: The organism selected for will be the type whose nutritional requirements are barely satisfied. Azotobacter, for example, grows best in a medium containing organic nitrogen, but its minimum requirement is the presence of N₂; hence, it is selected for in a medium containing N₂ as the sole nitrogen source. If organic nitrogen is added to the medium, the conditions no longer select for Azotobacter but rather for a form for which organic nitrogen is the minimum requirement.

When searching for a particular type of organism that is part of a mixed population, selective or differential media are used. Selective media inhibit the growth of organisms other than the one being sought. For example, Thayer-Martin agar is used to isolate Neisseria gonorrhoeae, the cause of gonorrhea, from clinical specimens. Differential media contain a substance(s) that certain bacteria change in a recognizable way. For example, colonies of E coli have a characteristic iridescent sheen on agar containing the dyes eosin and methylene blue (EMB agar). EMB agar containing a high concentration of one sugar will also cause organisms that ferment that sugar to form reddish colonies. Differential media are used for such purposes as recognizing the presence of enteric bacteria in water or milk and the presence of certain pathogens in clinical specimens. Table 5-2 presents characteristics of representative media used to cultivate bacteria.

Isolation of Microorganisms in Pure Culture

To study the properties of a given organism, it is necessary to handle it in pure culture free of all other types of organisms. To do this, a single cell must be isolated from all other cells and cultivated in such a manner that its collective progeny also remain isolated. Several methods are available.

A. Plating

Unlike cells in a liquid medium, cells in or on a gelled medium are immobilized. Therefore, if few enough cells are placed in or on a gelled medium, each cell will grow into an isolated colony. The ideal gelling agent for most microbiologic media is agar, an acidic polysaccharide extracted from certain red algae. A 1.5–2% suspension in water dissolves at 100°C, forming a clear solution that gels at 45°C. Thus, a sterile agar solution can be cooled to 50°C, bacteria or other microbial cells added, and then the solution quickly cooled below 45°C to form a gel. (Although most microbial cells are killed at 50°C, the time course of the killing process is sufficiently slow at this temperature to permit this procedure; see Figure 4-3.) Once gelled, agar will not again liquefy until it is heated above 80°C, so that any temperature suitable for the incubation of a microbial culture can subsequently be used. In the pour-plate method, a suspension of cells is mixed with melted agar at 50°C and poured into a Petri dish. When the agar solidifies, the cells are immobilized in the agar and grow into colonies. If the cell suspension is sufficiently dilute, the colonies will be well separated, so that each has a high probability of being derived from a single cell (Figure 5-6). To make certain of this, however, it is necessary to pick a colony of the desired type, suspend it in water, and replate. Repeating this procedure several times ensures that a pure culture will be obtained.

Alternatively, the original suspension can be streaked on an agar plate with a wire loop (streak-plate technique). As the streaking continues, fewer and fewer cells are left on the loop, and finally the loop may deposit single cells on the agar (Figure 5-7). The plate is incubated, and any well-isolated colony is then removed, resuspended in water, and again streaked on agar. If a suspension (and not just a bit of growth from a colony or slant) is streaked, this method is just as reliable as and much faster than the pour-plate method.

In the spread plate technique, a small volume of dilute microbial suspension containing ca 30–300 cells is transferred to the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod. The dispersed cells develop into isolated colonies. Because the number of colonies should equal the number of viable organisms in a sample, spread plates can be used to count the microbial population.

B. Dilution

A much less reliable method is that of extinction dilution. The suspension is serially diluted, and samples of each dilution are plated. If only a few samples of a particular dilution exhibit growth, it is presumed that some of the colonies started from single cells. This method is not used unless plating is for some reason impossible. An undesirable feature of this method is that it can only be used to isolate the predominant type of organism in a mixed population.

• An organism requires all of the elements in its organic matter and the full complement of ions required for energetics in order to grow. Nutrients are classified according to the elements they provide, including carbon source, nitrogen source, sulfur source, phosphorous source, and mineral sources.

• Growth factors are organic compounds that a cell must have to grow but that it is unable to synthesize.

• There must be a source of energy present to establish a proton motive force and to allow macromolecular synthesis. The three major mechanisms for generating metabolic energy are fermentation, respiration, and photosynthesis.

• Environmental factors such as pH, temperature, and aeration are important for growth. Most human pathogens are neutralophiles (grow best at pH of 6.0–8.0) and mesophilic (grow best at 30–37°C).

• Organisms vary widely in their ability to use oxygen as a hydrogen acceptor and in their ability to inactivate toxic by-products of aerobic metabolism. They may be grouped as obligate aerobes, facultative anaerobes, obligate anaerobes, microaerophiles, and aerotolerant anaerobes.

• Microbiologic media can be formulated to permit the growth of a particular type of microorganism present in low numbers (enrichment culture), identify specific types of microorganisms (differential medium), or isolate a specific organism from a mixed population (selective medium).