General Microbiology - Schlegel, H. 1987

Microbial growth
Elective cultivation methods

We owe our knowledge of The Diversity of microorganisms to two main circumstances. Certain microorganisms attracted attention long ago because they form colonies or large aggregates, or cause noticeable changes in their environment. Many of these easily detectable microorganisms lend themselves to direct isolation. It is straightforward to find conditions that support their growth. However, There are many other microorganisms belonging to various physiological groups that only became accessible for study after Winogradsky and Beijerinck developed enrichment culture techniques.

Enrichment cultures. The enrichment culture technique is both in principle and practice very straightforward. Enrichment requires conditions that allow a given Organism to outcompete all others. By carefully adjusting a range of factors (energy, carbon, and nitrogen sources, electron acceptors, gas atmosphere, illumination, Temperature, pH, etc.), specific conditions are created, and the medium is inoculated with a mixed population, such as that found in soil or sludge. The microorganism best adapted to this environment grows and displaces all other accompanying organisms. By repeated transfer into the same liquid medium and plating onto a solid medium of identical composition, the dominant (enriched) strain can be easily isolated. Frequent liquid-to-liquid transfers prevent the growth of concomitant organisms that might otherwise consume excretion products or even Cell autolysates from the primary culture. Samples from sites that already exhibit "natural enrichment" serve as the best inoculum material. For example, carbon monoxide-utilizing microorganisms can be isolated from gas works wastewater; Hemoglobin-utilizing Bacteria from slaughterhouse wastewater; and hydrocarbon-oxidizing forms from oilfield soils or petroleum sludge.

The enrichment culture method makes it possible to isolate microorganisms with virtually any combination of nutritional requirements—provided, of course, that the desired type actually exists in nature. It is particularly easy to establish selective conditions for highly specialized microorganisms. For instance, a mineral medium devoid of nitrogen compounds exposed to light is strictly selective for N₂-fixing cyanobacteria. If the same medium is supplemented with an organic energy and carbon source, *Azotobacter* will develop under aerobic conditions in the dark, whereas *Clostridium* will grow under anoxic conditions. To successfully obtain enrichment cultures, one must limit the provision of nutrients strictly to the minimal requirements of the target microorganism. If, for example, one wishes to isolate bacteria capable of oxidizing methanol or H₂ using nitrate or sulfate as an electron acceptor, access to O₂ must be excluded; otherwise, aerobic methanol- or hydrogen-oxidizing forms will dominate. Resistance or tolerance to acids, bases, high temperatures, or radiation can also be used for Selection. Finally, alongside "positive" selection, "negative" selection using selectively acting inhibitors is frequently employed. On a medium containing azide in the presence of O₂, lactic acid bacteria will grow, for example, while the growth of aerobic microorganisms is suppressed. Azide, cyanide, and H₂S exert a selective inhibitory effect on those aerobic organisms whose Respiration relies on Cytochromes. In medical Diagnostics, selective growth inhibition is used to detect *Corynebacterium diphtheriae* (using tellurite media) and pathogenic *Enterobacteriaceae* (Agar media containing bismuth). The Use of penicillin to select for auxotrophic mutants of *Escherichia coli* will be discussed further in Section 15.2.3. To suppress the growth of Gram-positive bacteria, penicillin is added to the nutrient medium. The Growth of filamentous Fungi, Yeasts, Protozoa, and other eukaryotes is inhibited by The addition of cycloheximide.

The inoculum used in an experiment may contain multiple strains with the same type of METABOLISM that differ only slightly from one another, such as in their pH optima and growth rates. If such material is used to establish an enrichment culture, the strain best adapted to the given conditions or the fastest-growing one will dominate; all others will be suppressed and remain unisolated. Therefore, whenever the goal is to isolate as many strains as possible growing under specific selective conditions, the inoculum should be plated directly onto agar plates. On solid selective media, strains for which the conditions are favorable form discrete colonies. When colonies are spaced sufficiently far apart, competition for nutrients cannot occur: slower-growing strains are not suppressed by faster-growing ones, allowing both types to be isolated separately.

Class="center">Table 6.3. Selective conditions for some bacteria

PI – pasteurized inoculum; YE – Yeast extract

Overview Table 6.3 lists the principal selective conditions for the growth of typical representatives of microbial groups with specific metabolic types.

Pure culture. A pure culture is defined as the progeny of a single cell (a clone). Obtaining a pure culture, unequivocally proving its purity, and protecting it from contaminating organisms is the primary task of a microbiologist. Pure cultures of microorganisms are, with rare exceptions, isolated On the surface or within solid nutrient media. The Procedure begins by separating a single cell from a cell population, and the colony arising from this cell must likewise remain isolated from other Cells and colonies. Aerobic bacteria are isolated using Koch's method—spreading a suspension over The surface of a medium in Petri dishes—or by employing the less laborious streak-plate method using a platinum loop on solidified agar (Fig. 6.3). Anaerobic bacteria are suspended in molten agar (45°C) and incubated without access to air (Fig. 6.4). Careful ISOLATION OF A single colony, resuspension in liquid medium, and repeated streaking or agar dilution make it possible to obtain pure cultures of the majority of microorganisms.

Fig. 6.3. Streak-plate technique for isolating a pure culture of aerobic bacteria. A drop of bacterial suspension is applied to solidified agar using a platinum loop. Successive streaking of the droplet reduces the culture density. The outermost, single colonies have grown with a high degree of probability from individual cells. The colony at the top of the image is a result of airborne contamination. (Photo by V. Lehmann.)

Pure cultures can also be isolated in liquid media if the target organisms numerically predominate in the initial material. By making serial dilutions of the suspension with nutrient medium, one can eventually ensure that only a single cell is isolated at the final dilution step. In this case, a clone—that is, a pure culture—will be obtained.

Fig. 6.4. Dilution series of a purple sulfur bacterium culture in semi-solid agar medium (0.8% agar) after one week of incubation in the light. The photograph illustrates the procedure for isolating a pure culture of anaerobic bacteria (the dilution-shake method). The agar Column is capped with a mixture of paraffin and liquid paraffin to exclude air. (Photo by V. Lehmann.)

Mixed cultures. Natural populations generally consist of a mixture of diverse microorganisms. Various Forms of interaction occur among them, such as competition for a shared substrate, commensalism, or mutualism (see Section 17.2). Mixed cultures are increasingly used to study these and Other types of interactions. Under defined, controlled conditions in both batch and continuous-flow cultures, it is possible to observe the sequential turnover (succession) of individual organisms and accumulated metabolic products. This, in turn, provides insight into the synergistic or antagonistic relationships between different organisms. Mixed cultures can be prepared by combining pure cultures. Research conducted on mixed cultures of defined composition helps elucidate the complex interactions of microorganisms in their natural habitats.

Both households and industries utilize not only pure cultures but also mixed cultures. Some of these have been termed "natural pure strains." Examples include sour dough, kefir, kombucha, and "pure yeast strains." Mixed cultures also play a major role in wastewater Treatment.