GENERAL MICROBIOLOGY - T.P. Pyrog - 2004

6. MICROBIAL GROWTH

6.3. MICROBIAL NUTRITION

6.3.8. Enrichment Culture Methods (Enrichment and Pure Cultures)

Many microorganisms are quite easy to isolate from their natural environments, and suitable conditions to support their growth can be selected without much difficulty. However, a vast number of microorganisms remained unknown until the enrichment culture technique was developed. The credit for this breakthrough belongs to our compatriot S.N. Winogradsky and the Dutch scientist M.W. Beijerinck.

Enrichment cultures. The enrichment culture method is conceptually straightforward. To enrich a specific Organism, conditions must be provided under which it can successfully outcompete others. By adjusting a combination of factors (sources of energy, carbon, and nitrogen, electron acceptors, gaseous atmosphere, illumination, Temperature, pH, etc.), specific conditions are established, and the medium is inoculated with a mixed population found, for example, in soil, mud, or Water. The microorganism best adapted to these specific conditions will grow and outcompete all accompanying organisms. Through successive transfers in the same liquid nutrient medium and plating onto an Agar medium of identical composition, the enriched strain can be readily isolated. The best inocula are samples collected from sites where 'natural enrichment' has already occurred. For instance, to isolate hydrocarbon-degrading microorganisms, soil samples from oil fields or petroleum separators are the ideal choice.

The enrichment culture method makes it possible to isolate microorganisms with virtually any combination of nutritional requirements, provided such organisms exist in nature. For example, a nitrogen-free mineral medium under illumination is selective for nitrogen-fixing cyanobacteria. If this same medium is supplemented with an organic source of energy and carbon, Azotobacter will develop aerobically in the dark, whereas Clostridium species will develop under anaerobic conditions. Frequently, 'positive' Selection is combined with 'negative' selection. For instance, in a medium containing sodium azide (a respiratory inhibitor) in the presence of oxygen, lactic acid Bacteria (facultative anaerobes) will grow, while the growth of aerobes is suppressed. Penicillin is added to the medium to inhibit the growth of Gram-positive bacteria, whereas the growth of micromycetes is suppressed by the antibiotic nystatin.

Pure culture. This is the progeny of a single Cell (a clone). Pure cultures of microorganisms are primarily isolated on agar media (either On the surface or embedded within). Pure cultures of aerobes are obtained using Koch's method (surface plating of serial ten-fold dilutions of a cell suspension onto a solid medium), while anaerobes are isolated by plating serial ten-fold dilutions of a cell suspension into molten agar (at 45 °C) and incubating them without access to air.