11-1 Introduction to isolation

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Many specific kinds of microorganisms can be obtained from natural habitats (soil, water, etc.) by the creation of an artificial environment for them in the laboratory which will enhance their growth over competing organisms. Characteristics of the organisms which give them special advantages over others are exploited in the formulation of culture media and the choice of incubation conditions. Ulti-mately we want to achieve the following:

  1. Satisfy the nutritional requirements of the desired organisms such that they grow well.
  2. Inhibit as many other organisms as possible.
  3. Enhance the detection of desired organisms as early in the process as possible. Some microbial groups have recognizable cultural characteristics (e.g., a certain type of colony as seen for Streptomyces, special pigmentation characteristics of colonies and broth cultures and/or recognizable cellular morphologies (e.g. peculiar cell shapes possessed by some photosynthetic bacteria and nitrogen-fixers, presence of endospores produced by Bacillus and related genera).
  4. Often the source material is inoculated into a broth medium which will encourage the prolifera-tion of the desired organism; this is called an enrichment. When an enrichment is formulated to sup-press the growth of undesired competitors, it is then a selective enrichment. A selective enrichment (such as what we will perform in in the for photosynthetics and the nitrogen fixers) increases the probability that colonies of the desired organism will be isolated upon subsequent streak-plating and not overrun too severely by others. When plate counts are to be performed and/or when the desired organism is relatively abundant, no enrichment is made and the source material is plated directly as we will see for the Bacillus.

In setting up any isolation procedure, four major considerations are made:

  1. Choice of suitable source material containing the desired organism.
  2. Possible pre-treatment of the inoculum such as heat-shocking or filtration of the source material.
  3. Choice of suitable media, usually but not always selective. Also whether an enrichment is needed prior to plating for isolation.
  4. Choice of suitable incubation conditions, e.g., anaerobic atmosphere, availability of light, high or low temperature.

Some examples of procedures not pursued in Experiment 11 are described as follows:

  1. One can enrich for cyanobacteria and algae by inoculating pond water into a simple mineral salts medium lacking a carbon source and incubating the medium under a fluorescent light at 25°C. As both types of organisms are photoautotrophs, they will grow, fixing carbon dioxide from the atmosphere. If a 37°C temperature were used, growth of algae would be inhibited, allowing growth of mostly cyanobacteria. As most cyanobacteria fix nitrogen, deletion of nitrogen-containing compounds from the medium will inhibit the non-nitrogen-fixing algae.
  2. It is possible to prepare an enrichment for microorganisms capable of degrading practically any naturally-occurring organic compound (and many synthetic compounds) by inoculating the source material into a broth medium containing the compound in question as the sole source of carbon and incubating in the dark (to discourage photoautotrophs). Theoretically, the only organisms able to initiate growth in such a medium are utilizing the compound, and isolation of colonies is done on the solid version of the medium.
  3. In the isolation of lactic acid bacteria (Lactobacillus, Streptococcus, etc.) which will be done in Experiment 12, competition from other organisms can be eliminated and growth of the lactic acid bacteria can be encouraged by a direct plating of the source material as follows:
  • The plating medium should be fairly rich, containing glucose and growth factors which are needed by these fermenting, fastidious bacteria.
  • As typical lactic acid bacteria are aerotolerant anaerobes and do not possess cytochromes, the incorporation of a selective agent which will inhibit respiring organisms can be done. Sodium azide interferes with the iron porphyrin moiety of one or more of the cytochromes and is usually added in a 0.02% concen-tration.
  • As strict anaerobes are also resistant to cytochrome inhibitors, aerobic incubation of the plates will result in their inhibition. (Sodium azide will not be expected to be an efficient inhibitor under anaerobic conditions as any cytochrome activity will be inhibited already.) Thus, exclusive growth of aerotolerant anaerobes will be attained.
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