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In 1872 scientists began to isolate and study microorganisms; generations of scientists have continued this pursuit. So why the interest? Of course there is the natural desire to understand the world around us and since microbes are part of our world we would like to understand them. Also there are practical reasons. The diagnosis and treatment of disease is often dependent upon the isolation and identification of the causative agent. Rapid screens have been developed to achieve this goal. Once isolated, bacterial pathogens can also be studied to help understand how they cause disease and how the body responds to infection. This information may then lead to better treatments. From a non-medical view point, investigation of microbes and their properties often has commercial applications. Bacteria are used in the production of many useful items such as antibiotics, food, and biological pesticides.

When designing an isolation protocol, many parameters need to be analyzed. One of the more important considerations is the need for a selective enrichment. This depends upon the population of the desired organism in the source material and the microbial community that surrounds them. If the bacteria are in high enough numbers to be easily detectable, it may be possible to plate directly onto a selective medium. If a low population is present, with or without large numbers of undesired bacteria, it is necessary to enrich for the desired organism using selective broth enrichment.

In overview, the process of enrichment involves inoculating a selective enrichment broth with the source material and incubating under conditions that will increase the population of the desired microorganism, while inhibiting the growth of unwanted microorganisms.

When designing or performing an enrichment, the goal is to take advantage of the microbe's biochemical and physiological properties so that its growth is favored. There are four main considerations.

1. Choice of inoculum. The source should contain the desired bacterium at a reasonable concentration to assure isolation.
2. Possible pre-treatment of the inoculum. Some microbes are resistant to certain environmental conditions and this property can be used to remove many competing contaminates. For example, endospore-forming organisms can be enriched for by heating a sample in a 70-80°C water bath for 10 minutes, which kills all vegetative cells, leaving only endospores. The heat shocked sample can than be used as a source for endospore-forming bacteria.
3. Choice of medium. Most enrichment media contain compounds that inhibit the growth of unwanted contaminants, while promoting the growth of the desired microbe. For example, Enteric Enrichment Broth contains bile salts (Oxgall) and brilliant green, both of which inhibit the growth of unwanted microorganisms. It is always a good idea to examine the medium used in each enrichment to learn the type of components used and their purpose. In other words, check the medium's recipe.
4. Suitable incubation conditions. The temperature, atmosphere (aerobic versus anaerobic), light intensity, etc., can be varied to favor the desired bacterium.

Once an organism has been enriched, it is streaked for isolated colonies on a suitable medium. (Typically this is the selective enrichment broth + 1.5% agar.) Eventually, the pure culture is stored in a manner to preserve it and then its properties can be studied.

Several examples and experiments follow to give you an idea of the nuts and bolts of enrichment and isolation procedures. There are probably as many enrichment protocols as there are microorganisms. No one protocol is the correct method for isolating a specific species. However, the rate of success and the ease of performing the protocol are important factors to consider. Here are some examples of enrichment protocols that take advantage of physiological properties of the desired organisms.

• Cyanobacteria and algae can be enriched for by inoculating pond water into a simple mineral salts medium lacking a source of carbon and energy and incubating them under a fluorescent light at 25°C. Being photoautotrophs, both organisms will grow, using CO₂ from the atmosphere as their carbon source and light as the energy source. Since many cyanobacteria can use N₂ gas as a nitrogen source and grow at 37°C (abilities not shared by the algae), they can be almost exclusively enriched for by eliminating nitrogen containing compounds from the medium and incubating at 37°C.
• Extreme halophiles grow in environments containing saturated salt solutions. Microorganisms of this type ( e.g. Halobacterium salinarium) can be enriched for by growing them in a rich medium containing peptone, yeast extract and 4.3 M NaCl. Very few other organisms are capable of multiplying in this concentration of salt.
• Thermophilic organisms capable of degrading starch can be isolated by use of a minimal medium containing starch as the sole carbon and energy source. After inoculation, the broth can be incubated at high temperature (65-85°C) and any organisms that grow will be of the desired type.

In this chapter we look at several enrichment protocols to familiarize the student with a few important groups and microbes and more importantly, aquaint students with the thought process of enrichment.

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