Definitive results from biochemical testing require

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Definitive Results from Biochemical Testing require that the Source Bacteria are from a Single Colony. Some of your Identifications will be complete to the Genus and Species Level while others will only be to the Genus Level. ____________________________________________ “Perhaps she would respond to an alternative form of persuasion.” What do you mean?’ I think it is time we demonstrated the full power of this station. Set your course for Alderaan.” © 1977 LucasFilm Ltd.
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Lab 1 Page 24 Gameplan for the Isolation of Pseudomonas sp. Pseudomonas is a Gram Negative, Rod-Shaped, Aerobic Bacterium that is Motile by means of Polar Flagella. Pseudomonas is Common, but not Abundant, in Soils. Thus, an Enrichment Step is necessary. An Enrichment relies on specific Metabolic or Physiological Capacities which can separate the Bacterium of Interest from other Bacteria that are likely to be found in a similar Environment. One of the most remarkable Physiological Properties of the Genus Pseudomonas is the very wide Range of different Organic Compounds that it can use as Sources of Carbon and Energy. Pseudomonas is one of the very few Bacteria that can utilize Benzoate as a Carbon Source. The Utilization of Benzoate requires Oxygen and Pseudomonas is Motile so it can migrate to the Surface of Non-Aerated Benzoate Media where the Oxygen Level is higher. These two Traits -- the Ability to utilize Benzoate and the Ability to migrate towards Regions of higher Oxygen Levels -- provide the Basis of our Pseudomonas Enrichment Strategy. A Soil Sample will be placed in a Test Tube of Medium in which Benzoate is the sole Carbon Source. This Test Tube will be left Stationary (Non-Aerated). This will soon result in an Anaerobic Environment at the Bottom of the Test Tube and an Aerobic Environment at the Air/Medium Interface at the Top of the Test Tube. Pseudomonas can migrate toward and remain in this Interface and utilize Benzoate in this Aerobic Environment. As a Result, Pseudomonas will frequently become the Dominant Bacterium in the Test Tube. 1. Enrichment A very small Amount of Soil will be added to a Test Tube of Sodium Benzoate Medium. The Test Tube will be incubated at 30°C for at least 48 Hours. Pseudomonas is one of very few Bacteria that can utilize Benzoate as a Carbon Source, and its Motility gives it the Advantage of being able to migrate to and remain in the Aerobic Air/Medium Interface at the Top of the Test Tube. Under these Conditions, Pseudomonas will outgrow other Soil Bacteria and will usually become the Predominant Species in the Test Tube. 2. Selection A Loopful of the Sodium Benzoate Medium used for Enrichment will be streaked onto a Sodium Benzoate Plate, which will then be incubated at 30°C for at least 48 Hours. A Gram Stain of Bacteria from an Isolated Colony should reveal Gram Negative Rods. A Wet Mount of Bacteria from an Isolated Colony should reveal Motile Rods.
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