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EXPERIMENT 4 ISOLATION OF SOIL BACTERIA: VIABLE TITER and PURE CULTURE Bacteria in soil occur singly and in aggregates. To estimate the number of bacteria in a gram of soil, the soil must be both diluted and mixed thoroughly so that the aggregates are broken up such that a suspension of single cells is achieved. The cell suspension is then serially diluted so that from some dilutions a reasonable number of cells (30 to 300) are dispensed into Petri plates. The samples in Petri plates are then mixed with sterile, molten (liquid) agar medium which is then allowed to solidify. This method of plating is called pour plating. In a later experiment, we will use spread plating. Upon incubation each cell will give rise to a colony either in the agar or on the agar surface. It is possible that a colony could have arisen from two or more cells that stuck together. Thus a colony forming unit (CFU) may have originated from one or more cells. The viable titer is determined by counting colonies (CFU's) and multiplying by the dilution factor. This method only counts living cells as dead cells do not reproduce to form colonies . The viable titer is determined from countable plates: plates from dilutions that yield at least 30 colonies (so that a statistically significant number has been counted) and less than 300 colonies. When a plate has more than 300 colonies, there is such crowding that fast growing bacteria overwhelm slow growers: the fast growers either remove nutrients or produce inhibitory end products before slow growers can form a visible colony. Appendix V reviews dilution problems and how to calculate dilutions, dilution factors and titers. Later, isolated colonies will be examined for the types of cells and one will be restreaked to obtain a pure culture. A pure culture is defined as the progeny from one cell. Actually we will be making an axenic culture from a clone (colony). Assuming that one cell could have given rise to the colony, we call these pure cultures even though we have no technical proof of that. Proof of pure culture involves showing that all the colonies on the restreak are identical and Gram staining these to demonstrate all the cells in the resulting colonies are identical and the same as those on the original plate.
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This note was uploaded on 03/06/2011 for the course MCB 2000 taught by Professor Gantar during the Spring '08 term at FIU.

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