2-5 Spread plates and dilution plating

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An absolute requirement for a microbiologist is to be able to determine the concentration of microorganisms in a given sample. Various particle-counting devices, spectrophotometric methods and microscopic techniques have been used to count cells. However, one drawback to these methods is that they count dead as well as living cells. The most common method of enumerating viable cells is the plate-count method. Diluting microorganisms and placing them into petri plates (or plates) for incubation is another essential technique for working with microorganisms.This method suffers from some problems. First, only those organisms which can grow on the medium, and at the temperature and atmospheric conditions of incubation, will divide and develop into colonies. Second, each colony may not represent the progeny from one cell, as two or more cells (those in clusters, chains or otherwise close to one another) can give rise to one colony. For these reasons the counts obtained from the plate-count method are given as the number of colony-forming units (CFU's) per ml (or gram) rather than the number of cells per ml (or gram). Despite these drawbacks, the plate-count method is a powerful means by which concentrations of viable organisms may be estimated. Also, if it is desirable to count a specific subgroup of microorganisms in a sample, selective media or special incubation conditions can often be used to encourage the growth of only this class of organism. More information is available in the chapter on quantitative microbiology.

As microbial quantitation involves the use of pipettes"(or micropipettes) in preparing dilutions and inoculating plates, the beginning student in microbiology must become familiar with their use.

Due to the possibility of ingesting pathogens and toxic liquids, mouth-pipetting is forbidden in the laboratory! Pipettes are filled and subsequently emptied by the use of propipettes or other pipette bulb. Pay close attention to the demonstration of their use.

Important Safety Consideration: When fitting the pipette and pipette bulb together, use very gentle pressure!! Do not jam these items together! (Force is usually not the answer, a good general rule to live by in this lab and in life for that matter.) The glass pipette will probably break and possibly cause severe injury. Handle the pipette only at the top inch or so.

For volumes of 5 ml of less, micropipettes are often the tool of choice. Instruments are available that are capable of dispensing 5 ml all the way to less than 1 µl (one one-millionth of a liter). Micropipettes have made it possible to miniaturize many experiments and greatly decrease the cost of running them. They are also easy to use and can dispense volumes quickly, increasing the number of experiments that can be performed in a set amount of time. Micropipettes are the tool of choice for small volumes.

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