Basic Media Making 2009

Basic Media Making 2009 - Basic Laboratory Procedures...

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Basic Laboratory Procedures Involved in Media Making: The majority of laboratory operations utilized in the in vitro propagation of plants can be easily learned. One needs to concentrate mainly on accuracy, cleanliness, and strict adherence to details when performing in vitro techniques. I. Medium Stock Solutions Before plant tissue culture formulations were commercially available, mineral salt mixtures were prepared as stock solutions ranging from 10 to 100 times the final concentrations used in the medium. Stock solutions can be prepared as two solutions, one containing all of the macronutrients and one containing all of the micronutrients. These solutions must be kept fairly dilute (10-20X) in order to avoid precipitation of calcium and magnesium phosphates and sulfates. A more common method is to arrange mineral salt stock solutions according to the ions they contain. A series of solutions containing the inorganic components of the medium is prepared; precise combinations may vary from lab to lab. Using this method, salt stocks can be prepared in 100X concentrations. Iron-EDTA chelate is prepared from iron sulfate and Na-EDTA by mixing the proper amounts of the two compounds with water and then autoclaving. This stock must then be stored in a dark container. The prepared stock solutions are usually stored in the refrigerator. Although the initial cost of chemicals may be substantial, overall the ongoing cost of using stock solutions is probably less than that of using prepared mixes (unless you factor in labor). However, the stocks must be prepared over and over when they are used up, and each time this is done potential error is introduced. It is best if one person consistently prepares the stocks. Most people no longer make up stock solutions of the inorganic medium components. Stocks solutions may be useful if many different media are made in the lab or frequent changes in concentrations are made in individual components. II. Prepared Mixes Several companies sell prepared salt and vitamin mixtures as powders. These are easily handled by adding the proper amount of powder to water. The mixtures can be purchased as complete media or as salts alone. The packs often contain the necessary ingredients for one liter of medium. The contents are extremely hyroscopic once a pack is opened, so it is best to use the contents all at once. A small amount of precipitate may be seen in media prepared using either of these methods. This results from iron being displaced from the chelate over time as media is stored. It doesn’t appear to be deleterious.
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III. Organic Addenda Most organic addenda are added in relatively low concentrations, too low to be weighed out accurately. Therefore, stock solutions ranging from 100 to 1000X final
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Basic Media Making 2009 - Basic Laboratory Procedures...

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