BISC209: Serial Dilution: Difference between revisions
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3. Using your P200 micropipet, transfer 100 μL of a 1:100 dilution to the 0.9 ml diluent in the tube labeled 10<sup>-3</sup>, mix well by vortexing or shaking vigorously.<BR><BR> | 3. Using your P200 micropipet, transfer 100 μL of a 1:100 dilution to the 0.9 ml diluent in the tube labeled 10<sup>-3</sup>, mix well by vortexing or shaking vigorously.<BR><BR> | ||
4. Using a new tip, transfer 100 μL ml of the 10<sup>-3</sup> dilution to the 0.9 ml diluent in the tube labeled 10<sup>-4</sup>. Mix well. This makes a 10,<sup>-4</sup>.<BR><BR> | 4. Using a new tip, transfer 100 μL ml of the 10<sup>-3</sup> dilution to the 0.9 ml diluent in the tube labeled 10<sup>-4</sup>. Mix well. This makes a 10,<sup>-4</sup>.<BR><BR> | ||
5. Continue sequentially in this manner until you have carried the dilution to an appropriate end, usually 10<sup>-7</sup> or 10<sup>-8<sup>.<BR><BR> | 5. Continue sequentially in this manner until you have carried the dilution to an appropriate end, usually 10<sup>-7</sup> or 10<sup>-8</sup>.<BR><BR> | ||
6. On your bench | 6. On your bench find a glass spreader and a petri dish of alcohol. The spreader is sterilized by briefly dipping it in the alcohol and passing it through the flame of your Bunsen burner. (The Bunsen burner is not used to heat sterilize the spreader but only for igniting the ethanol.) After the spreader has been flamed, it should be moved through the air as little as possible. In order to cool the spreader, the cover of the Petri dish is lifted just enough to get the spreader under it. Briefly touch the spreader to the agar avoiding the dispensed dilution (touching the hot spreader to the microbial solution may kill cells.) <BR><BR> | ||
Follow these steps each time you dispense an aliquot of your serial dilutions to a destination dilution plate. Use the spreader to gently push the dispensed diluent two or three times clockwise around the dish, and then several times counterclockwise. Pressing too hard will cause the microorganisms to collect at the edge of the spreader, resulting in uneven distribution. | |||
7. Use a flamed glass spreader, spread a 1ml (or 0.1ml) sample of the 10<sup>-5</sup> dilution on the agar surface of the appropriately labeled dish. <BR><BR> | 7. Use a flamed glass spreader, spread a 1ml (or 0.1ml) sample of the 10<sup>-5</sup> dilution on the agar surface of the appropriately labeled dish. <BR><BR> | ||
8. Repeat for 10<sup>-6</sup> and 10<sup>-7</sup>dilution.<BR><BR> | 8. Repeat for 10<sup>-6</sup> and 10<sup>-7</sup>dilution.<BR><BR> | ||
9. Allow any moisture to be absorbed into the agar, invert and incubate, at the appropriate temperature.<BR><BR> | 9. Allow any moisture to be absorbed into the agar, invert and incubate, at the appropriate temperature.<BR><BR> | ||
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Revision as of 09:53, 11 January 2010
Serial Dilution Technique :
This technique is useful for obtaining countable numbers of bacterial colonies by diluting a highly concentrated culture. The protocol described here is for serial 1:10 dilutions and can be modified for any dilution series desired. Generally, the last 3 dilutions are plated in duplicate or triplicate. This procedure is based on using a 1/100 stock (e.g. Soil Extract of 1 gram with diluent added to 100 ml)
1. Label 5 tubes 10-3, 10-4 etc. through 10 -7.
2. Label destination plates of appropriate solid medium with dilution and identifying information. Because your goal is to obtain 30-300 well isolated colonies on a plate, generally only the 10-5 through 10-8 dilutions are plated. If your soil is exceptionally rich or poor the choice of dilutions will need to be modified. Often dilutions are plated in duplicate or triplicate.
2. Pipet 0.9 ml of sterile water using the same sterile 1 ml pipet into the 5 tubes labeled in step 1.
3. Using your P200 micropipet, transfer 100 μL of a 1:100 dilution to the 0.9 ml diluent in the tube labeled 10-3, mix well by vortexing or shaking vigorously.
4. Using a new tip, transfer 100 μL ml of the 10-3 dilution to the 0.9 ml diluent in the tube labeled 10-4. Mix well. This makes a 10,-4.
5. Continue sequentially in this manner until you have carried the dilution to an appropriate end, usually 10-7 or 10-8.
6. On your bench find a glass spreader and a petri dish of alcohol. The spreader is sterilized by briefly dipping it in the alcohol and passing it through the flame of your Bunsen burner. (The Bunsen burner is not used to heat sterilize the spreader but only for igniting the ethanol.) After the spreader has been flamed, it should be moved through the air as little as possible. In order to cool the spreader, the cover of the Petri dish is lifted just enough to get the spreader under it. Briefly touch the spreader to the agar avoiding the dispensed dilution (touching the hot spreader to the microbial solution may kill cells.)
Follow these steps each time you dispense an aliquot of your serial dilutions to a destination dilution plate. Use the spreader to gently push the dispensed diluent two or three times clockwise around the dish, and then several times counterclockwise. Pressing too hard will cause the microorganisms to collect at the edge of the spreader, resulting in uneven distribution.
7. Use a flamed glass spreader, spread a 1ml (or 0.1ml) sample of the 10-5 dilution on the agar surface of the appropriately labeled dish.
8. Repeat for 10-6 and 10-7dilution.
9. Allow any moisture to be absorbed into the agar, invert and incubate, at the appropriate temperature.
