Silage protocols: Difference between revisions
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#Vortex to mix. | #Vortex to mix. | ||
#Perform five more 1:10 serial dilutions by adding 100ul of the previous dilution to 900ul water. | #Perform five more 1:10 serial dilutions by adding 100ul of the previous dilution to 900ul water. | ||
*you will use the last three of these dilutions for plating (1:10<sup>4</sup>,1:10<sup>5</sup>, 1:10<sup>6</sup>) | ::*you will use the last three of these dilutions for plating (1:10<sup>4</sup>,1:10<sup>5</sup>, 1:10<sup>6</sup>) | ||
#Plate 100ul of each of these three final dilutions in triplicate | #Plate 100ul of each of these three final dilutions in triplicate | ||
*this means you'll use a total of nine plates. | *this means you'll use a total of nine plates. | ||
#Calculate the total count per 1g of silage. | #Calculate the total count per 1g of silage. | ||
*for 1:10<sup>4</sup> dilution multiply the plate count by 10<sup>5</sup> to get cfu/g silage. | ::*for 1:10<sup>4</sup> dilution multiply the plate count by 10<sup>5</sup> to get cfu/g silage. | ||
*for 1:10<sup>5</sup> dilution multiply by 10<sup>6</sup> | ::*for 1:10<sup>5</sup> dilution multiply by 10<sup>6</sup> | ||
*for 1:10<sup>6</sup> dilution multiply by 10<sup>7</sup> | ::*for 1:10<sup>6</sup> dilution multiply by 10<sup>7</sup> | ||
*if a plate has more than ~300 colonies,then forget about that plate. | ::*if a plate has more than ~300 colonies,then forget about that plate. | ||
#Average results | #Average results | ||
Revision as of 17:01, 8 June 2011
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Introduction
These protocols are use for the analysis of any forage material, but especially silage.
pH Measurement
- Weigh out 5g of silage.
- Add this material to 45ml of nanopure water
- Put in the shaker for 30 mins.
- Measure pH by submerging the probe in the liquid.
Plate Count
- Add 25g of silage to 25ml of TE Buffer in a small blender
- Blend 2X for 10 seconds (each time).
- Weigh 2g of this mix and add to 8ml DI water (This the first 1:10 dilution of your original sample).
- Vortex to mix.
- Perform five more 1:10 serial dilutions by adding 100ul of the previous dilution to 900ul water.
- you will use the last three of these dilutions for plating (1:104,1:105, 1:106)
- Plate 100ul of each of these three final dilutions in triplicate
- this means you'll use a total of nine plates.
- Calculate the total count per 1g of silage.
- for 1:104 dilution multiply the plate count by 105 to get cfu/g silage.
- for 1:105 dilution multiply by 106
- for 1:106 dilution multiply by 107
- if a plate has more than ~300 colonies,then forget about that plate.
- Average results
Notes
References
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