Team:SDU-Denmark/protocols

From 2010.igem.org

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(Protocols)
(Flagella staining)
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===Flagella staining===
 
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1.1
 
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Day 1: The bacteria were grown in 5 ml-LB media ON. The solutions used for staining were prepared.
 
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Solution I:
 
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The following components were added in the listed order:
 
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• 5 g of tannic acid was dissolved in 9.65 ml distilled water.
 
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• 150 µl  9% FeCl3
 
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• 100 µl 1% NaOH
 
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• 200 µl formalin
 
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Solution II:
 
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• 2 g silver nitrate was dissolved in 10 ml distilled water
 
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• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.
 
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Day 2: The bacteria were boosted in 5 ml LB-media to ensure that they were in the exponential growth phase when used for staining. They were diluted to approximately OD=1.
 
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Preliminary bacteria work:
 
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• The bacteria were centrifuged 15 min at 4000rpm
 
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• The pellet was resuspended in LB-media to an OD of 3.
 
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Staining protocol: 
 
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• A clean glass slide was used and Poly-L-Lysin was added onto a small area. 
 
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• 20 µl of the bacteria solution was plated on the slide and allowed to air dry.
 
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• The slide was flooded with solution I and allowed to stand for 30 min before it was washed with distilled water.
 
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• Solution II was added and was incubated at room temperature for 10 min and was washed with distilled water.
 
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• The slide was flooded with a carbol-fuchsin solution and air-dried before washed with distilled water.
 
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PBS were added to the area containing the bacteria and they were covered with a cover slide. The slides are now ready for examination under the microscope.
 
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1.2
 
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Day 1: Bacteria were platede on agar plates and incubated at 37 degrees ON.  The staining solutions were prepared.
 
-
Solution I:
 
-
The following components were added in the listed order:
 
-
• 5 g of tannic acid was dissolved in 9.65 ml distilled water.
 
-
• 150 µl  9% FeCl3
 
-
• 100 µl 1% NaOH
 
-
• 200 µl formalin
 
-
Solution II:
 
-
• 2 g silver nitrate was dissolved in 10 ml distilled water
 
-
• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.
 
-
Day 2: A bacteria colony was dissolved in LB-media .
 
-
Staining protocol: 
 
-
• A clean glass slide was used and Poly-L-Lysin was added onto a small area. 
 
-
• 20 µl of the bacteria solution was plated on the slide and allowed to air dry.
 
-
• The slide was flooded with solution I and allowed to stand for 30 min before it was washed with distilled water.
 
-
• Solution II was added and was incubated at room temperature for 10 min and was washed with distilled water.
 
-
• The slide was flooded with a carbol-fuchsin solution and air-dried before washed with distilled water.
 
-
PBS were added to the area containing the bacteria and they were covered with a cover slide. The slides are now ready for examination under the microscope.
 
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Revision as of 19:46, 18 October 2010