Erickson:Flea Crushing

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(New page: Flea Crushing Beforehand, gather infected fleas into an microcentrifuge tube and place in -80 freezer to kill fleas. Remove fleas from freezer. Sterilize fleas with two was...)
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Flea Crushing
Flea Crushing
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     Beforehand, gather infected fleas into an microcentrifuge tube and place in -80 freezer to kill fleas.
+
     *Beforehand, gather infected fleas into an microcentrifuge tube and place in -80 freezer to kill fleas.
-
     Remove fleas from freezer.
+
     *Remove fleas from freezer.
-
     Sterilize fleas with two washes of 95% ethanol (add ethanol to tube and pipet up and down to wash fleas).
+
     *Sterilize fleas with two washes of 95% ethanol (add ethanol to tube and pipet up and down to wash fleas).
-
     Remove ethanol from fleas and wash with sterile water.
+
     *Remove ethanol from fleas and wash with sterile water.
-
     Dump fleas on an empty, sterile Petri plate and remove remove as much water as possible.  Spread the fleas out and allow them to
+
     *Dump fleas on an empty, sterile Petri plate and remove remove as much water as possible.  Spread the fleas out and allow  
-
     dry for 15 minutes.
+
     them to dry for 15 minutes.
-
     While the fleas are drying, label and number microcentrifuge tubes, one for each flea.
+
     *While the fleas are drying, label and number microcentrifuge tubes, one for each flea.
-
     Using wide bore pipet tips, measure out and place 20 uL of glass sand in each tube.
+
     *Using wide bore pipet tips, measure out and place 20 uL of glass sand in each tube.
-
     Using ethanol-sterilized forceps, transfer one flea to each tube, sterilizing the forceps each time you transfer fleas infected  
+
     *Using ethanol-sterilized forceps, transfer one flea to each tube, sterilizing the forceps each time you transfer fleas infected  
     with a different substance.
     with a different substance.
-
     Stick the end of a 1000 uL plastic pipet tip in a flame to soften the plastic.  Remove from the flame and allow the plastic to  
+
     *Stick the end of a 1000 uL plastic pipet tip in a flame to soften the plastic.  Remove from the flame and allow the plastic to  
     cool or press it against the inside of a microcentrifuge tube to blunt it (just make sure that there is no longer an opening  
     cool or press it against the inside of a microcentrifuge tube to blunt it (just make sure that there is no longer an opening  
     at the end of the tip).
     at the end of the tip).
-
     Use the tip to grind a flea into the glass sand.  Leave the pipet tip in the glass sand.  Use a new tip for each flea.
+
     *Use the tip to grind a flea into the glass sand.  Leave the pipet tip in the glass sand.  Use a new tip for each flea.
-
     Add 500 uL of broth (PBS or TB) to each tube, rinsing off and discarding the melted pipet tips.
+
     *Add 500 uL of broth (PBS or TB) to each tube, rinsing off and discarding the melted pipet tips.
-
     Vortex each tube and plate 100 uL of each flea onto its own plate (TB with any necessary antibiotics).
+
     *Vortex each tube and plate 100 uL of each flea onto its own plate (TB with any necessary antibiotics).
-
     Incubate plates at 30 degrees C.
+
     *Incubate plates at 30 degrees C.
-
     Save fleas in 4 degree C fridge.
+
     *Save fleas in 4 degree C fridge.
-
     After 24 hours count the colonies on the plates.
+
     *After 24 hours count the colonies on the plates.

Revision as of 18:52, 1 July 2009

Flea Crushing

    *Beforehand, gather infected fleas into an microcentrifuge tube and place in -80 freezer to kill fleas.
    *Remove fleas from freezer.
    *Sterilize fleas with two washes of 95% ethanol (add ethanol to tube and pipet up and down to wash fleas).
    *Remove ethanol from fleas and wash with sterile water.
    *Dump fleas on an empty, sterile Petri plate and remove remove as much water as possible.  Spread the fleas out and allow 
    them to dry for 15 minutes.
    *While the fleas are drying, label and number microcentrifuge tubes, one for each flea.
    *Using wide bore pipet tips, measure out and place 20 uL of glass sand in each tube.
    *Using ethanol-sterilized forceps, transfer one flea to each tube, sterilizing the forceps each time you transfer fleas infected 
    with a different substance.
    *Stick the end of a 1000 uL plastic pipet tip in a flame to soften the plastic.  Remove from the flame and allow the plastic to 
    cool or press it against the inside of a microcentrifuge tube to blunt it (just make sure that there is no longer an opening 
    at the end of the tip).
    *Use the tip to grind a flea into the glass sand.  Leave the pipet tip in the glass sand.  Use a new tip for each flea.
    *Add 500 uL of broth (PBS or TB) to each tube, rinsing off and discarding the melted pipet tips.
    *Vortex each tube and plate 100 uL of each flea onto its own plate (TB with any necessary antibiotics).
    *Incubate plates at 30 degrees C.
    *Save fleas in 4 degree C fridge.
    *After 24 hours count the colonies on the plates.
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