Team:EPF Lausanne/Project/Materials Methods Drosophila

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=Materials and Methods=
=Materials and Methods=
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The Flies.
 
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Working with flies: We put the drosophila to sleep with CO2 and then manipulated them with fine paintbrushes and tweezers on a box were we could add CO2 at will.
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==The Flies==
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Females / Males: For each experiment we worked exclusively on female flies to have the least variability. We chose to work on females rather than males because the females eat more, as they have to lay eggs, are bigger and have a higher metabolism. We hence assumed that by working with females, we could achieve an higher uptake of bacteria.
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====Working with flies:====
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Oregon / Relish: We worked on the drosophila WT Oregon. We also conducted some experiments with the drosophila mutant relish, which is immunodeficient, to see if the basic immune system of the WT flies is harmful to asaia.
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We put the drosophila to sleep with CO<sub>2</sub> and then manipulated them with fine paintbrushes and tweezers on a box were we could add CO<sub>2</sub> at will.
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The Infection
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====Females / Males:====
 +
For each experiment we worked exclusively on female flies to reduce the  variability. We chose to work on females rather than males because the females eat less randomly, as they have to lay eggs, are bigger and have a higher metabolism. We hence assumed that by working with females, we could achieve an higher uptake of bacteria.
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We prepared aliquots of bacteria with OD 100. We then put 50ul of the solution on filters that we placed over the medium which the flies feed on. Therefore the flies take up the bacteria whenever they eat.  
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====Oregon / Relish: ====
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We worked on the Drosophila wild type Oregon strain. We also conducted some experiments with the immunodeficient  Drosophila mutant Relish, to determine  if the innate immune system of Drosophila is harmful to asaia.
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Before infecting the flies we starved them for approximatively two hours in order to ensure that they would  immediately take up the bacteria, and all at approximately the same time.
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==The Infection==
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The Bacterial Controls
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The bacterial cultures were concentrated by centrifugation to obtain a sample with the optical density measured at 600 nm was 100 (OD600=100). We then put 150 ul of the solution on filters that we placed over the medium which the flies feed on. The flies then take up the bacteria whenever they eat.
 +
 
 +
Before infection the flies were starved for two hours to ensure and synchronize their food uptake..
 +
 
 +
==The Bacterial Controls==
We infected a set of flies with different types of bacteria whose reaction in the drosophila’s gut is are well known, and therefore use these as controls.
We infected a set of flies with different types of bacteria whose reaction in the drosophila’s gut is are well known, and therefore use these as controls.
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Positive control (Bacteria that should persist in the gut of the drosophila).
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<b>Positive control</b> (Bacteria that should persist in the gut of the drosophila).
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Pseudomonas entomophila (Pe)
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<br>Pseudomonas entomophila (Pe)
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Erwinia carotovora carotovora 15 (ECC-15)
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<br>Erwinia carotovora carotovora 15 (ECC-15)
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Negative control (Bacteria that do not persist in the gut of drosophila).
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<b>Negative control</b > (Bacteria that do not persist in the gut of drosophila).
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Gac: non persistent Pe mutant
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<br>Gac: non persistent Pe mutant
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Time of measurements
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==Time of measurements==
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3h: Three hours after the infection we verified that the flies were actually properly infected. We expected to observe the presence of all types of bacteria  (asaia, positive and negative control) as the flies would not have had time to produce an immune response against these bacteria.
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<b>3h: </b>Three hours after the infection we verified that the flies were actually properly infected. We expected to observe the presence of all types of bacteria  (asaia, positive and negative control) as the flies would not have had time to produce an immune response against these bacteria.
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24h: We should start observing a decline in the negative control whilst the positive control persists. We can also start observing the tendency of asaia.
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<b> 24h: </b> We should start observing a decline in the negative control whilst the positive control persists. We can also start observing the tendency of asaia.
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48h: The positive control should still persist and the negative control should have almost disappeared. We can then compare asaia to the controls and conclude on the persistency of asaia.  
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<b> 48h: </b> The positive control should still persist and the negative control should have almost disappeared. We can then compare asaia to the controls and conclude on the persistency of asaia.  
bove.
bove.
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Latest revision as of 20:27, 26 October 2010



Contents

Materials and Methods

The Flies

Working with flies:

We put the drosophila to sleep with CO2 and then manipulated them with fine paintbrushes and tweezers on a box were we could add CO2 at will.

Females / Males:

For each experiment we worked exclusively on female flies to reduce the variability. We chose to work on females rather than males because the females eat less randomly, as they have to lay eggs, are bigger and have a higher metabolism. We hence assumed that by working with females, we could achieve an higher uptake of bacteria.

Oregon / Relish:

We worked on the Drosophila wild type Oregon strain. We also conducted some experiments with the immunodeficient Drosophila mutant Relish, to determine if the innate immune system of Drosophila is harmful to asaia.

The Infection

The bacterial cultures were concentrated by centrifugation to obtain a sample with the optical density measured at 600 nm was 100 (OD600=100). We then put 150 ul of the solution on filters that we placed over the medium which the flies feed on. The flies then take up the bacteria whenever they eat.

Before infection the flies were starved for two hours to ensure and synchronize their food uptake..

The Bacterial Controls

We infected a set of flies with different types of bacteria whose reaction in the drosophila’s gut is are well known, and therefore use these as controls.

Positive control (Bacteria that should persist in the gut of the drosophila).
Pseudomonas entomophila (Pe)
Erwinia carotovora carotovora 15 (ECC-15)


Negative control (Bacteria that do not persist in the gut of drosophila).
Gac: non persistent Pe mutant

Time of measurements

3h: Three hours after the infection we verified that the flies were actually properly infected. We expected to observe the presence of all types of bacteria (asaia, positive and negative control) as the flies would not have had time to produce an immune response against these bacteria.

24h: We should start observing a decline in the negative control whilst the positive control persists. We can also start observing the tendency of asaia.

48h: The positive control should still persist and the negative control should have almost disappeared. We can then compare asaia to the controls and conclude on the persistency of asaia. bove.

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