# Ventral Nerve cord ( VNC) regenration in earthworms

Ventral Nerve cord ( VNC) regenration in earthworms
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September 5th 2019

Earthwom group

Long term objective :- to study ventral nerve regeneration in earthworms ( e.fetida)
Short term objective:- to perform rapid escape response assay (RER)

Immediate objective:- to maintain cup cultures

Today’s plan

Today I’m going to perform RER assay with 15 earthwoms.

In this assay We’ll take 30 testubes with 2 test tubes for each eartworm.
In one test tube we’ll take distilled water
In other we’ll have 200mM NaCl solution

First we add the eathworm in distilled water and start timer of two minutes after 2min we transfer it to NaCl and put timer of two minutes.

Expectations :-
The eartworm will not come out of disstilled water as it contains no ions in two minutes
And in NaCl it will come in two minutes .

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Please provide the rationale behind choosing a particular concentration of 200mM of NaCl for the assay and is it not possible like when you put the earthworm in distilled water there is a chance that solutes from the inside the body will come out as there is no ions in the water @baviskarritik

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@bivasnag @Arunan
So here’s the explaination, as we want to used hypertonic solution in testube so that the eartworm comes out of it and also is not dead we use slight hypertonic solution.
In isotonic solution :- the salt concentration is 0.9%
Where for 200mM we first make 1 molar solution.
Q) How to make one molar solution?
• We take 58.4 grams of NaCl and dissolve it in 1000ml water and hence this is our one molar solution. ie; 5.84% in 1 molar
To make 200mM we dilute 1molar solution 80times. ie; 20ml 1M NaCl solution + 80ml distilled water
We know that
1M = 1000mM
Therefore, if 1M NaCl solution has 5.8% salt
100mM = 0.58% salt
200mM = 1.16% salt
Now when isotonic condition has 0.9% salt
200mM is just above the range of isotonic solution and is slightly hypertonic hence we use 200mM solution.

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Any reference? How do you know that the isotonic condition for earthworm is 0.9%?

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@baviskarritik
Why are you selecting 2 mins time interval? why not 1min?
What is the rationale behind the experiment design?
Which earthworms you are using?
How many earthworms do you have in stock? Please provide photos of your earthworm cultures? Are you also maintaining vermicultures for stock earthworms? Pictures please.
How many will be needed for this experiment?
How many runs will you have per earthworm?
How many runs you expect will be sufficient to prove earthworms escape from NaCl?

How is it connected to ventral nerve cord regeneration?

I don’t have any specific reference @Akshitha Wikipedia is the reference.
Saline (medicine) - Wikipedia

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2mins we put them in distilled water, so if it stays in dw for 2 mins then we transfer it to NaCl test tube. That is why we select 2mins of time interval.
We are using E.fetida earthworms
For this earthwom we have 15 earthworms and is stock we have 4stock cup culture 10earthworms in each cup.
We will perform 3 runs with non lesioned earthworms and 3 runs with lesioned earthworms. 3 runs are enough I guess

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