snail habitutioon & learning

[Pooja Chouhan]

dear all,

Hypothesis- Land snails ( Cepaea hortensis) are capable of learning
and can exhibit habituation.

Here in CCT Neuro lab we are performing the behavioral study of
snail(model system) for explaining some facts of learning. Our study
is going in two ways—
(1)Viewing Habituation (imp. Feature of cognition-non assosiative
learning) in snail.
(2)Verifing operant conditioning (a form of learning).

In first part of experiment------

Hypothesis-
Habituation is a phenomenon that is widespread across the
animal kingdom, it helps them survive by balancing responses to either
harmful stimuli or stimuli with uncertain significance. No animal can
afford to ignore potentially dangerous stimuli; the process of
habituation is a compromise between saving energy and the animal
saving itself. We can habituate snails to survive in laboratory
conditions. In relation, the snails will not be afraid after a while
of no harmful stimuli but when the stimulus is first presented the
snails should cover in an attempt to preserve it’s own life. These
habituated snails can be used in learning & memory based experiment.

Method-

Research design-
We have natural habitat ( fresh & flowing water, mild climate with
high humidity, litter etc.) for pulmonate snails (cepaea hortensis) in
our university garden. We collected 6 healthy snails from there on
July 4,2010. In lab we kept them for two days in litter-containig(2-3
inches) flower-pot(30*15cm). To provide humidity we poured some water
in litter everyday.
After two days (July 6), snails were cup cultured. To perform cup
culture we took two snails(less population density) in one plastic cup
containing- wetted filter paper & some pieces of cabbage leaves.
Plastic cups then covered with cotton cover. (For maintaining the
moisture we add some water in every 12 hours.)

in cup 1. snail1(small) & snail2(slight larger) cup2. snail3(small)
&snail4 (large) cup3. snail5 (small) & snail6 (large)

Research method-
we are habituating snails in laboratory conditions, (temp-22’C, dim
light, cup culture) feeding them cabbage (4 equal pieces of cabbage
leaf) every day (timing 11.00-11.45am). We are replacing filter paper
& cleaning cups in every two days. (For maintaining the moisture we
add some water in cup as in every 12 hours. )We are providing stress-
free (noiseless, low light, regular feeding, not being touched)
enviornment.

Snail’s growth factors-
Many factors can affect the habituation experiment .
1. Strength of stimulus: If a stimulus is too light or too strong,
habituation may be masked.
2. Population density-
3. Stress- Noise,Light,Vibration,Unsanitory conditions.
4. Feeding
5. Temrerature & moisture

Measured Variables-
1.Food eaten 2. Moving 3. Climbing 4. Fecets 5. Looking
active or not 6. Astivation

Obsertations-
We are observing habituation potential of snails in laboratory
conditions.
July,6
-Snail 5&6 were observed in coupled condition. All were active.
Astivation condition was not seen.
July,7
-All snails were active. Astivation condition was not seen.
July,9
- snails 3&4 were in astivation condition(on July 8,sunday. Neuro lab
was close. Snails were not feed.)
-other snails were active.
July,10
-In cup2 food was eaten completly. in cup 3 snail5 was sitting on
shell of snail6.
-All snails were active.
July ,11
-snail 3 & 6 were not moving. Other snails were active.
July,13
-snail 1,3,4,5 were in astivation condition.(on July 12. Neuro lab was
close due to holiday. so Snails were not feed.)
July14,
- some holes were seen at the opening of shell in snail 1,2,3,4,6.
- all snails were active.
July,16
-One spot was present on the shell of snail 3.
-all snails had been eaten food completely. Snails had been eaten some
part of filter paper along with cabbage.
July17,
-snail1&2 were climbing on the wall of cup. all snail were active.
july18,
All snails were active and healthy.they had eaten food completely.
(experiment is in progress.......)
Result estimation - All 6 snails are alive, active & healthy till
today. Hopefully ,All basic characteristic features(taken food using
rodula- thousands of microscopic tooth-like structures located on the
ribbon like tounge called rodula , movement, fecets, climbing,
astivation) of snails were observed during habituation experiment (in
process).


In second part of experiment------

Initiation- Any relatively permanent change in behaviour (for
behavioural potential) resulting from experiences is called learning.
Here we are trying to explain OPERANT conditioning (one
form of learning in which behavior is maintained or changed through
concequenses) by observing olfaction & gustatory behavior of snail.
As we know that snails are very sensitive and ideal for olfaction
responce. Land snails have four tentacles, two upper having two eyes
on top and two lower act as olfactory organs. Both set of tantacles
are retractable in land snails. Snails show olfactory response to the
environment- positive responce in favourable
conditions (food) and negative responce in unfavourable conditions
(bad smell, stress, water).

Hypothesis-
As we know that snails are very good in olfaction behaviour. They
likes to eat fruits & leaves or plants ,apple, beans, citrus, clover,
lettuce, rip cherry, tomatoes, cabbage etc . Snails have nervous
system. We can show learning in snails. (steps- habituation,
potential to habituation, study of olfaction behaviour of snail).
Natural food odours elicit different behavioural responses in snails.
Snails can either protract toward a stimulating cabbage odour or can
retract. Olfactory behaviour and feeding experiences can be operantly
conditioned as below-

1.Confirmatory test-1(olfactory)

control-water stimulation- cabbage
extract

if organism show positive responce to stimulus then-

2.Confirmatory test-2(gustatory)

control-water stimulation- various conc. of citric
acid(0.01M,0.05M,0.1M,0.5M,0.75M)

if organism show negative responce on a particular conc. (due to
bitter test of citric acid) to stimulus then-

3.Learning test( olfactory & gustatory)

control-cabbage extract stimulation- optimal conc. of
citric acid +cabbage extract

Required materials-
- Healthy snails
- Olfactometer
-Glass plates for moving the snails.
-Distilled water
-Beaker
-Micropippete
-Dropper
- Packed black Box with one side open
-Stopwatch
-freshly prepared cabbabe extract
-Various conc. Of citric acid(0.01M,0.05M,0.1M,0.5M,0.75M)
We are performing the experiment on Olfactometer-
- We are using one organism during the one test (20 readings).
Diameter of olfactometer is 10.1 cm.

Process-
To design the experiment we make statement (given on top);
predict hypothesis (as given above); select two parameters – control
(water), stimulation (cabbage extract); and replica(two healthy, cup
cultured landsnails (Cepea hortensis).
As we know that humid condition is required for snail growth but it
keep away from water(repulsion). So we are using water as control in
initial confirmatory tests.
..........................

All the steps of process will be post in next mail..
From
jaiprakash kushvah,
Pooja Chauhan
CCT Neuro lab
-07 batch

[Sam Cct]

 jaiprakash kushvah,
 Pooja Chauhan

Great Report.....................!!!!!!!!!!!!

Now that's what i call almost preciseness ...................

great work.......

  

[MC Arunan]

Dear  Jaiprakash & Pooja,

 

It is very nice to hear from you. But it is too difficult to read and understand 14 days of work in one shot! I am sure even for writing, you must have found it difficult! (This is why I insist that you should post your reports daily  if not the very second day, by pressing the reply or reply all button, for maintaining the continuity of your postings and mine as well as others' comments.

 

I suppose you are trying to do

1. Habituation experiment and

2. Operant conditioning experiment

 

You have to write the methods of the experiments separately and convince us that what you measure is actually telling you what assume to measure.

 

In this write up, mostly because both questions are attempted together, you have not been able to convince us  either.

Most importantly, though you have attempted, the method of experiment is not clear .

 

The point is that when you write a method that you have followed in your experiment, anybody who reads it should be able to follow it with out any difficulty and repeat it in her or his lab!

 

I will not be making separate comments to each line. I would request you to rewrite each experiment separately so that we will able to make sense separately.

 

I am sorry, I am asking you do this, but, once now you learn these tricks of science, you also will become masters in science! This stage is the most demanding stage. But you can pick up these with in very short time.

Cheers!

Arunan

[mohit saraf]

thnx bos........

[Himanshu Joshi]

Dear Jaiprakash  et. al
Nice report, But I wanted it to be posted in modules but any ways Good.
I will provide you some nice tips for documenting experimentation.

1. Always use tables like you had taken 6 snails and 14 days reading so make 15*6 table to report this if you have to report in bulk.you can use google docs and send it to our group.

2.Report your work daily so one can grab much from the report.

3.
Always have a precise hypothesis.In your comments you had mentioned the following to be the hypothesis

"Hypothesis-
As we know that snails are very good in olfaction behaviour. They
likes to eat fruits & leaves or plants ,apple, beans, citrus, clover,
lettuce, rip cherry, tomatoes, cabbage etc . Snails have nervous
system.  We can show learning in snails. (steps- habituation,
potential to habituation, study of olfaction behaviour of snail).
Natural food odours elicit different behavioural responses in snails.
Snails can either protract toward a stimulating cabbage odour or can
retract. Olfactory behaviour  and feeding experiences can be operantly
conditioned as below-"

Where is your hypothesis ? This seems to be an essay on snail's behavior

4.Confirmatory test are quite O.K you had written all tthe conc. for citric acid. but in your  learning test optimal conc. taken cannot do justice to your experiment .Be precise and write the accurate conc.of citric acid you had taken for learning exp.

5. The batch in the end written is not at all important to write in scientific documents.The name itself signifies who is performing it.
This means no one from any other batch had helped you  in this work.

I am sorry to write all this but it was the need of the hour so that one can lead towards the excellence in science.

Himanshu Joshi

[mohit saraf]

APPLECATION:- Copper Peptides in the Restorative Secretion of Snails Help to Reinforce Skin Healing and Minimize Post Burn Scars.

guys what do u think about this?

can we make it in our lab?

 

[MC Arunan]

Dear all,
Waiting for more comments and a rewriting from Pooja and Jaiprakash one by one, on the question of
1. Studies on Habtuation
2. Studies on Olfactory behavior
3, Studies on associative conditioning/ learning
4.  (?)Operant conditioning!!!

In fact, I suggest you should write a protocol for studying olfactory behavior in the snail that you have.
By the way, did you identify this snail scientifically, using taxonomically salient characteristics? If not specify that clearly some where, even though you suspect that it is Cepaea hortensis. (Did you notice that there is a way for writing the scientific name: either the genus name and the species name have to be underlined or should be in italics).
 Looking forward to.

Cheers!
Arunan

[jai prakash]

Dear sir ,
We are sending a rewrite on snail habitutation responding for
your questions.
Rewrite on snail habitutation
>>link-
"https://docs.google.com/document/edit?id=1ZOmfIRIWn-
TQcULFfCDM7jai0RrTHvu76lfyXgbebdI&hl=en#"

Answer for the question of
1. Studies on Habtuation
link-
"https://docs.google.com/document/edit?id=1ENHFMNxrw3oZb_Dzl-
djQnogp2CH9EYQShyIA_mUiXA&hl=en#"
"https://docs.google.com/document/edit?
id=1SsaTmU3HQ_bTQ4TNaNjRpng84pvAmtkpcE1kqSoNjDw&hl=en#"


Answer for the question of

2. Studies on Olfactory behavior

As we know that snails are very sensitive and ideal for
olfaction responce. Land snails have four tentacles, two upper having
two eyes on top and two lower act as olfactory organs. Both set of
tantacles are retractable in land snails. Snails show olfactory
response to the environment- positive responce in favourable

conditions (food- fruits & leaves or plants ,apple, beans, citrus,
clover, lettuce, rip cherry, tomatoes, cabbage etc.) and negative

responce in unfavourable conditions (bad smell, stress, water).

Hypothesis-
As we know that snails are very good in olfaction behaviour.

Snails have nervous system. Natural food odours elicit different

behavioural responses in snails. Snails can either protract toward a
stimulating cabbage odour or can

retract. Further, we can show learning though olfactory behavior.

Required materials-

- Healthy snails
- Olfactometer
-Glass plates for moving the snails.
-Distilled water
-Beaker
-Micropippete
-Dropper
- Packed black Box with one side open
-Stopwatch
-freshly prepared cabbabe extract

Process-

We are performing the experiment on Olfactometer-
- We are using one organism during the one test (20
readings).
Diameter of olfactometer is 10.1 cm.

Selecting two parameters –
(a) control(water), (b)
stimulation (cabbage extract);

As we know that humid condition is required for snail growth but it

keep away from water(repulsion). So we are using water as control.
1. We took one snail during the experiment.
2. Snail was placed at the middle of circle at initiation of
each reading.
3. As the snail tantacles touch the circumference , we stop the
stop-watch.
4. Glass plate was covered with black box (one side open- to
observe the snail's behaviour). So that behaviour not affect by
stress(noise & light).
>>>> for each reading we changed glass plate( snails have mucus
glands in its foot region. When snail moves on glass plate secretion
of these mucus gland makes a path during movement).
5..After every 10 readings we gave 2 minutes rest to
the snail.
>>>>> if snail did not reach at the circumference within 2
minutes,we made the dot on olfacto meter at that place.
>>>> some times if snail did not moves(2 minutes)or show
very slight displacement we canceled the reading.

Result-

Experiment 1-
(snail was kept upward direction during whole
experiment)

- Out of 20 readings snail went 12 times towards
to stimulation.Olfaction ratio was 0.6 in negative.In this
experiment once we interchanged the control and stimulation, an
interesting thing happened -snail show the olfactory responce
towards the cabbage extract. So that one point of our hypothesis-
snail good at olfaction, is proved.
********** Snail went to cabbage extract because it like
the cabbage(food material).smell of cabbage attracts the snail.
********** Olfaction ratio =>C-S/C+S
=4-16/4+16
= - 0.6
NOTE:- snail touched the cabbage extract 3 times.


Experiment 2-
(snail was kept in both direction(upward and
downward) direction during whole experiment)
-To confirm our first experiment result we
performed second experiment
-We got the value of olfaction ratio 0.4 in
negative this prove the olfaction behaviour of snail towards
the cabbage extract.

*********** Olfaction ratio =>C-S/C+S
=6-16/6+16
= - 0.4

From,
Jai Prakash Kushvah
Pooja Chauhan
CCT neuro-Lab

[MC Arunan]

Dear Jai Prakash & Pooja,

Let me first congratulate for attempting to study Olfactory behavior in snail using the simple Glass Plate Olfactometer Assay.

Let me tell you some correct things  about your experiment and also some  major problems  in your understanding of the experimental design. (If the experimental design is not clear, your experiment is wasted. There fore it is important to correctly understand the objective and the design of the experiment

1. The correct thing is that you seem to be using a test arena of about 10 cm diameter drawn on a white paper and running the animal on a glass plate kept over this.
And that after every run you are changing the glass plate to a fresh clean dry one.

2. But the most obvious wrong thing is that you are calculating the positive olfactory behavior as -0.6 or -0.4 etc. (This you are doing because you have not discussed your design of the experiment thoroughly to understand it). The minus reading you are getting for a positive olfactory response because you are using the wrong formula to calculate the Olfactory response Index is  Olf I = C-S/C+S

 It should have  been Olf I = S-C/S+C.

In the latter case, you would have got the positive index for attractants and negative readings for repellents.

May I request you to write to us your Design of the Experiment in detail, so that you understand the experiment after discussion with us.

Let me again wish you well for attempting this experiment in Snail Olfactory Behavior studies.

It is very important to understand the experiment thoroughly!
Please write the detail method, including the detail of the requirements.
All the best.
Arunan

[Himanshu Joshi]

Dear JP and Pooja,
I guess you are now in a position to design your objective and a healthy experimental design.

Let me provide share some  knowledge on Hypothesis

Hypothesis is a tool by which one can provide explaination for a observed phenomenon.The explaination may or may not be correct.If explaination is correct then it becomes theory.

Scientific hypothesis is based on previous observations that can not be truly  explained by availabe theory(scientific)

A hypothesis requires more work by the researcher in order to either confirm or disprove it. In due course, a confirmed hypothesis may become part of a theory or occasionally may grow to become a theory itself.

Objective must be loud and clear such that, it must be supported by your experimental design.


Writing the requirements of the experiment is to show the nearly all things required are thought in advance.(There might be some things that may be required on or after starting the experiments)This supports the famous saying "PREVENTION IS BETTER THAN CURE ".


At last you must Know
Why you are doing this ?
For what you are moving your snails here and there??
What are you trying to prove by your so called experiment?

By this you can get the exact idea of
1.Objective(goal).
2.Reason of doing experiment.

Its some hint time.

While attempting a study on Olfactory behavior on an olfactometer, one must consider following points.
1.The Olfactory stimulus must be kept in the experimental chamber so as a differentiated smell must exist.i.e as we move towards stimulus the smell's magnitude must increase with distance travelled.

2.So One must take care that the smell must be not as sharp that it can be felt every where in the experimental chamber.i.e Apply your brains to overcome this problem and design your experiment accordingly..


ALL THE BEST
Himanshu Joshi
Neurolab CCT

[MC Arunan]

Dear Himanshu,

 This is simply great that you have taken it upon to do this process of PeerTeaching-Learning.  All good teachers are students too; all good students are teachers as well!

 

This, infact, is the best form of learning and teaching! This can be complete only with Jai Prakash and Pooja as well as others in the CCT group joining in the discussion.

 

Yes, organisms normally detect a gradient of chemicals before 'deciding' to orient to  a stimuls. This type of questions like if a snail is supposed to move towards, then what all makes it move, are the right way in dealing in science!

 

These finer points actually make experiments interesting. If from what you do, you do not get to raise more questions that may look a wee bit sterile an effort.

 

So raising of relevant questions, is the test of a good experiment well done!

 

ACIVE INVOLVEMENT IS WHAT SCIENTIFIC EXPERIMENTATION TRAINS YOU FOR!

Ones you do  an experiment well, you cannot be uninvolved in life!

 

Looking for some comments from others as well as more comments from Himanshu.

Cheers!

Arunan

[jai prakash]

Dear sir,

             Thank you.
 >>We have corrected the olfactory response index formula as olf i= s-c/s+c.
      
>> Method of  Glass Plate Olfactometer Assay: (On model system- Snail (Cepaea hortensis) )

 1. We are performing the experiment using Olfactometer.
  -Designing of Olfactometer: Olfactometeris a test arena of 10.1 cm diameter drawn on a plane white paper used to measure olfactory response of snail ( in our experiment ).On the one side of the arena we have region for control(water) & on another side for stimulus.
-We write all the  necessary informations of experiment on the olfactometer-paper.
i.e.
Date of experiment
Name of species
No. of readings
Control
Stimulus
Observation time
Diameter

*** Diameter of arena can vary according to size of snail.
***size of snail (used in experiment)- 2cm

2. We are running the animal on a glass plate(25*25 cm) kept over olfactometer .
***We are using one organism during the one test (20 readings).

3. Control (water) & stimulation were dropped on glass plate using dropper at their regions as drawn in olfactometer.

***(As we know that humid condition is required for snail growth but it
keep away from water(repulsion). So we are using water as  control.)
    
4. We took one snail during the experiment.

5. Snail was placed at the middle of circle on glass plate at initiation of each reading.( At center of the circle snail's head region was kept upward direction & in next time down ward direction, alternatively)

6. As the snail tantacles touch the circumference , we note time taken by snail to reach at circumference from center of olfactometer. and marked that one on olfactometer
.
7. When snail was running ,glass plate was covered with black box (one side open- to
observe the snail's behaviour). So that olfaction behaviour not affect by
stress(noise & light).

 ***for each reading we changed glass plate by new clean one.( snails have mucus

glands in its foot region. When snail moves on glass plate secretion of these  mucus  gland  makes  a  path  during  movement).

8..After  every  10  readings  we  gave  2  minutes  rest to the  snail.
*** if  snail did not  reach  at  the  circumference  within  2

minutes,we  made  the  dot  on  olfactometer  at  that  place.

***some times  if  snail  did  not  moves(2  minutes)or  show

very  slight  displacement  we  canceled the  reading.

Results-

Experiment 1-

***Control- Water          Stimulus- cabbage extract

(snail  was kept  upward  direction during  whole experiment)

- Out  of  20  readings  snail  went  12  times  towards to  stimulation. Olfaction index was 0.6 in  positive. this show the attractant olfaction behavior of snail towards the cabbage extract.
-  In  this experiment once  we  interchanged the  control  and  stimulation, an interesting  thing  happened -snail  show the  olfactory  response towards  the  cabbage extract. ********** Snail  went  to  cabbage  extract  because  it  like the  cabbage (food material). smell  of  cabbage  attracts  the  snail.
**********     olf i =>  s-c/s+c
                         =16-4/16+4
                        =  + 0.6

        NOTE:- snail  touched  the  cabbage  extract  3 times.


Experiment 2-

***Control- Water          Stimulus- cabbage extract

(snail  was kept  in both  direction(upward  and downward) direction during  whole  experiment)

-To  confirm  our  first  experiment  result  we performed  second experiment.
 -We  got the  value  of  olfaction  index 0.4  in positive . this show the  attractant olfaction  behaviour  of  snail  towards the  cabbage  extract.

 *********** olf i =>             s-c/s+c
                                     =16-6/16+6
                                     =  + 0.4


Experiment 3-
***Control- Water          Stimulus- Withania Somnifera extract

-We  got the  value  of  olfaction index   0.1 in negative.   this show the repellent olfaction  behaviour  of  snail  towards the  Withania Somnifera  extract.

**********olf index           = s-c/s+c
                                     =9-11/9+11
                                     =  -0.1

Material used in Experiment-

-Healthy,cup cultured snail (snail give response for 20 readings)
-Clean glass plates (25*25 cm) used  for running the snail over olfactometer
-Distilled Water as  control
-Beaker
-Dropper
-Packed black box with one side open
-Stimulus
-Stopwatch for recording time duration

From,
Jai Prakash Kushvah
Pooja chauhan
CCT- Neuro Lab

[MC Arunan]

Dear Jai,

My comments are in red. Please, especially, read the last part with care!!!

All the best.

Arunan

On Thu, Sep 9, 2010 at 3:22 PM, jai prakash <jpcct...@gmail.com> wrote:

Dear sir,

             Thank you.
 >>We have corrected the olfactory response index formula as olf i= s-c/s+c.
      

>> Method of  Glass Plate Olfactometer Assay: (On model system- Snail (Cepaea hortensis) ) Are you sure of the identification of the species?MCA

 1. We are performing the experiment to measure olfacory behavior of the snail using an Olfactometer .
  -Designing of Olfactometer: Olfactometer is a test arena of 10.1 cm (why 10.1cm and not 10cm)MCA diameter drawn on a plane white paper used to measure olfactory response of snail ( in our experiment ).On the  on one side (immediately outside of the arena) we have region for control(water) & on the opposite side another side for stimulus.
-We write all the  necessary informations of experiment on the olfactometer-paper, below the arena.
i.e.
Date of experiment

Name of the scientist:

Name of species

Size or number of whorls of the shell:

No. of readings:
Control: (C)
Stimulus (S)
Observation time: (I suppose you give 2 minutes and within which if the animal does not touch the circulference, you will not be counting that reading, right?)MCA
Diameter (The diameter of the arena is good to be fixed. I guess, if you take all snails of a certain age by defining the number of whorls, you do not change the arean diameter! In fact, it will be better to fix all these, initially during standardization)MCA

 

Olfacotry Response Index Olf RI= S-C/S+C =

.

 

 

*** Diameter of arena can vary according to size of snail.???MCA pl see above
***size of snail (used in experiment)- 2cm (Define it by the number of whorls and also of an approximately similar shell sizeMCA)

2. We are running the animal on a glass plate(25*25 cm) kept over olfactometer .
***We are using one organism during the one test (20 readings).

3. Control (water) & stimulation (stimulus _%cabbage (W/V) ), a drop each were dropped (palced) on glass plate using dropper at their regions, immediately outside the arena, as drawn in olfactometer.

***(As we know that humid condition is required for snail growth but it

keep away from water(repulsion). So we are using water as  control.) No. No No!!!

You rae using water simply because you have made your cabbage extract in distilled water and that distilled water will not have any odour or smell)MCA So it is a control for the odourant stimulus. In case you are diluting your stimulus, say, benzaldehyde with paraffin oil, you will use paraffin oil as the control.

Control in this case means something you are sure which does not act as a olfactory stimulus.

 

In facrt, you should do a series of tests, some time soon with Distilled water on both the sides (DW vs DW)to see that the distribution of intersects are equal meaning if 30 runs are done it should be 15 and 15 on either half of the arena.
This is a very important  control vs Control experiment to be discussed at the causerie while showing your results!

 

    
4. We took one snail during the experiment. One snail (snail no.RLS01 Rajasthan Local Snail, if you are not sure of the species) was run for 20 timesMCA

5. Snail was placed at the middle of circle on clean, dry glass plate at initiation of each reading.( At center of the circle snail's head region was kept upward direction & in next time down ward direction, alternatively) You have to give ditrection of orientation of the anterior of the sanil as north, south,east or west!  And always the direction should be orthogonal to the the stimulus-control axisMCA

6. As the snail  touches the circumference , we note time taken by snail to reach at circumference from center of olfactometer. and marked that one on olfactometer There is no need to mention the time unless that is part of your research question; otherwise  snails that do not reach the arc with in the stipulated 2 minutes will not be counted.MCA
.

7. When snail was running ,glass plate was covered with black box (one side open- to

observe the snail's behaviour). You have to explain about the chamber in detail. Is it made of glass??? etc Will there be any possibility of smell from that? Will there be any possibility of directed light that will creat a bias in your reading etc MCASo that olfaction behaviour not affect by
stress(noise & light). Noise is not the important aspect.  Directed light can creat problems! So except on top all other sides will be dark and the light on top will be uniform and diffused! But the most important aspect is that the animal gets oriented in its movement towards the stimulus because of the GRADIENT FORMATION OF THE ODOURANT MOLECULES!!!

reading we changed glass plate by new clean one.( snails have mucus

glands in its foot region. When snail moves on glass plate secretion of these  mucus  gland  makes  a  path  during  movement).

8..After  every  10  readings  we  gave  2  minutes  rest to the  snail.
*** if  snail did not  reach  at  the  circumference  within  2

minutes,we  made  the  dot  on  olfactometer  at  that  place.

***some times  if  snail  did  not  moves(2  minutes)or  show

very  slight  displacement  we  canceled the  reading. No reading will be counted if the animal does not reach the circulference with in 2 minutes.MCA

Results-

Experiment 1-

***Control- Water          Stimulus- cabbage extract  (You have to explain the way you made it W/V etc!)

(snail  was kept  upward  direction during  whole experiment) No you have to keep it ideally ones North next time South or accordingly!! Please see above!MCA

- Out  of  20  readings  snail  went  12  times  towards to  stimulation. Olfaction index was 0.6 in  positive. this show the attractant olfaction behavior of snail towards the cabbage extract.
-  In  this experiment once  we  interchanged the  control  and  stimulation, an interesting  thing  happened -snail  show the  olfactory  response towards  the  cabbage extract. ********** Snail  went  to  cabbage  extract  because  it  like the  cabbage (food material). smell  of  cabbage  attracts  the  snail.
**********     olf i =>  s-c/s+c

                         =16-4/16+4  =12 / 20
                        =  + 0.6

        NOTE:- snail  touched  the  cabbage  extract  3 times. You should not allow the snail to touch the stimulus or control!!! This means the stimulus & control space should be 5 mm away from the arc.MCA


Experiment 2-

***Control- Water          Stimulus- cabbage extract

(snail  was kept  in both  direction(upward  and downward) direction during  whole  experiment)
-To  confirm  our  first  experiment  result  we performed  second experiment.

 -We  got the  value  of  olfaction  index 0.4  in positive . this show the  attractant olfaction  behaviour  of  snail  towards the  cabbage  extract.

 *********** olf i =>             s-c/s+c
                                     =16-6/16+6
                                     =  + 0.4


Experiment 3-

***Control- Water          Stimulus- Withania Somnifera extract Why did you use this? Unless you calibrate the instrument with DW vs DW and DW vs Cabbage etc by several repeats and also calculate the mean Olf RI and SD in each case we cannot say what it does wth an unknown or test substance like W somnifera. This is the difference between research and regular practicals!!!!! This is also why after the experiment some one is going to believe us!!!! That is when the excitement comes!!!

All the best.

-We  got the  value  of  olfaction index   0.1 in negative.   this show the repellent olfaction  behaviour  of  snail  towards the  Withania Somnifera  extract.

**********olf index           = s-c/s+c
                                     =9-11/9+11
                                     =  -0.1

Material used in Experiment-

-Healthy,cup cultured snail (snail give response for 20 readings) You have to number the animals and say which snail was used in which experiment. Are you culturing in the cup? Or just rearing it for a few days before the experiment. Culturing means they should be eating etc, if so what do they eat etc to be mentioned in each case. Are the animals starved for 20 hours before the experiment?

-Clean glass plates (25*25 cm) used  for running the snail over olfactometer
-Distilled Water as  control
-Beaker
-Dropper
-Packed black box with one side open
-Stimulus
-Stopwatch for recording time duration

[Himanshu Joshi]

Dear JP et al,

Yes, you can answer all these points.
Waiting for answers!!

Himanshu Joshi
Neurolab CCT

[jai prakash]

Dear sir,   
   Thank you for correcting the write up & guiding the right way of experiment.

 

>Are you sure of the identification of the species?

 
     Ans. yes we sure in identification of snail species  (Cepaea hortensis).

Snails which we using-
1. These are air-breathing land snails, which we collected from university garden.
2. These are  white lipped, medium sized, pulmonate snails. One snail is  brown lipped.
3. The Shell dimensions of snails varies from  1.5 cm to 2 cm.
4. Shell of snails are yellowish with brown banding.

   

       >>-We write all the  necessary informations of experiment on the olfactometer-paper, below the arena.

i.e.
Date of experiment

Name of the scientist:

Name of species

Size or number of whorls of the shell:

No. of readings:
Control: (C)
Stimulus (S)
Observation time:

 

>>(I suppose you give 2 minutes and within which if the animal does not touch the circulference, you will not be counting that reading, right?)

      No sir, it is not like that. If snail has some reasonable displacement(3-4 cm) from the initial point but could not touch the circumference within 2 min, we  made a dot at that place. but if snail not showing any displacement or showing a slight displacement within 2 min , we are not counting that reading.
  
                              

 

>>Diameter (The diameter of the arena is good to be fixed. I guess, if you take all snails of a certain age by defining the number of whorls, you do not change the arean diameter! In fact, it will be better to fix all these, initially during standardization)

         yes sir, we have fixed the diameter of arena in all all experiments.
   
        " We count the whorls of snail but we have no idea of defining their ages by counting the Whorls.???"

 

>>***size of snail (used in experiment)- 2cm  (Define it by the number of whorls and also of an approximately similar shell sizeMCA)   having 3 whorls.
 

***(As we know that humid condition is required for snail growth but it

keep away from water(repulsion). So we are using water as  control.) No. No No!!!

You rae using water simply because you have made your cabbage extract in distilled water and that distilled water will not have any odour or smell)MCA So it is a control for the odourant stimulus. In case you are diluting your stimulus, say, benzaldehyde with paraffin oil, you will use paraffin oil as the control.

Control in this case means something you are sure which does not act as a olfactory stimulus.

 

In facrt, you should do a series of tests, some time soon with Distilled water on both the sides (DW vs DW)to see that the distribution of intersects are equal meaning if 30 runs are done it should be 15 and 15 on either half of the arena.
This is a very important  control vs Control experiment to be discussed at the causerie while showing your results!

      yes sir, We made the experiment DW Vs. DW  initially, we have 10 readings.  2 times  out of 10 snail went straight,3 times went  one half of arena &4 times another side of arena.


 

 

    

>>7. When snail was running ,glass plate was covered with black box (one side open- to

observe the snail's behaviour). You have to explain about the chamber in detail. Is it made of glass??? etc Will there be any possibility of smell from that? Will there be any possibility of directed light that will creat a bias in your reading etc MCA

   Ans. chamber within which the we are performing the experiment is made of 5 equal(25*25 cm)glass plates. all the plates are covered with black paper excepting one side(for taking observations). when we perform the experiment we keep away  the directed light  from the observation side.

>>experiment -1

***Control- Water          Stimulus- cabbage extract  (You have to explain the way you made it W/V etc!)

 

cabbage extract-
     we crush some cabbage leaves(without weighting it) in mortar with pistol by adding 9-10 drop of distilled water.
     after that we filter the cabbage using whatsman filter paper. We took 3 ml of cabbage extract in our experiment for stimulus.

 

Experiment 2-

***Control- Water          Stimulus- cabbage extract

(snail  was kept  in both  direction(upward  and downward) direction during  whole  experiment)
-To  confirm  our  first  experiment  result  we performed  second experiment.

 -We  got the  value  of  olfaction  index 0.4  in positive . this show the  attractant olfaction  behaviour  of  snail  towards the  cabbage  extract.

>>-Healthy,cup cultured snail (snail give response for 20 readings) You have to number the animals and say which snail was used in which experiment. Are you culturing in the cup? Or just rearing it for a few days before the experiment. Culturing means they should be eating etc, if so what do they eat etc to be mentioned in each case. Are the animals starved for 20 hours before the experiment?

     Ans. yes . we are cultering the snails in the cups. At present time we have 4 snails in 3 cups.
     (1) snail S-1 & snail S-2 in Cup No .1
     (2)snail S-4 in Cup No. 2.
     (3) snail L-1  Cup No. 3 .
     We are feeding snails with Cabbage leaves regularly. We are changing the leaves by new fresh leaves daily and are changing the filter paper by new one within two days. 
     All these snails are active & healthy.  
** our 1st & 2nd experiment, olfaction behaviour was observed on snail S-1.
   
 *** in one  experiment of olfaction behaviour we use the W. Somnifera  extract as stimulus because of its smell.
Before the Experiment we have checked that W. Somnifera does not affect the snail by feeding it for one day.  we provide crushed W. Somnifera  by keeping it in cup No. 3 (snail L-2). & took observation continuously for 6 hours. snail came to W. Somnifera 6 times but not touched to it . At this time snail show nice tentacles activity. After 4 hrs snail ate Withania extract. snail show un conciousness for 3 minuts . But after some time it become active & not show un conciousness.
     


From,
JAI PRAKASH KUSHVAH
POOJA CHAUHAN
MOHIT SARRAF
CCT NEURO-LAB.

[MC Arunan]

Dear all,
You are not trying to understand the meaning of scientific experiments. I am sorry to say that.
See the following from your latest mail:

"cabbage extract-
     we crush some cabbage leaves(without weighting it) in mortar with pistol by adding 9-10 drop of distilled water.
     after that we filter the cabbage using whatsman filter paper. We took 3 ml of cabbage extract in our experiment for stimulus.

"
Please tell me, from "9-10 drops of distilled water used for crushing the cabbage , how on earth, you will mange to get "3 ml" of cabbage extract for your experiment as stimulus?
All this after filtering through filter paper!!!

I should stress this fact again: When you report an experiment it should be in such a way that any one reading that should be able to reproduce the experiment and get the result.

If you look at it you will realize that you have not addressed all my questions  satisfactorily. You have not try to understand the rationale of my questions. I would like you guys to go over my questions again (and not just explain away) and try to understand the problems in that and corrct so that others can repeat it.

For example, when I ask whether the animal is identified, what I mean is there is a method in identifying an organism by looking at salient features of the Phylum, Class, Order, Family, Genus and then Species. Each is based on crucial and salient characters.
(Till that time we may work with the animals, but won't call them by that specific name, unless an expert has identified them or that we have identifed them in a scientific way. In this case, you may be right. But do you know how right you are? On what basis you can convince a critic?

This is also true about the counting of what is S and what is C. To me, there is no point in you marking or counting the run that stopped after partial movement within the stipulated time of one minute or two minutes and ended some where in the middle of the arena!
 Only those intercepts you have to count as either S or C depending upon which half of the arc of the arena the snail  intercepted.

So, I am not happy with the answers. Please go over my questions again and try to write a new protocol.
Arunan

On Thu, Sep 16, 2010 at 1:11 PM, jai prakash <jpcct...@gmail.com> wrote:

Dear sir,   
   Thank you for correcting the write up & guiding the right way of experiment.

 

>Are you sure of the identification of the species?

 
     Ans. yes we sure in identification of snail species  (Cepaea hortensis).

Snails which we using-
1. These are air-breathing land snails, which we collected from university garden.

2. These are  white lipped, medium sized, pulmonate snails. One snail is  brown lipped. There can be pulmonates of different shapes. You did not factor in that. Is it that you have been identifying it or that you have consulted an expert taxonomist.MCA

3. The Shell dimensions of snails varies from  1.5 cm to 2 cm.
4. Shell of snails are yellowish with brown banding.

   

       >>-We write all the  necessary informations of experiment on the olfactometer-paper, below the arena.

i.e.
Date of experiment

Name of the scientist:

Name of species

Size or number of whorls of the shell:

No. of readings:
Control: (C)
Stimulus (S)
Observation time:

 

>>(I suppose you give 2 minutes and within which if the animal does not touch the circulference, you will not be counting that reading, right?)

      No sir, it is not like that. If snail has some reasonable displacement(3-4 cm) from the initial point but could not touch the circumference within 2 min, we  made a dot at that place. but if snail not showing any displacement or showing a slight displacement within 2 min , we are not counting that reading.

Your reasonable displacement may not be your friend's reasonable one and will not be reasonable according to me or Himanshu. In other words, if you think movement of 3-4cms is enough you may change the diameter of the arena accordingly!Because, in Science we do not change the goal Posts accoording to out idea of reasonableness each time! MCA

  
                              

 

>>Diameter (The diameter of the arena is good to be fixed. I guess, if you take all snails of a certain age by defining the number of whorls, you do not change the arean diameter! In fact, it will be better to fix all these, initially during standardization)

         yes sir, we have fixed the diameter of arena in all all experiments.
 

  
        " We count the whorls of snail but we have no idea of defining their ages by counting the Whorls.???" So, fix all organisms of the same number of whorls. You should also know how to count the number of whorls from a particular axis!MCA

 

>>***size of snail (used in experiment)- 2cm  (Define it by the number of whorls and also of an approximately similar shell sizeMCA)   having 3 whorls.
 

***(As we know that humid condition is required for snail growth but it

keep away from water(repulsion). So we are using water as  control.) No. No No!!!

You rae using water simply because you have made your cabbage extract in distilled water and that distilled water will not have any odour or smell)MCA So it is a control for the odourant stimulus. In case you are diluting your stimulus, say, benzaldehyde with paraffin oil, you will use paraffin oil as the control.

Control in this case means something you are sure which does not act as a olfactory stimulus.

 

In facrt, you should do a series of tests, some time soon with Distilled water on both the sides (DW vs DW)to see that the distribution of intersects are equal meaning if 30 runs are done it should be 15 and 15 on either half of the arena.
This is a very important  control vs Control experiment to be discussed at the causerie while showing your results!

      yes sir, We made the experiment DW Vs. DW  initially, we have 10 readings.  2 times  out of 10 snail went straight, 3 times went  one half of arena &4 times another side of arena.


 

 

    

>>7. When snail was running ,glass plate was covered with black box (one side open- to

observe the snail's behaviour). You have to explain about the chamber in detail. Is it made of glass??? etc Will there be any possibility of smell from that? Will there be any possibility of directed light that will creat a bias in your reading etc MCA

   Ans. chamber within which the we are performing the experiment is made of 5 equal(25*25 cm)glass plates. all the plates are covered with black paper excepting one side(for taking observations). when we perform the experiment we keep away  the directed light  from the observation side.

>>experiment -1

***Control- Water          Stimulus- cabbage extract  (You have to explain the way you made it W/V etc!)

 

cabbage extract-
     we crush some cabbage leaves(without weighting it) in mortar with pistol by adding 9-10 drop of distilled water.
     after that we filter the cabbage using whatsman filter paper. We took 3 ml of cabbage extract in our experiment for stimulus.

 

Experiment 2-

***Control- Water          Stimulus- cabbage extract

(snail  was kept  in both  direction(upward  and downward) direction during  whole  experiment)
-To  confirm  our  first  experiment  result  we performed  second experiment.

 -We  got the  value  of  olfaction  index 0.4  in positive . this show the  attractant olfaction  behaviour  of  snail  towards the  cabbage  extract.

>>-Healthy,cup cultured snail (snail give response for 20 readings) You have to number the animals and say which snail was used in which experiment. Are you culturing in the cup? Or just rearing it for a few days before the experiment. Culturing means they should be eating etc, if so what do they eat etc to be mentioned in each case. Are the animals starved for 20 hours before the experiment?

     Ans. yes . we are cultering the snails in the cups. At present time we have 4 snails in 3 cups.
     (1) snail S-1 & snail S-2 in Cup No .1
     (2)snail S-4 in Cup No. 2.
     (3) snail L-1  Cup No. 3 .
     We are feeding snails with Cabbage leaves regularly. We are changing the leaves by new fresh leaves daily and are changing the filter paper by new one within two days. 
     All these snails are active & healthy.  
** our 1st & 2nd experiment, olfaction behaviour was observed on snail S-1.
   
 *** in one  experiment of olfaction behaviour we use the W. Somnifera  extract as stimulus because of its smell.

Before the Experiment we have checked that W. Somnifera does not affect the snail by feeding it for one day.  we provide crushed W. Somnifera  by keeping it in cup No. 3 (snail L-2). & took observation continuously for 6 hours. snail came to W. Somnifera 6 times but not touched to it . At this time snail show nice tentacles activity. After 4 hrs snail ate Withania extract. snail show un conciousness for 3 minuts . But after some time it become active & not show un conciousness. All this is problematic as a scientific experiment. In science you do not do the experiment to get the result, any how. Each step of the method has to be fool-proof!MCA