Moina as an Epigenetic model organism

drishtantmkawale · October 18, 2020, 8:13am

Yes, this is a matter of worry because in the previous experiments, a distinct colour change from colourless to (pale-yellow) coloured Moina was seen on the 3rd day itself when 6drops of milk were added in 250mL of dechlorinated water on alternate days.

Apologies for not describing this clearly in the writeups.

In Test-1/2/3 cups, 2 drops (everyday), 4 drops and 6 drops (both on alternate days) of milk respectively are being added. In Test-2 and Test-3, milk is being added on alternate days because in the initial stages, as the number of Moina are less (say 5-10), adding 4 and 6 drops daily will result in a nutritional load in the cups. There will be more nutrients in the cups which will have the ability to harbour more bacteria. This may result in a sudden decrease in the levels of dissolved oxygen in water in the cups and Moina won’t be able to produce enough of Haemoglobin in this short span of time and can die. If there are enough of Moina (say ~30-40) and then if they are exposed to the condition of adding milk daily, then we can expect atleast 50% of them to survive.

This is being said based on the earlier experience of adding 6 drops of milk in 250mL of DC water daily wherein the result was that Moina died on the second day without showing any colour change.
Although this was observed just a couple of times (which means there is a possibility of this as a chance event), further experimentation on the same parameter (6drops daily) can be done to see the results this time.

drishtantmkawale · October 18, 2020, 7:44pm

CUBE Nerul Home Lab
Moina Model
Update for 17th Oct, Saturday (apologies for the delay)

Breaking News
Still no prominent colour change!!

It was the fourth day (made on 14th Oct) of the setup and still no colour change in Moina from the test cups (having 4 and 6 drops of milk) was seen!
Moina in the control cups having 1 drop of milk in 250mL of DC water are seen to be colourless.

https://drive.google.com/file/d/1uqimDIJlP-_4Nm96qUI6Jj-Y2TAMXcdq/view?usp=sharing
Video evidence showing Moina from the control, test1, test2 and test3 cups respectively.
The control Moina is colourless (as expected) but the rest of the Moina that were subjected to 2 (daily), 4 and 6 drops (on alternate days) of milk are colourless too! All of them are gravid. The expectations were to see a colour change in Moina from the test cups.

Magnification: 5.5x via digital zoom.
Date and Time: 18th Oct, 1.10am
Place: Nerul, Navi Mumbai
Data Collector: Drishtant MK.

I used a new method for taking this video of Moina samples.
In this, a phone’s flashlight was kept at the bottom above which there was a transparent writing pad above which I kept a ruled-paper and above that there was a glass slide on which Moina were kept in a drop of water (from the cups).

There was no light directed from above.

What could be a possible reason(s) (the devil) for not seeing a colour change even after 4days?
Let me mention that test1 is added with 2drops of milk daily and cups test2 and test3 are added with 4 and 6drops of milk on alternate days. All of this is added in 250mL of dechlorinated water.

Expectations for 18th Oct:
Control: To see ~20 colourless Moina in both the cups C1A and C2B.

Test1: ~20 Moina should be there in T2A out of which ~5 should have developed a very light yellow colour and the rest being colourless. The cup T2B will have no Moina in it.

Test2: ~7 Moina should be there in cup T4A out of which 1 (parent Moina) should have a pale yellow colour and rest colourless. As there is just a single gravid Moina in cup T4B, ~10 Moina are expected out of which 1 (the parent) should have a pale yellow colour.

Test3: ~20 Moina should be there in both the cups T6A and T6B out of which ~5 should have a distinct yellow colour.

drishtantmkawale · October 20, 2020, 12:46pm

CUBE Nerul Home Lab
Moina Model
Update for 19th Oct

Breaking News
Moina are flourishing but no colour change still!

Moina in six (out of the eight) cups have increased in number but there is no colour change seen in Moina from the test cups.

Above is the growth curve of Moina starting from 14th Oct till 19th Oct.
It is indicating the growth of Moina as the days are passing.

Observations made on 19th Oct 2020:

Control: ~70 and ~60 colourless Moina in cups C1A and C1B. As there is an increase (from ~40 to ~60-70) in the number of Moina, ~40 in both cups are young ones. Expectations for both the cups have been met.
Test1: ~60 colourless Moina in cup T2A with ~30 being young Moina. No colour change seen in Moina. Cup T2B has no Moina.
Test2: ~50 colourless Moina in cup T4A with ~30 young Moina. Cup T4B has no Moina.
Test3: Both the cups T6A and T6B have ~50 Moina with none of them showing a colour change.

In all the cups except the control C1A and C1B and two of the test cups, expectations for numbers have met but the expectations for colour change haven’t been met.

https://drive.google.com/file/d/1W8ypgRzGYRiPNBXIL1m-5tsEPMm7qBg-/view?usp=sharing
Video showing Moina from the control, test1, test2 and test3 cups respectively. All of them seen to be colourless.
Now that we have figured out the possible reason for not seeing a colour change, I think we should not expect the Moina to be coloured.

Magnification: 2.1x via digital zoom. No external light has been flashed so as to avoid interference with the actual colour of the Moina.
Date and Time: 20th Oct, 1.38am
Place: Nerul, Navi Mumbai
Data Collector: Drishtant MK.

drishtantmkawale · October 20, 2020, 12:52pm

I think the problem is in the design of the experiment.
The expectations were to observe the first colour change on the third day.
This is for Moina from the test cups in which 4drops of milk and 6drops of milk is added on alternate days.

But later I realised that these expectations were seen when the same experiment was done using 500mL plastic bottles instead of plastic cups.

The design should not have included the expectations of the plastic bottles as they are not being used here.
Using plastic cups in place of plastic bottles is showing a major difference.

And this is possibly a GOOFUP!
Expecting an observation that is true for another design and not really true for the current design!
Missing a small detail (bottles–>cups) has changed the outcome of the experiment.

The bottles that were used were in earlier experiments were of 500mL volume.
If we visualise a typical bottle, it will have a narrow mouth. The bottles were covered with a cotton plug.
Having a narrow mouth as well as covering the mouth with cotton plug minimises the entry of air.

And that is what we want!!
As a low oxygen condition is supposed to get created after addition of 6drops of milk (in 250mL DC water), we will not want oxygen (air) to enter the bottle because if oxygen is entering inside the bottle, it will get dissolved in the water and the low-oxygen condition won’t be low oxygen anymore.
But in the cups, the opposite thing is happening altogether.
The plastic cups have a broad mouth and are not covered with cotton plug but just a cotton cloth is being used to cover the cups.

As a result, oxygen through the air will be entering the cups and will be dissolving in the water, because of which the low-oxygen condition doesn’t persist!

And if a low-oxygen condition isn’t created in the water, then Moina will be having oxygen and Moina won’t produce extra-haemoglobin!

karnamdpdurga · October 21, 2020, 5:58am

Cool!
So basically you claim that hypoxia (low oxygen) condition is not created, as the containers were broad mouthed and not plugged. btw, I believe cotton plugs don’t make it air tight, and still allow oxygen transfer! That is the reason fruit fly culture use them.

Is there a way to estimate the effect of this? Does it depend on the area of water surface exposed or area of the mouth of the container!

Does anyone any idea?

-DP

karnamdpdurga · October 21, 2020, 6:07am

@drishtantmkawale Does this thread have an answer? The title suggests so! You too seem to have taken part in it.

-DP

drishtantmkawale · October 21, 2020, 7:07pm

Correct! And I agree to you too that cotton plugs do not make it airtight and will allow oxygen transfer. That is why it is preferred to wear cotton clothes in summer! To allow passage of air!

If we think logically or even visualise the broad mouth of the cups and the narrow mouth of the bottles, there is a considerable difference in both.
Even though cotton-plugged, can we not say that air-passage in the bottles will be comparatively less than that of the cups?
In addition to this, 4 and 6drops of milk was added on alternate days in two of the test cups. A ~24hour time period would be enough for the oxygen-level rise.

A way to estimate the effect of what @karnamdpdurga? (Sorry for missing it)

It will depend on both; the area of the water surface exposed as well as the area of the mouth of the container.
The first point of entry of air will be the mouth of the container and then the surface of the water.

shitalsalunke2503 · October 22, 2020, 9:30am

Sir please send the link of today’s class

drishtantmkawale · October 29, 2020, 6:26pm

CUBE Nerul Home Lab
Moina Model

Summary of the experiment with Moina.
Objective: To observe a colour change in Moina when they are kept in low-oxygen conditions.
Date of Setup preparation: 14th Oct 2020
Result: The objective wasn’t achieved according to the expectations. The expectations were to observe the first colour change in Moina [kept in the cups having 250mL of dechlorinated water+6drops of milk (on alternate days)] on the third day i.e. on 17th Oct. But no colour change was seen in Moina.
Below is the link of the video from the 3rd day.
https://drive.google.com/file/d/1hgg8cKph8Ja0rPe4Sj4TLcGAQXm03ryt/view?usp=sharing

Two of the test cups (with one replicate each) having 4drops and 6drops of milk (milk added on alternate days) were added with milk on 14th, 16th, 18th, 20th Oct whereas the remaining test cup(s) (one replica) were added with 2drops of milk daily. Moina in none of the test cups showed a colour change from colourless to coloured.
The control Moina (in cups) were added with 1drop of milk daily. Moina in control cups remained colourless throughout and are still colourless.

No colour change was seen in Moina from all the test cups and the control Moina remained colourless.
Meanwhile no colour change was seen, number of Moina in the cups increased as well as decreased i.e. they followed a growth curve.
The above growth curve [X-Axis: number of days from 14th to 28th Oct, Y-Axis: number of Moina in all (8) the cups aggregate] tells us about different phases of growth of Moina.

From 14th to 16th Oct, the number of Moina remained constant more or less–> Lag Phase.
From 17th to 23rd Oct, the number of Moina increased–> Exponential Phase.
From 23rd to 25th Oct, the number of Moina remained constant–> Stationary Phase.
From 25th Oct till now, the number of Moina has been decreasing that marks the onset of death phase.

This growth curve is more or less similar to a bacterial growth curve.

Explanation of the observations:
As there was no colour change seen in Moina from any of the test cups at any point (totally opposite of what was expected), I think there are two reasons for this both because of an improper design of the experiment.
i) The design had 8 cups in total that were divided into 4 sets of two each. Control and Test1 cups were added with 1 and 2drops of milk daily. The Test2 and Test3 cups were added were 4 and 6drops of milk on ALTERNATE DAYS.

This is where the devil was, I think. As the expectations were to observe the first colour change in 6drops of milk set on the 3rd day (17th Oct), milk should’ve been added everyday so that a low-oxygen condition would’ve been maintained. But as 4 and 6drops of milk were added on alternate days, the low-oxygen condition didn’t sustain. In addition to this, the cups have a broad mouth and were covered with a cotton cloth, oxygen kept on diffusing in the water due to which the dissolved oxygen level of the water didn’t really decrease till such an extent, where Moina would’ve started the production of extra-haemoglobin.
That is why the test Moina appeared colourless throughout.

ii) The expectations of observing a colour change in Moina (from the test cups) on the third day were not of this experiment. This can be expected when 6drops of milk are added in 250mL of DC water daily and not on alternate days. Also, in the same experiment, plastic bottles having a narrow mouth plugged with cotton were used. Plastic bottles having a narrow mouth and plastic cups having a broad mouth do make a difference in the giving entry to oxygen inside. Here, the difference that was seen was the bottles having Moina+water+6drops of milk daily, colour change was seen on the third day whereas we saw no colour change in this experiment.

GOOFUP:
Not designing an experiment with proper test sets.
This improper design of the experiment with expectations from another experiment led to an unexpected outcome of this experiment!

I learned that while designing an experiment like this, we should keep in mind the smallest of things such as the size of the container, the drops of milk that are being added and whether expectations are according to the setup…

Celebration:
Learning from the above goofup, another design of the experiment has been planned, this time with proper test sets.
Keeping in mind the expectation of observing a colour change in Moina, this experiment will give results.
The design will be shared later tomorrow.

Till then, everyone please suggest improvisations on the design.

ravi312 · November 7, 2020, 6:44am

Can we approximate from the image that moina is around 1mm long?

drishtantmkawale · November 7, 2020, 3:56pm

Moina through 8x magnification via digital zoom.

drishtantmkawale · November 8, 2020, 6:48pm

Yes we can!
In the earlier posts, photos/videos of Moina with a ruler (scale) beside have been posted.

drishtantmkawale · January 14, 2021, 1:08pm

Moina Model Concept Map made with the collaboration of CUBists from all over India during the CUBE STEM National Meet (Virtual) 2020

drishtantmkawale · January 19, 2021, 10:37am

CUBE Nerul HomeLab
Moina macrocopa JSK1 Model System

Breaking News
Two bottles full of Moina!

Research Question: Why do Moina undergo a colour change when subjected to low oxygen conditions?
Long-term objective: To study the effect of histone deacetylases-inhibitor Valproic Acid on the colour of Moina due to Haemoglobin protein.
Objective: To culture and maintain Moina in the HomeLab.

https://drive.google.com/file/d/1TaTXfFOMR-vGGNvF4z3zk_uE9-fPbb_n/view?usp=sharing

The above video shows ~150 colourless Moina in two of the culture bottles L4 and L5.
Currently, there are five culture bottles L1 to L5 having 250mL of dechlorinated water, 1 drop of milk is added daily and 5 Moina were added initially.
L1 to L3 made on 1st Jan 2021 and L4, L5 were made on 12th Jan 2021.

Date of Video: 19th Jan 2021
Place: Nerul, Navi Mumbai, MH
Taken by: Drishtant MK

Achievement so far: I alongwith my collaborator CUBists have successfully cultured/been culturing Moina since the lockdown started i.e. for almost 300+ days now!
Our CUBists @manmasih @Manjuel, @Rechel_tirkey @Ram (from Ranchi, JH) and Abhijith Vinod (from Kolenchery, Kerala) have been culturing Moina in their HomeLabs even before the start of lockdown!
Let us continue this stint for the time to come!
First step would be updates… followed by discussion.
Looking forward to hear from you alongwith updates
@Zahra_R17 @saida786110 @saswathy679 @arunimakunju99

Smitra2021 · March 17, 2021, 7:30pm

@drishtantmkawale Thank you for narrating all the experiments and results in such an amazing fashion. It is really very exciting and interesting.

drishtantmkawale · April 8, 2021, 6:29pm

CUBE Nerul Homelab
Moina Model System

Breaking News
Moina fit and fine all throughout an ~3000kms journey down-south!

Courtesy: Google Maps

Above is the map of my 15day journey wherein I took alongwith me the model organism Moina macrocopa JSK1 and was successful in maintaining them throughout with fewer hiccups wrt. culturing, etc. and the journey involved shifting my Nerul homelab to temporary Car and Train homelabs too!
Two 500mL bottles having 250mL of DC water + ~30 Moina each were taken along out of which Moina survived in only one of the bottles. Their feeding pattern wasn’t fixed as a result of which 1drop of milk was added just four times in 15days.
When I returned back, ~30 Moina were remaining in the bottle (that was taken along) out of which I made two new bottle cultures.

Video
And here I am updating after quite sometime😅
Above is a video showing movement of Moina alongwith alot of Rotifers accompanying them in the 250mL DC water and are fed with 1drop of milk on alternate days.

Date: 8th April 2021, Thursday
Place: Nerul, Navi Mumbai
Video by: Drishtant MK

Lydia · July 29, 2021, 5:27am

Interesting CUBE Chat here! Good record of all your observations!

How do we calculate the size of an organism or anything that we observe under a microscope?
Context2Curriculum! Moina model system or any other model system → connected to microscopy.

drishtantmkawale · July 30, 2021, 1:05pm

All the collaborative stuff here!
I’ll share my experience of calculating the size of Moina. I bought a laser light (red-coloured one used in presentations, etc.) and took the lens (convex) out of it.

This one

Steps
i) I attached this lens to the rear camera of my mobile phone (via a stick tape).
ii) Further, I harvested Moina out of my stock culture through a dropper and placed a few of them on a glass slide.
iii) Held a light source (another phone’s torchlight) below the glass slide.
iv) Simultaneously, I also placed a ruler scale under the glass slide as a reference scale.
v) Click!
Took a photo from above by doing the required adjustments. No digital zooming was done.
Well, this is not it . We’ll have to process the image to measure the approximate length/size of the organism.
That can be done by using the ImageMeter application for mobile phones.
Here’s the link to a video that will introduce you to the app.

Results

Fig.1 All of them from the same mother, of the same age but of varying lengths. This shows that Moina too, show polymorphisms (variations).

Fig.2 A 36hour-old (young

) juvenile

Fig.3 A gravid one

The pictures may not be resolute and clear but I am improving upon that.
All of it will be possible when collaborators will try it out in their homelabs and give feedback whether it worked out there.

Rest, I think this inexpensive jugaad can be used for every model system in CUBE provided that a reference scale and a bit of meticulousness is maintained while doing it.

What do you say? @Lydia

Lydia · August 1, 2021, 9:24am

This is great!
Maybe, you can share a ray diagram kind of to show how the whole setup looks.
What we can also do is, we can share this with our collaborators and they too can follow this. We need to get reproducible results with the help of our collaborators. The size of different model systems can be measured using this method.

I’m not sure if everyone has a laser light lens in their house. Is there by any chance, an alternative for this one component?

drishtantmkawale · August 1, 2021, 11:33am

Yes agreed!

There is an alternative, but its an expensive one
Some YT videos suggest the lens CD-DVD reader (the centrepiece in the picture below)

One can use the above lens if they have a damaged DVD player.

Apart from this, I’ll prefer that you should look for this laserlight lens which cost me Rs.60 (after alot of bargain. Eventually I was going to break it ).
Less than a $ it’ll cost us’all!

@ravi312 had come up with this interesting thread. Do give a visit