Building a micro-aquarium using the ditch-spacer slide technique

One of my long-term aspirations is to possess a micro-aquarium, where microscopic life is constrained in a small slide volume that I could watch for days and see aspects of the cycles of the various life forms. How cool would that be? Yet, it all only seemed like a fancy… until this May, where at the  National Geographic Bioblitz, I had the wonderful opportunity to meet Alice Dai, Jim Cybulski, Matt Bull (from the Prakash Lab) and super-user Matt Rossi. Alice showed us this wonderful PVC material that she was playing with, where she could punch in a hole and fill it with liquid sample and hermetically seal it with PVC at both ends. Around the same time Manu showed how he solved the fixed focus problem and also demonstrated dark field with a table lamp (see https://microcosmos.foldscope.com/?p=16093). At that very moment, I knew that a life-long dream was very close to being fulfilled. So here is a preliminary hack taking what I saw with Alice a bit further towards a self-contained system. My first goal was to get a system where 1) critters dont get smashed in and are freely mobile, 2) the system is stable and doesn’t move around when the foldscope pans from one field to another, 3), the system is sealed to prevent water evaporation.

Here’s what I did :
Materials: 1) Glass slide, 2) thick-PVC (0.016 inches/0.4 mm thick), 3) thin-PVC (0.004 inches/0.1 mm thick, product number 8562K11). See the figure for methods.

ditch-slide-collage
1. Cut a rectangular ditch in the thick-PVC and place it on a glass slide. Press to get a nice seal. 2. Fill the ditch with the liquid sample (Keep some space, don’t fill it up completely). Put a thin-PVC cover on top and press this along the edge of the ditch so that it seals into the thick-PVC.

This is what I would like to term the basic setup. Even this basic setup is very stable: Critters dont get smashed in, liquid samples don’t leak out and the coverslip is very stable. The sample remains in the ditch for as long as you want it. Following are the organisms I saw on the first day from a pond water sample. This is from the same spot I observed last year. My goal is to identify as many organisms as possible from this pond. Note, for unknown organisms, I am using a convention that Manu and I decided upon in an older post ( see comments in https://microcosmos.foldscope.com/?p=16599 for convention).

1. Ceratium (SAR->Dinoflagellate). This is my best guess for now, but looks very likely. Looks great in dark field.


2. Wolfia; Duckweed (Viridiplantae -> Araceae). Hard to believe that this is an angiosperm, but it does makes a flower and seed.


 


3. Spirogyra (viridiplantae -> Charophyta): Asexual reproduction in these algae is by fragmentation. The fragmentation pathway is clearly seen in the video. I suspect the fragmenting cell apoptoses and the surrounding filaments break off. Note that cell in the middle of the fragmenting alga shows an altered chloroplast morphology.


4. Mougeotia (viridiplantae -> Charophyta): Similar to Spirogyra. Note fragmentation points.Thanks Dr. John Hall (algal expert) for the identification.


5. Trachelomonas (viridiplantae ->Chlorophyta)
Looks like a moving period. Seen often in freshwater samples. Thanks Dr. John Hall (algal expert) for the identification.


6. Euchlanis-like Rotifer (Metazoa > Rotifera). Rotifer-16915-6-LI


7. Rotifer type II (Metazoa > Rotifera);  Rotifer-16915-7-LI. Note the carapace, that looks like cape when it swims about.

8. Cyclops-like Copepod (Metazoa> Crustacea): Note I also observed the nauplis larval form ( see beginning of euplotes video).


9. Flatworm (Platyhelmenthis); Flatworm-16915-9-LI. Stenostomum-like flatworm


10. Unknown Diatom (SAR supergroup > Stramenopiles->Chromista->Bacillariophyta); Diatom-16915-10-LI. They are the little projections lining the charophyte.


11. Euplotes-like spirotrich (Ciliophora); Ciliate-16915-11-LI
Moves like a Euplotes, but not sure if it is the same species. Did not obviously see the cirri, but the contractile vacuoles are really neat.


12.Unknown Ciliate; Ciliate-16915-12-LI (Ciliophora)


13. Halteria (Ciliophora): Note Saltatory motion of the ciliate.


14. Flagellate (Eukaryota); Flagellate-16915-14-LI. Very fleeting glance.

Now I need to set this up for a few days and see what happens. Smaller volumes makes for a different set of problems. Some more modifications of this basic setup would include sealing this, or perhaps using Agar to make the ditch, which might permit gas exchange. Lots to play with, and I urge you to try this at home. A complete set of the raw videos and pictures can be accessed at https://goo.gl/photos/7mjdsCyhVf2T7LKc9. I have also uploaded some of these to inaturalist.

11 Comments Add yours

  1. Saad Bhamla says:

    Wow! Just wow!

    This is awesome Laks. I really like the naming format for the unknown ciliates. I will follow it as well in future samples.

    This is one of those posts, that i will keep coming back to – fantastic!! :)))

    Saad

  2. Manu Prakash says:

    @Laks: love the micro-aquarium dream come true. The slides are a game changer. I also see; when you pan – you don’t see any flow in the slide which means that the sample does not get sheared – which is a big plus.

    What a fantastic collection of organisms. Number 12 is a big puzzle for me – since I don’t know anything like that that jumps. Manu

  3. laksiyer says:

    Hi @Manu and @Saad. I am pleased with this setup but it still needs some optimization. After 24 hours all the animals were dead and barring Halteria and the spirogyra , the water wasnt too active. It suggests to me that the seal was a bit too good for these critters and I need to find a way for gas exchange to keep animals alive. In my next experiment, I am going to peel the thin PVC off after observations (it comes off very well) and leave the slide in a humid chamber (petridish with water). The corollary to this is that if you want to view critters in anoxic water this setup is pefect, which reminds me I have a bottle which is over a year old releasing the most noxious of H2S when shaken. Need to sample it. Also, am actively searching for other material as coverslip. There are these commercial plastic coverslips that will be tried next. What is the most transparent and thin material that I could use other than glass that is cheap.

  4. Saad Bhamla says:

    @laks – when i was at hopkins and I wanted to do long-term imaging on slides, i used glass slides, made a well out of silicone sealing grease and placed a plastic coverslip. The organisms – in this case, a dozen marine larvae – were happy and swimming for 4 days. Now, of-course, the silicone sealing grease is messy, and tricky to use using foldscope, since it doesn’t withstand too much pressure (will squish), but just sharing as a thought.

    Saad

  5. laksiyer says:

    @Saad thats a great tip. I could use silicone grease on the thick PVC and use a plastic slide or thin PVC as colverslip and see how it goes. Also need to try VALAP as seal. Wonder what those breathable contact lenses are made of and if this could be used as cover.

  6. Saad Bhamla says:

    Silicone is the answer – it’s extremely permeable to oxygen – so contact lenses are made from it as well.

    Saad

  7. Matthew Rossi says:

    Curious: why should oxygen permeability be regarded as a necessary and good thing in the aquarium setup? In Laks’s arrangement, shouldn’t the plants provide the necessary oxygen to keep every other critter swimming?

    1. laksiyer says:

      @Matt. I think most animals need a stable concentration of dissolved “air”. At those micro-volumes, the fluctuations of dissolved O2 concentrations might be too much to bear for the critters. This seems to be my only explanation. After a few days, while the Spirogyra is alive, it too isnt doing too well. All others have given up the ghost, including the duckweed. I am going to try Silicone sealing soon. Actually, there are thin, ultra-transparent silicone sheets available too.. just that I havent found a cheap source, else, the coverslip should be made of that. Perhaps @Saad, @Manu, @Alice or @Jim might be able to tell us of a cheap source. The previous source I got from @Alice.

  8. djbeede says:

    Hey Laksiyer,
    Really enjoying what you’re up to here. Any updates on your wet slide research?
    Thanks,
    David

  9. djbeede says:

    Hey Laksiyer,
    One more thing.
    I’d like to come up with a wet slide system optimized for finding “swimmers” in contaminated drinking water sources. I have access to a vinyl cutter so could make these PVC slips pretty easily. Is there an ideal shape of the reservoir opening? Would an oval for instance work any better than a rectangle? I did a quick sketch of a 12″x12″ layout.
    http://davidbeede.com/WetSlideTemplate1.jpg
    Appreciate your sharing your experiences.
    David Beede

    1. laksiyer says:

      Hi David. I have been using this technique for all my wet mounts and it works very well for me. Further, I can go back to a slide and to a sample even after a day. Vinyl cutter sounds really exciting. I have been using a blade. The size of the reservoir shouldnt matter at all. Initially, I wanted to make it as big as possible to see a wide field which would hold a lot of water. However, sometimes fitting it all into a couple of field-of-view worth is also advantageous, especially if you are taking time-lapse photos. So I have also punched out holes with a belt-hole puncher. The problem with small holes and thus small volumes is surface tension and it is easy to lose the drop if you open and close the cover repeatedly. The oval design is fantastic, more so because you know exactly what kind of volumes you need, which might help in quantitative analysis (swimmers per 100 microlitres for example). Tell me more about these vinyl cutters, sounds interesting. also looking forward to what you see in these systems.— Best

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