Water Critters Technique

[PeglegOS]

How do you prepare for photos of the denizens of the water world.

Do you use well slides, flat slides, special slides? Do you use cover slips? Where do you find your samples? What kind of lighting and/or scope do you use?

Maybe your answers will help newbies (like me ), and get them rolling.

[Charles Krebs]

Wow!.... where to start!

There are probably as many ways of working as there are people doing this. I’m glad you asked this question. Hopefully you will get a variety of responses. I too would like to learn how others approach this. When you learn how others work you always pick up new ideas. Sort of surprising no one has posed this question before. I hope this will grow into a very useful thread.

There are many different scenarios for collecting, depending on what I’m after and where I am.
I'll approach it from a “general” standpoint… the way I usually work casually when at home.

I have a small, nearby natural pond that is quite productive. Samples are collected from the edge of the pond in large peanut butter jars ( ). I try to get a very small amount of the muck at the bottom and a goodly amount of the vegetation. Then I fill the jar with pond water. I will sometimes grab a handful or two of vegetation and squeeze the water out into the jar as well. I have a stiff aquarium net with a two foot handle that I use to reach out and scoop up the vegetation so I don’t need to wade in.

When I get home, I pour the contents into a rectangular glass baking dish and keep it near a window (but not in direct sun light). One corner is pointed toward the window. Then I take a small square plastic container and squeeze out some of the vegetation. I prop up the small container so that any sediment gradually settles into a corner.

Now, depending on the time of year and type of specimens that might be present I start looking for appropriate material to transfer to a slide. After things have “settled out” (an hour or two) I put on my reading glasses and examine the corner of the baking dish near the window. Many subjects are phototropic, and will aggregate in the corner near the window light. If I can discern movement, or a slight green or yellowish “tinge” to the water, I know there is good subject matter there. I use polyethylene eye-droppers. (I originally used glass, but got tired of trying to clean them. If you purchase poly droppers through a biological supply house in quantity they are very cheap, and can be tossed when they are dirty or “contaminated”). I will take a couple drops from the corner of the dish and place them on a flat slide. I always use cover slips. The amount of water transferred is somewhat important. Too little and it will not spread to the cover slip edges, and can “crush” your critters. Too much and the cover slip will “float” and jiggle. After a few tries you get an idea of the correct amount. I typically opt for a little too much. It will evaporate while I scan the slide. (A little can be "sucked out" if need be by placing a piece of lens tissue or paper towel along the edge of the cover slip). I will often keep an eyedropper filled with pond water next to the microscope. If there happens to be relatively “thick” specimens on the slide, they can be crushed as the water evaporates and the cover slip “drops”. If I notice this, a small drop of water placed at the edge of the coverslip will be drawn under by capillary action… extending the useful observation time for the slide. (I don’t use well slides too often. If I have large specimens I will occasionally make up my own well slide by gluing a very thin nylon washer to a slide.)

If there is no “gathering toward the light” I look elsewhere. Typically it can be frustrating to try to find subjects in “open water”. They tend to be found on/near the vegetation or detritus. I will take a few drops of the sediment from the corner of the small bowl mentioned above, and examine that. This sediment, and that in the larger dish, is the source of many subjects. One difficulty can be if there are tiny particles that keep the cover slip “lifted” more than desired. Usually there is little problem with a 10X or even 20X. But on a slide where the cover slip is “held up” by hard debris, it can be difficult or impossible to get a quality image on things that have settled to the bottom with a 40X and higher.

So mine is sort of a “shotgun” approach. I look through quite a few slides. On occasion I will transfer part of the sample to a Petri dish and study it through a low powered stereo scope, looking for appropriate subjects to transfer to the slide. This is done more often when I am looking for a particular subject. But I’m impatient and like exploring a wet mount slide, so I don’t do this as frequently as others might. You can see some very interesting things when scanning with a 10X that might not even be noticed under a low power stereo.

When I pack it in for the night I will sometimes carefully “float” a cover slip or two on the waters surface in my sample dishes. The next day these are carefully lifted out and placed on a slide. Frequently, interesting things will have attached themselves to the cover slip and can be examined nicely right up against the cover slip.

Probably enough for now.

Charlie

[Ken Ramos]

I don't think anyone could have explained it better Charlie.

[Thomas Ashcraft]

Peg,

There is a lot of good dialogue on the Yahoo Microscope email list. It is a good place to ask questions or just research through the message archives which I think are accessible even if you don't join the list.

http://tech.groups.yahoo.com/group/Microscope/

Good luck with your process. It is a lot of learning by trial and error and asking questions. - Tom

[PeglegOS]

Peg,

There is a lot of good dialogue on the Yahoo Microscope email list. It is a good place to ask questions or just research through the message archives which I think are accessible even if you don't join the list.

http://tech.groups.yahoo.com/group/Microscope/

Good luck with your process. It is a lot of learning by trial and error and asking questions. - Tom

I joined the list yesterday. Learning is just part of the fun.

[PeglegOS]

Thanks Charlie. Many people will benefit from your information. Not to mention marvel at your results.

[Walter Piorkowski]

Hi Peg,

My collection and bulk storage techniques are very similar to those that Charles has mentioned. However I do not squeeze out the vegetation but treat it as if I don’t want to create a disturbance. Usually having a jar with some sediment and of course water, submerged along side the vegetation that I will cut from its surroundings or roots. I then move the material into the jar with it never having left the water. The large number of critters capuered is amazing. Another technique I use is to take a soup spoon and just touch the water surface. Transferring the water droplet captured directly to my slide. Different types of organisms seem to be captured by this method as a large number live within 2-3 mm of the surface.

I try whenever possible to collect the fine fibrous algae but my favorite is the water mosses which have a leafy structure but are so small that they squish nicely on a slide. Do not pass up collecting in cold fast moving streams. I wade in and cut samples from the long strands of algae wiping about in the current attached to rocks. These provide critters too.

Back in the lab I have two observing techniques. One is to transfer a water droplet to a flat 1x3 slide or to a concave glass lens. I then observe directly with water immersion objectives. This method has to be monitored closely as to not let too much water evaporate as you observe. I will have a syringe of distilled water or an eye dropper of rain water to add as needed. Working with these objectives requires cooperation from every one in the house as someone walking nearby will create quite a vibration.and disturb the image.

My favorite method however is to make a semi-permanent micro aquarium. I collect clear acetate sheets whenever I see one. They come in a variety of thicknesses and can easily be cut. I will cut a square piece to fit on the glass slide that I will be using and then cut a circular hole in the center to create a donut with square outer corners. Working cleanly with a syringe I apply petroleum jelly to both sides and press it down against the slide. A small droplet of the specimen water is placed in the center and followed by a small piece of vegetation cut while it is under the water surface. A minute amount of additional water is added so that when a cover slip is jellied and added to the top the water fills but does not squeeze out of the chamber I have created. If you take great care and create a total seal this aquaria will last for months. None of this is new its in many books on the subject. I will of coarse match the cell thickness to the specimen.

I have found acetate as thin as .002 inches which allows for observation with objectives as high as 50X. Oiled objectives of higher magnification are more problematic but I am experimenting with stress relieved Mylar to act as a cover slip to allow even those to be used.

I always keep the micro aquaria in indirect sunlight so that the vegetation that I have trapped will continue to grow. Its am amazing thing to watch along with the critter amongst them. Eventually your critters will die off but the you can spend another couple of weeks observing the bacteria! Have fun and good hunting.

Walt

[DaveBH]

This is a great thread 8>) After the rain, I've been retrieving standing water from open containers around the back yard. All contain a range of little beasties feeding on dead insect carcasses and rotting vegetation, and in some cases on each other.

This is all new to me, and to be able to view this 'other world' by microscope is staggering. My next step, as a photographer, I can already see will be more challenging!

I'd like to hear more about collecting and maintaining live specimens. Also great would be to see a chart showing the relative sizes of the little beasts and the best magnifications for viewing them.

thanks!

DaveBH

[Linden.g]

After I've inspected a pond sample for a while I'll often add "micro Algae grow" ( http://florida-aqua-farms.com/shop/micro-algae-grow/ ) and cultivate the sample in a shallow try under a lamp. I often find that several different species bloom in the sample in large numbers at different times of incubation. Algae tend to grow first and then preditory species take over to feed on these blooms later. I can keep samples evolving in this way for a couple of weeks. Here is an example http://www.photomacrography.net/forum/v ... hp?t=18912
Linden

[Linden.g]

When I pack it in for the night I will sometimes carefully “float” a cover slip or two on the waters surface in my sample dishes. The next day these are carefully lifted out and placed on a slide. Frequently, interesting things will have attached themselves to the cover slip and can be examined nicely right up against the cover slip.

Charlie
Thanks for the floating cover slip tip, I tried it last night and it worked well with many Vorticella attaching themselves to the surface. Keeping the desired water layer thickness bacame much more of a challange due to the lack of large particles but it does allow you to control this as needed

Linden

[NikonUser]

One way to control water depth is to position the coverglass onto 4 spots of grease: lanolin, vaseline, candle wax. Start off with depth greater than you need and gently push down on the corners of the coverglass until you get the desired depth of the water.
Some illustrations here:
http://www.photomacrography.net/forum/v ... ht=lanolin

[DaveBH]

I have a few times inadvertently got the front of a regular objective wet with pond water, and enjoyed a few guilty moments during which the lens seemed to perform extremely well 'sub-aqueously'.

To date I've noticed no obvious negative effects on the lenses so mistreated, but it got me thinking about the possibility of using a set of relatively cheap objectives for pond water immersion, first perhaps treating the cement around the front lens with silicon grease, or other sealant, to prevent or delay any potential damage.

Has anyone else tried this?

DaveBH

[Charles Krebs]

Dave,

There have been a fair number of "dipping" objectives made over the years. In some cases attachable waterproof caps are used over the front of the objective. These have typically been are relatively low power objectives.

Most of the Big 4 now make hugely expensive high NA water immersion objectives for studying live cells in solution.

I have a couple of older far more modest water immersion objectives and frankly very rarely use them. Subjects are much more easily followed, focused and photographed when they are under a coverslip.

I have a few times inadvertently got the front of a regular objective wet with pond water, and enjoyed a few guilty moments during which the lens seemed to perform extremely well 'sub-aqueously'.

A low power, low NA objective might do this.... as long as it lasts . Most better quality objectives will have some sort of moisture "sealing" at the front element, but don't count on it! If water does creep inside you can experience internal condensation and possibly set the stage for fungus or other nasties.

If you had an inexpensive objective that was "dispensable" it could work. (Don't use a silicone grease however).

[DaveBH]

Yes, keeping the little buggers under a cover slip does solve a lot of problems, especially with DOF. And the fact that you don't use you WIs all that much probably tells me all I need to know. Thanks, Charles.

DaveBH

[discomorphella]

I use both Charlie and Walter's methods for my pond samples. I also use a version of Linden's technique and will spike a pond culture with a few mls of strong hay infusion (approx. 100 gm of hay boiled in 500 ml of pond water, you can also add a few lentils or wheat grains to it as well) to make it grow out. One thing that I really like to do is pipet a sample of 10 ml or so from my pond jars into a small (60 mm diameter) petri dish, and examine it under a stereoscope and pick out the organisms that I really want to culture and innoculate them into a small flask or petri dish of hay infusion or alga-gro or homebrew algae culture medium. Then I can get a really dense culture of protozoa or algae for fixing and staining for permanent mounts.

David