Rotifer Testudinella patina - common name “Turtle rotifer” 400X DIC microscopy. Note the two eyes.© Robert Berdan
Aquatic invertebrates are some of the strangest and most beautiful organisms on the planet. Many appear alien-like and can be found living in bird baths, eaves troughs, puddles, ponds, lakes, rivers and oceans. Some organisms thrive in open water others crawl through the mud or attach themselves to aquatic plants, algae and even live in the film of water in various lichens and moss. You can find thousands of species in a nearby pond and a microscope will let you examine these tiny creatures.
Water beetle larva Darkfield and polarizing microscopy 50X© Robert Berdan
Collecting Aquatic Invertebrates
To collect aquatic invertebrates start with a few plastic jars and some rubber boots. Some tools that will be helpful for collecting include a golf ball retriever which extends and holds small plastic bottles for sampling. I also use an aquarium net, plankton net, wide mouth plastic bottles and a white plastic tray. When I collect the water I often pour it first into a white plastic tray so I can see what I have caught. Sometimes there will be large insect larvae including dragon flies, beetles, and damsel flies. Mosquito larvae and Chaoborous (midge fly larvae) are easy to pick out once you see them wiggle. Another useful tool for collecting is a turkey baster that looks like a giant pipette and cost $2 at Walmart. To collect Diatoms I use an old tooth brush to clean the slime off rocks into a dish or jar. Collecting is fun and can even be done during winter by cutting a hole in the ice. By keeping a small aquarium or making a hay infusion you can view microorganisms throughout the year (see Hay infusion ref. below). You may find unique organisms in different ponds and the abundance of different species can change from week to week (M. Kreutz and W. Foissner, 2006).
Hydra oligactis (brown hydra) photographed with DIC microscopy 50X© Robert Berdan
When filling jars with pond water keep the samples cool during transport. When I get back to my home lab I open the lids to allow fresh air inside. Some species are sensitive to reduced oxygen as the water warms up. If possible examine your catch within hours as some species will die while others feeding on the dead organisms may increase in number. To improve your chances of seeing interesting organisms include pieces of plant material in the jars and on your microscope slides. Also some organisms like amoeba tend to settle on the bottom of the jar so use a pipette and include a bit of sediment and detritus. Larger organisms like Hydra can often be seen by eye and selected with a glass pipette. To help select specific species I place some of the pond water in plastic petri dishes available from Amazon or science supply stores and examine the water using a stereomicroscope. I use a glass pipette to select worms, insect larvae, rotifers, cladocerans and large ciliates which are placed on microscope slides with a glass coverslip for examination with a microscope. A stereomicroscope also lets you observe their behavior and how they swim in three dimensions.
Amoeba feeding on Diatoms DIC (Differential Interference contrast) microscopy© Robert Berdan
Ciliate Frontonia sp viewed with three different forms of microscopy. Frontonia are closely related to members of the genus Paramecium. Diatoms and other algae can be seen inside the cell.© Robert Berdan
Mounting Invertebrates on Microscope Slides
To avoid crushing some organisms on the microscope slide I put small drops of vacuum grease or Vaseline using a syringe or toothpick on the microscope slide to support the coverslip. I then push the coverslip down lightly to trap organisms so they are immobilized. I also draw water out from under the coverslip using a small piece of paper towel to immobilize the organisms, but if too much water is drawn some organisms will be crushed. With a little practice you can trap rotifers and other fast moving organisms in place for photomicrography or drawing. Other tools like a centrifuge can help concentrate organisms. I found a used centrifuge on Kijjii for $75 and a new mini-microfuge on Ebay for $50.
Calanoid Copepods are found in open water on lakes and ponds. Viewed with A) Bright field microscopy B) DIC microscopy C) Polarizing microscopy D) Polarized light and full wave 550 nm retardation filter.© Robert Berdan
Methods to View Aquatic Invertebrates
To view the organisms you can use any light microscope. The most frequently used objectives will be 4X, 10X, 20X, 40X and 60X magnifications. Bright field microscopy works fine for most subjects, but keep in mind that many specimens like amoeba, ciliates and various worms are transparent. Look for an organism’s movement and search the surfaces of algae. Most bright field microscopes can be adapted for darkfield using special filters or by putting a small coin on a filter over the light source. Experiment with different sized coins with different objectives. You can add a set of Rheinberg coloured filters which can be homemade (M. Shaw 2014) or purchased on Ebay for about $30 and are worth the investment for any microscope. These filters will introduce colour to rotifers, cladocerans and other organisms (J. Rheinberg 1896, R. Berdan 2017).
Volvox sp forms spherical colonies containing up to 50,000 cells that form a hollow ball. It's made up of individual flagellated somatic cells and smaller number of germ cells. Viewed by a) Brightfield microscopy b) Darkfield microscopy c) Phase contrast microscopy d) DIC microscopy e) Rheinberg lighting f) fluorescence microscopy. 200X.
Rotifer Synchaeta pectinata by combined Darkfield and Rheinberg lighting© Robert Berdan
You shouldn’t be able to see organisms in clean drinking water. Some water sources like rain puddles and bird baths often require the organisms to be concentrated in order to detect them. To see transparent organisms more clearly one of the best and affordable microscopy methods is phase contrast. Phase contrast makes cells and their organelles like the nucleus and mitochondria appear dark. Phase contrast also creates halos that can reduce the resolution slightly, but it significantly increases their visibility and is widely used to examine living cells. Frits Zernike, a Dutch physicist received the Nobel Prize in Physics in 1953 for his invention of the phase-contrast microscope.
Ciliate viewed by phase contrast microscopy© Robert Berdan
Oblique lighting is an economical alternative to DIC that produces a similar effect and is created by illuminating the sample by only a portion of the light coming through from the condenser (C. Sanchez et.al. 2017). I achieve oblique lighting by offsetting the phase condenser opening or the filter holder below the condenser to partially block the incoming light. It requires a bit of experimentation to achieve good results.
Diatom Gomphonema geminatum A) Brightfield microscopy B) Oblique lighting C) DIC microscopy. Each image is a focus stacks of 6 images to increase the depth of field.© Robert Berdan
Diatom frustule made of silica, this one has been cleaned. Diatoms are used as filters in the beer industry, silver polish and for sensing in nanotechnology, molecular separation and molecular biology.© Robert Berdan
Stylaria lacustris - 2.5X objective and DIC© Robert Berdan
Stylaria lacustris is a common worm found in pond water that has a very long proboscis and transparent body. It has remarkable regenerative abilities. This is a panoramic photo stitched from 5 separate images and taken with 2.5X objective and DIC microscopy.
Rotifer Brachionus quadridentatus Phase contrast microscopy 400X© Robert Berdan
Rotifer Stephanoceros fimbriatus one of the most beautiful rotifers - Darkfield microscopy© Robert Berdan
Cladoceran found in open water of a pond Polyphemus pediculus – Darkfield microscopy© Robert Berdan
Fluorescence microscopy can also be used to observe plankton. Some subjects like algae and desmids are autofluorescent, while other organisms can be made fluorescent by staining with Acridine orange and other fluorescent dyes. Another alternative is to stain invertebrates with vital dyes. Examples include: Bismark brown, Methylene blue, Neutral red at low concentration to increase contrast (R.L. Howey 2020) You can also feed some organisms like paramecium colourful particles like carmine which are taken up and stored in vacuoles (R.L. Howey, 2000).
Spirogyra is a filamentous charophyte green algae named for the helical or spiral arrangement of the chloroplasts which are autofluorescent when viewed with an Epifluorescence microscope. 400X© Robert Berdan
Fixing, Staining and Anesthetizing Microorganisms
When researchers are unable to examine plankton specimens shortly after collecting they will often preserve the samples in 4% Formalin or 70% alcohol. You can also fix and stain the organisms with 0.1 to 0.5% lugol’s iodine to increase their visibility. Fixation and staining may alter the shape, size and appearance of the organisms making them more difficult to identify. I don’t recommend preserving the specimens unless you can’t view the specimens for a few days or longer. Live organisms are more interesting to study and easier to identify. Similarly methods intended to slow down microorganisms can alter their shape and behaviour. One other technique used by some researchers to anesthetize rotifers and worms is to put them in carbonated mineral water -the carbon dioxide in the water will temporarily anesthetize them.
Live Diatom Navicula sp DIC microscopy© Robert Berdan
Paramecium caudatum surrounded by smaller ciliates, DIC microscopy 400X© Robert Berdan
Documenting Your Observations
To make records of your specimens you can take photographs with through a dedicated microscope camera or digital single lens reflex (DSLR) camera attached to your microscope. If you don’t have a camera you can draw images of the specimens. Some microscopes have devices to assist drawing e.g. a camera lucida. Many science publications include drawings to show identification features of the organisms more clearly. If you plan to make videos use the correct adapter when using dedicated cameras and DSLR cameras for the best videos from which you can also extract pictures using Adobe Photoshop or other image editing software.
Although considered one of the best microscopic methods for viewing ciliates and other small micro-organisms, Differential Interference Contrast microscopy (DIC) can be extremely expensive and can easily add $10,000 or more to the cost of a microscope. Affordable alternatives to DIC, without the same 3–D appearance, and offering equal high resolution, are Polarization and Phase Contrasting methods. Polarizing microscopy can also add colour to birefringent specimens and alter the background with retardation filters. Polarizing filters can be added to any light microscope, though it requires a bright light source. Oblique lighting can simulate the appearance of DIC microscopy and can be implemented at no additional cost by blocking part of the light going through the condenser (C. Sanchez et. al. 2018). Motic plans to release its DIC microscope series later this year but has many affordable selections of Polarized and Phase Contrast options to achieve visually stunning images.
Rotifer Synchaeta pectinata viewed with A) Brightfield B) Darkfield C) Phase contrast and D) DIC microscopy.© Robert Berdan
A pond can support thousands of different species and there is always the thrill of finding something new. The internet offers science papers, aquatic guides to pond life to help you identify your catch. Some of the organisms found in ponds are used by scientists to undertake research into health, nutrition, aging and regeneration. Science is a process of learning and discovery where the microscope is an important tool. Owning a microscope allows you to perform science experiments and provides an opportunity to make personal discoveries all year long.
Robert Berdan Ph.D. for Motic Microscopes
C. Sanchez et. al. (2018) Oblique illumination in microscopy: A quantitative evaluation.
Micron 105:47-54. doi: 10.1016/j.micron.2017.11.006. Epub 2017 Nov 16. https://europepmc.org/article/med/29179008
R. Berdan (2017) Rheinberg Filters for Photomicrography
M. W. Shaw (2014) Your Microscope Hobby: How to Make Multi-coloured filters: Rheinberg, Polarizing, Darkfield, and Oblique.
Fresh Squeezed Publishing, Kindle Edition available on Amazon.ca
Rheinberg filters from Ebay.com
M. Kreutz and W. Foissner (2006) The Sphagnum Ponds of Simmelreid in Germany: A Biodiversity Hot-Spot for Microscopic Organisms. Protozoological Monographs Vol. 3, 1-267. Shaker-Publishers.
R. L. Howey (2000) Vital Staining for Protozoa and Related Temporary Mounting Techniques. Micscape Magazine.
W. Foissner and H. Berger (1996) A user-friendly guide to the ciliates (Protozoa, Cilophora) commonly used by hydrobiologists as bioindicators in rivers, lakes, and waste waters, and their ecology. Freshwater Biology 35: 375-482.
J. Rheinberg (1896) Original Note on Coloured Illumination "Rheinberg filters"
Hay infusion Procedure – Carolina Biological Teacher Resources: