Clive’s Corner #10: Sample Chambers for Plankton
Clive’s Corner by Clive Bagshaw
A feature of MicroNews, Clive’s Corner is a place created for the sharing of knowledge, tricks, and tools. The Corner is where you read about clever microscopical hacks - and submit your own. Clive’s Corner is the namesake of SFMS member Clive Bagshaw, who has spent a lifetime looking into microscopes - including 50 years studying protein reactions.
One of the most popular subjects for amateur microscopists are live plankton samples obtained from fresh or sea water sources. In the monitoring process, it is common practice to get a quick overall impression of the sample in a Petri dish on a stereo microscope and then take a closer look at a slide sample under a compound scope. My Amscope 305 stereo scope has 10x and 30x options, which are fine for larger zooplankton, but it is hard to identify smaller phytoplankton species at these magnifications. On the other hand, with a compound scope it is possible to view live samples at 100x or more magnification, but a much smaller sample volume can be explored on a slide. Consequently, rarer plankton may well be missed when monitoring relative abundance of species. Why not use a Petri dish on a compound microscope?
There are a couple of obvious reasons why not. First, a Petri dish is not a comfortable fit in the slide calipers found on most compound microscopes (Figure 1a). Second, even if the dish did fit, would you want to run the risk of dunking the objective lens into seawater? If not, then another option is to use a spacer slide or a commercial chamber (more on this below). However, with care, a Petri dish can be used on a compound scope, although it is probably wise to stick with objective lenses in the $20 price range (e.g. Amscope 4x and 10x achromats) – the typical budget for a Corner procedure – in case of an accident.
To control the movement of a Petri dish on a compound microscope stage, the slide caliper device must be removed and substituted with a custom holder. Such a holder can be cut out from a yoghurt pot lid with scissors (Figure 1b,c). The mounting holes in the calipers can be used as a guide for drilling mounting holes in the lid. The small ridges around the edge of the lid are sufficient to raise it above the stage and make contact with the Petri dish a few millimeters above its base (Figure 1d). As a result, the Petri dish will move with the holder, unlike with the calipers whose clip tends to slide under the Petri dish. It is best to choose a Petri dish with low wall height. While this will clear the working distance of a 4x objective, that of a 10x one will likely be below the Petri dish wall. It is therefore important that the stage is fully lowered before swinging the 10x objective into place over the sample. All objectives above 10x magnification are best removed from the microscope turret to avoid collisions with the dish. The user should be familiar with the microscope such that the operation of the focus and stage controls are “automatic” as to the direction of movement. The final precaution is to have a bottle of distilled water to hand, so that if the objective makes contact with the sample, it can be quickly rinsed.
Petri dishes with diameters in the region of 5 to 6 cm will require about 4 to 6 ml of sample to give around a 2mm depth of water. This is the minimum depth, limited by the surface tension of water but is within the working distance of 4x and 10x objective lenses. Although these objectives normally specify a coverslip thickness of 0.17 mm, the aberrations introduced by the absence of a cover slip are small for low power (low NA) objectives. In any event, looking at live plankton specimens requires water depths of at least around 100 µm, so the resolution is already compromised compared with specimens fixed in 1.54 refractive index media for which most lenses are designed. The whole dish may be scanned in “lawn mower” fashion by translating the stage back and forth and from side to side. The back- and forth-movements may be limited by the stage micrometer to around 2 cm, so to explore the complete dish, it will need to be rotated within the holder. Move the stage slowly, so that if the objective barrel touches the side wall, it can quickly be brought to a halt. Practice with a distilled water sample first. Care is required when assessing plankton abundance in a Petri dish because standing waves usually concentrate organisms in the center of the dish.
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Figure 1. (a) Original stage calipers for holding a slide. (b) Yoghurt pot lid cut to hold 6 cm Petri dish. (c) Multipurpose lid cut to hold a 3” slide or 6.5 cm Petri dish. (d) Plankton sample in dish showing working distance with 10X objective lens (about 4 mm from the water surface when viewing specimens lying on the bottom of the dish).
For those who don’t want to risk using a Petri dish on a compound microscope, there are alternatives, although the volume that can be explored is likely much smaller. Silicon isolators/spacers allow a coverslip to be stuck on top of a conventional slide which protects the objective lens from damage. Silicon isolators come in different thicknesses allowing higher power objectives with lower working distances to be used. However, commercial brands are expensive for single use, and with multiple use the sample tends to leak around the edges with the risk of stage corrosion, or damage to the condenser lens beneath. A cheaper spacer can be made using rubber O-rings which are available at hardware stores or on-line in various thicknesses for less than a dollar a piece. A smear of Vaseline around the O-ring provides a good seal which will hold the water sample for several days (see video). Slides and coverslips should be cleaned with soap and water after use to remove the Vaseline. Other slide spacer designs are described on-line. Finally, depression (concavity) slides give room for motile plankton to swim around, but the total sample volume explored is around 1/100th that of a Petri dish. Depression slides can be used with up to 20x objectives, but above this power, some specimens in the center of the cavity may be beyond the available depth-of-field. For quantitative analysis of plankton populations, any of these slide chamber options can be used in conjunction with 1 mm adhesive grids (Figure 2d).
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Figure 2. (a) Commercial 1 mm silicon spacer. (b) 2.5 mm thick rubber O-ring held with Vaseline. (c) A depression slide. (d) Slide with adhesive 1 mm grid.
Dedicated chambers for microscopy are also available commercially. Sedgewick-Rafter chambers allow for quantitative analysis of 1 ml samples. The MicroSafari Aqua kit comes with an added bonus of having a gas permeable surface on one side, allowing plankton samples to remain viable for several weeks. The chamber is made of acrylic and requires a working distance of at least 1 mm. The company founder, Saroosh Hedayati has been involved with the Society and exhibits at CuriOdyssey in San Mateo.