Getting started with microscopy

I talked about algae last week at an event organised by Durham Wildlife Trust (part of the publicity for The Natural History of Upper Teesdale) and I promised them a post about how to get started in microscopy and, more specifically, to start discovering more about algae.  I have illustrated the post with some of Chris Carter’s spectacular images of algae to whet your appetites for exploring the world of freshwater algae …

Broadly speaking, the natural historian wants a microscope for one of two tasks: to make small things bigger or to make invisible things visible.   There is not really a sharp dividing line between these two categories, as the illustrations of Cladophora filaments in “Summertime blues …” show.   You might start out looking at a handful of green slime pulled from your garden pond, but then you might see smaller algae growing on those filaments that you want to examine too.   The good news is that you should be able to get hold of a reasonable microscope with the capacity to magnify up to 400 times for about the same outlay as a digital SLR camera.   That should let you see all but the smallest algae.   If you are sure that your interests lie mainly in “making small things bigger” then you should consider a low power dissecting microscope (these are probably the best way of introducing children to microscopy, as there is a smooth transition between the tangible but small object that has piqued their interest and the larger, more detailed image that they see when they peer through the microscope’s eyepieces).

However, even though a basic microscope need not cost a fortune, good microscopes are expensive so my advice to a beginner is to search out a rerfurbished second hand microscope.  In north east England, I can recommend JB Microscopes but Google should help you find dealers elsewhere in the country.   A reasonably local supplier is necessary because you should really try out a microscope before you buy.   There are reputable mail order suppliers (e.g. Brunel Microscopes) but I would not want to spend a large sum on a piece of equipment that I had not had a chance to use first.

A colony of the diatom Meridion circulare.  The image at the top of the post shows desmids from the genus Micrasterias.   Both photographs by Chris Carter. 

If you are on a limited budget, I suggest you go for a good basic microscope with the option to fit a camera at a later stage.  It is possible to take a reasonable photograph by pointing a digital camera (or even a smartphone) down the microscope’s eyepiece and it is better to put up with the shortcomings of these images than to sacrifice the quality of the microscope itself.

Once you have your microscope, you will also need slides, coverslips, forceps, some plastic Pasteur pipettes, a couple of needles, a scalpel and some collecting tubes.  You can buy all of these from Brunel Microscopes and NHBS, both of whom cater for both the amateur and professional markets.   They also sell boxes of prepared slides, which are a good way to get some experience at using a microscope.

The microscopic world generally lacks the type of user-friendly well-illustrated identification guides that help us identify wild flowers, birds, butterflies and so on.   Most books are aimed at the academic market and are, consequently, expensive.   If you want to get started with freshwater algae, one useful resource is this guide to the larger algae found in rivers: RAPPER_manual_version1.7_May2016.  It was produced to accompany a method for rapid assessment of streams and rivers and, as the journey towards formal publication has stalled, I am happy to make it available here.

Hydrodictyon reticulatum, the water-net, photographed by Chris Carter.  500 mm (micrometres) is half a millimetre.

Useful websites include AlgaeVision and the Diatom Flora of Britain and Ireland.  As most freshwater algal genera are found throughout the world, Diatoms of North America is also a useful resource.

The Freshwater Biological Association have affordable booklets on the identification of desmids and diatoms and there is an AIDGAP key, too, for freshwater diatoms.   The latter is badly in need of updating but, people assure me, is still useful for beginners.

There are plenty of other online resources, but l would recommend visiting the website of the Quekett Microscopical Club, a long-established group of enthusiasts whose interests span the whole realm of natural history and optics.   www.microscopy-uk.org.uk is also worth a visit.   Both websites will help you as you start your explorations of the hidden worlds of nature.

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Grazing on algae …

I comment on the role that grazers play in controlling algal biomass in rivers in these posts and this is the time of year when I, myself, take a more participatory role.   As it is spring, Lemanea fluviatilis is thriving in our rivers (the cleaner ones, at least) and I could not resist grabbing a couple of handfuls whilst out in the field recently for culinary purposes.

This time, I followed the routine I described in “More from the Lemanea cookbook … ” and washed, air-dried and then cut-up some Lemanea filaments into short lengths (they need to be about a centimetre long, otherwise they can form clumps).   My experience is that the fishy taste of Lemanea is a fine complement to freshwater fish, so decided to use it in a warm potato salad which I then served underneath a salmon fillet seasoned and sprinkled with dill and then wrapped in foil and baked with a couple of knobs of butter.

The warm potato salad needs a mayonnaise made from one egg yolk and about 150 ml of olive oil into which a couple of tablespoons of lemon juice are stirred, along with salt and pepper.   Add a generous handful of dried Lemanea to this and leave to soften for about 20 minutes, and also add a teaspoon of capers and a small handful of land (or water) cress.  Cook and drain enough new potatoes for two, then cut these into small chunks and stir the mayonnaise and algae mixture into these.   Divide between two warmed bowls and place half the salmon fillet on top of each.  Finally, add a few fresh pea shoots as a garnish, along with a wedge of lemon, and serve.

Definitely worth repeating.

Warm potato salad with lemon and Lemanea, served with salmon fillets.

Cypriot delights …

I could not return from my visit to Cyprus without an algal sample and a fine opportunity presented itself early last week when we visited the Avgás Gorge, on the Akámas peninsula at the west coast of Cyprus, just north of Pathos.  This is a spectacular limestone ravine whose steep sides offered welcome relief from the Mediterranean sun.  At points, as in the photograph above, the ravine narrowed to just a few metres wide, reminiscent of the siq which guards the entrance to Petra except that instead of exquisite carvings we stumbled across a Russian team conducting a glamour shoot.

A small stream made its way down the gorge.  The presence of woody debris at intervals suggested a considerable head of water during the winter months but, at this time of year the water has reduced in power, tumbling across a series of boulders into stagnant pools, interspersed with short runs shaded either by the high cliffs or the vegetation that flourished away from the harsh glare of the sun.

In the sections where water was still running there were clumps of Chara tangled up with filamentous algae, with plenty of bubbles of oxygen as evidence that both were busily photosynthesising away.  The filamentous algae was a coarse unbranched filament that was clearly a relative of Cladophora but which did not match any of the genera or species that I had encountered before (see “Fieldwork at Flatford” for similar situation).   There were pebbles and cobbles between these clumps, their surfaces criss-crossed by the galleries of caseless caddis larvae – probably Psychomyiidae, according to Richard Chadd.

Chara growing in the stream at Avgás Gorge in western Cyprus, April 2018.

Cladophora or a relative growing in the stream at Avgás Gorge in western Cyprus, April 2018.  The scale bar is 50 micrometres (= 1/20^th of a millimetre). 

There were a number of diatoms present too, the most abundant of which was a chain-forming Ulnaria.  Unfortunately, despite having just co-authored a paper on Ulnaria from Cyprus, I cannot name the species, as I saw no cells in valve view.  I will have to return to this subject once I have prepared a permanent slide from the sample that I brought back.   The chloroplasts in the illustration below are not in a very healthy state because the sample lived in a fridge for almost a week before I was able to get it under a microscope.  Had I looked at this sample 20 years ago, I would have assumed that I was looking at a species of Fragilaria, as most keys then stated categorically that Synedra (the former generic name) were solitary rather than chain-forming.  However, we now know that there are several Ulnaria species that form chains although most that I see in my regular haunts in the UK do not.   Our paper states that the species we describe form short chains although, as we worked from cleaned samples collected by other people, I now wonder if that was an artefact of the preparation process and whether these, too, formed longer chains in their living state.

A chain of Ulnaria from the stream at Avgás Gorge in western Cyprus, April 2018.  Scale bar: 20 micrometres (= 1/50^th of a millimetre).

Though it is rare for me to stray into describing the invertebrate life of streams, the Psychomyiidae are actually an important part of the story here, as the larvae graze algae from the surface of the stones.  They create a silk tube and this, in turn, becomes covered with fine sediment to create the galleries that are visible in the picture.   We know that invertebrates can change the composition of the attached algae by grazing but I sometimes wonder if the caseless caddis larvae also change the composition by creating myriad patches of very fine sediment across the rock surface.   If you look closely you can also see a couple of Simuliidae (blackfly) larvae.  These attach themselves to the rock by a circle of hooks at their last abdominal segment (I think that is “bum” in entomological language) and then use fan-like structures around their mouths to filter tiny particles from the water.  However, I have also seen Simuliidae larvae bent double on rock surfaces in order to hoover up particulate matter and algae that live there.

That, I thought, was just about enough natural history for one day.   I put my toothbrush and bottle back into my rucksack and made my way back down until, turning the corner, I stumbled across the Russian glamour models.   I’ve often written about the similarities between freshwater ecosystems in different parts of Europe but you don’t often see a bikini – or less – in April in my cold, damp corner.

Galleries of Psychomyiidae larvae on the top of a limestone cobble from the stream at Avgás Gorge in western Cyprus, April 2018.

Reference

Cantonati, M., Lange-Bertalot, H., Kelly, M.G. & Angeli, N. (2018).  Taxonomic and ecological characterization of two Ulnaria species (Bacillariophyta) from streams in Cyprus.   Phytotaxa 346: 78-92.

Wallace, I. (2003).   The Beginner’s Guide to Caddis (order Trichoptera).  Bulletin of the Amateur Entomologists’ Society 62: 15-26.

Letter from Cyprus

Travel can be one of the most rewarding forms of introspection ….
Laurence Durrell, Bitter Lemons, 1957

When I stepped off flight EZY1973 from Manchester to Paphos on Saturday night I passed a personal milestone. Arriving in Cyprus means that I have now visited all 28 Member States of the European Union. Starting with (West) Germany in 1972 on an exchange visit before the UK was even a member of the European Economic Community, followed shortly after by a family holiday to southern Austria (where my father had been stationed just after the war) with a day trip to Slovenia (then part of Yugoslavia), the number started to increase in the late 1990s when I became involved in the work of CEN, the European Standards Agency and, from the mid-2000s onwards, with the intercalibration exercise associated with the Water Framework Directive. A few years ago I made a list and realised just how many I had visited, after which, I have to admit, my choice of conference and holiday destinations was driven by this rather childish whim. Latvia, Malta and Bulgaria, all subjects of posts on this blog, were ticked off, leaving just Cyprus. This year, a family holiday to celebrate my mother’s 80th birthday provided the opportunity and, after some shameless lobbying, we had booked a villa near Paphos via AirBnB and were on our way.

How Europe has changed in the 47 years since my first overseas trip. Twelve countries were behind the Iron Curtain, three of the remainder were right-wing dictatorships. Two have merged (East and West Germany) whilst seven have become disentangled from previous relationships (the Baltic States from the USSR, Slovenia and Croatia from the former Yugoslavia and the two former constituents of Czechoslovakia from each other). Cyprus, from where I am writing, was in political chaos in the early 1970s. A former British colony whose territory was argued over by Greece and Turkey, it was soon to be split into two, separated by a buffer zone. I used to browse my Collins World Atlas assuming national borders to be fixed and immutable; the older and wiser me wonders where (and when) the next changes will come from.

The intercalibration exercise, in particular, was an opportunity for an exchange of ideas and I counted co-authors from 23 of the 28 EU states on my publication list. Looking back, these papers show remarkable consistency in some aspects of ecology across Europe whilst, in other respects, I am much more cautious about assuming that knowledge gained in my damp corner of north-west Europe can be applied to warmer and more continental regions. This publication list includes, incidentally, two papers about Cyprus, despite never having either visited before or having a native Cypriot on my list of co-authors. In the first paper, we worked with an Austrian employed by the Ministry of the Environment but, for the second, the samples were collected and analysed by Italians and Germans whilst I helped out with data analysis. Scientific colonialism is not, perhaps, dead?

My favourite? I don’t think I should single one of the 28 out. The food and culture of the warm lands of the Mediterranean basin draw me but I think that the parched summer landscapes would lose their appeal if I was there for too long. I find the grey, damp climate of my own corner of Europe wearisome but the greenness of the Spring and Summer, and the Autumn colours almost compensate. My ideal, in other words, seems like it should be a semi-nomadic existence but that, too, would pale with time. The truth is that, for me, elsewhere, being wanted, is always more wondered at ….

Natural lenses …

The photograph above is as about as far from Andreas Gursky’s careful constructions, described in the previous post, as it is possible to get.  It is a close-up of a green algal floc Heather noticed whilst on a walk around a local nature reserve.   I guess it fits the general description of “decisive moment” except that it takes a special sort of observer to find any interest at all in such an unprepossessing habitat.

Under the microscope, the floc turned out to be composed of filaments of Spirogyra, with a single helical chloroplast.  Members of this genus (and related genera such as Mougeotia) produce copious mucilage so are always slimy to the touch.  However, this mucilage makes it difficult for the waste gases produced by photosynthesis to diffuse away, leading to the production of bubbles within the mucilage mass.   The interest, today, however, was that these air bubbles are acting as tiny lenses through which it is possible to make out the individual filaments of Spirogyra.

The green floc beside a footpath in Crowtrees local nature reserve from which the other images in this post were derived. 

I should add the caveat here that the photograph was taken with the “super macro” facility of our Olympus TG2 camera but the end-product is, nonetheless, impressive.   It also offers us an insight into the world of the very earliest microscopists.  Anton van Leuwenhoek’s microscopes consisted of a metal plate which held a tiny sphere of glass which acted as a convex-convex lens capable of up to 266x magnification to a resolution of little  more than a micron (1/1000^th of a millimetre) (follow this link for more details).  To give an idea of what he might have seen with this, the right hand image below used 400x magnification.

That, however, only tells us part of the story of Anton van Leuwenhoek’s genius.   Whilst we should not underestimate the skill required to make the lenses and their mounts, the other essential element is curiosity.   Curiosity is, itself, multifaceted: in a few weeks we will probably make a trip out to an old quarry where we know we will find several species of orchids, and maybe some excursions to locations new to us but where others have reported interesting assemblages.  That’s one type of curiosity.  However, simply looking harder at the habitats all around us involves a different type of curiosity: a recognition that there is more to know even about things we think we already know about.   The former broadens our experiences, the latter deepens them …

The algal floc at Crowtrees local nature reserve in close-up: left: an extreme macro view of a single bubble from the image at the top of the post and, right: filaments of Spirogyra photographed under the microscope.  Scale bar: 20 micrometres (= 1/50^th of a millimetre).

3 minutes 59.4 seconds …

Back in 1995 I interviewed a number of eminent people about their first academic publications as part of an occasional series for the Times Higher Education Supplement.  I wrote about one of the more daunting of these in “An encounter with Enoch Powell”.   The hour or so I spent with Sir Roger Bannister, who died a couple of days ago, could not have been more different.   He was best known for three minutes 59.4 seconds on a running track in Oxford in May 1954 but went on to have a successful career as a neurologist and eventually became master of Pembroke College, Oxford.  He was, despite this sporting and academic prowess, one of the most charming people I have met.

One of the secrets of his success on the running track was that he was, to all intents and purposes, a sports scientist before that term had been coined.  He took time out from his medical degree to do research on the physiology of breathing and, more particularly, how the point of exhaustion could be delayed by feeding his subjects with different concentrations of oxygen.   As a medical student working in straightened times just after the war, his first task was to build his own equipment, including the treadmill on which he and an assortment of colleagues and friends ran in order to generate the data he needed.  Building this kit involved trips to RAF bases to strip meters and other parts from decommissioned bombers (John Hapgood, former Archbishop of York and also a physiologist by training told me a very similar story).

Whilst his experiments were not directly relevant to his running (his actual training time amounted to less than an hour a day), there was, clearly, a benefit from understanding how his body worked.  However, whilst a runner cannot alter the concentration of oxygen that he breathes, a mountaineer can, and Bannister’s work was used by the team that conquered Everest the following year (he commented that he was surprised at how unfit some of the Everest team were by the standards of track runners).

Whatever his other achievements, however, it was that afternoon in Oxford in May 1954 that defined Roger Bannister.  Three minutes 59 seconds works out at just over a quarter of Andy Warhol’s quotient of fifteen minutes of fame and would have ensured Bannister’s place in the history books.   However, as the obituaries in the newspapers show, he achieved far more than that in his life.   And he was a gentleman too.

If you have the patience to battle with News International’s paywall, you can read my original article by following this link.

More about Platessa oblongella and Odontidium mesodon

As my last post used the conventions of figurative art to describe algal ecology, I thought I would stick to graphs – science’s very own school of abstract art – for this one.   I spent some time in “Small details in the big picture” discussing the ecology of Platessa oblongella (including P. saxonica) but without saying very much about the types of streams where these species were found.  So I am going to take a step away from the Ennerdale catchment in this post and, instead, collate environmental data a large number of sites to get a broader understanding of their habitat preferences.  As these species are often associated with Odontidium mesodon (see “A tale of two diatoms …”), I will summarise the preferences of this species at the same time (but see Annex 1 for a graph of this species’ preferences for still versus standing water).

The first set of graphs show the response of these species to pH and alkalinity and establish both as species typical of circumneutral soft water.  Platessa oblongella can be abundant in more acid conditions (i.e. to the left of the green vertical lines) but most of the records where it is abundant have pH values between 6.5 and 7.5.   Note that P. oblongella can also be found in humic waters, where lower pH thresholds apply (see Annex 2).

Distribution of Odontidium mesodon and Platessa oblongella (including P. saxonica) to pH and alkalinity in UK streams.   Vertical lines for pH indicate threshold values that should support high (blue), good (green), moderate (orange) and poor (red) ecological status classes.  See Annex 2 for more explanation.

The second set of graphs shows how these species respond to inorganic nutrients.   Both are most abundant when inorganic nutrients are present in low concentrations, though the trend is stronger for phosphorus than it is for nitrate-nitrogen.   The graphs for Platessa oblongella, however, both have a few outliers.   I have seen P. oblongella in a few situations where I did not expect it – I remember finding it in the Halebourne, a stream draining heathland around Aldershot and Bagshot in Surrey, where the water was well buffered (mean alkalinity: 61.3 mg L^-1 CaCO₃) and nutrient concentration were high (mean total oxidised nitrogen: 4.01 mg L^-1; dissolved phosphorus: 0.25 mg L^-1) and Carlos Wetzel and colleagues note some other anomalous records from the literature in their paper (cited in my earlier post), including a few from high conductivity and even brackish environments.   So we should treat these plots as indicative of the ecological preferences rather than definitive.

Distribution of Odontidium mesodon and Platessa oblongella (including P. saxonica) to nitrate-N and dissolved phosphorus in UK streams.   Vertical lines indicate threshold values that should support high (blue), good (green), moderate (orange) and poor (red) ecological status classes.  See Annex 2 for more explanation.

The final pair of plots show how the relative abundance of these two species changes over the course of the year.  These plots show the months when each taxon is abundant, by the standards of that taxon.  Because Platessa oblongella tends to be very numerous in samples, the threshold for this taxon (the 90^th percentile of all records) is higher than that for O. mesodon.   This reveals a very clear pattern of O. mesodon thriving in Spring whilst P. oblongella is abundant throughout the year, but with a slight preference for summer and autumn.  We need to reconcile these patterns with the observations in A tale of two diatoms that show that P. oblongella is associated with thinner biofilms than O. mesodon and try to work out whether season is driving the patterns or whether the seasonal patterns are the manifestation of other forces.   My suspicion is that P. oblongella is a classic pioneer species but also has a low-growing prostrate habit which means that it should be resistant to heavy grazing, which may confer an advantage in the summer and autumn when grazers are most active.  However, I may be getting ahead of myself, as we are in the process of analysing data on grazer-algae interactions in the River Ehen and Croasdale Beck that may throw more light on this.  There are clearly more layers to this story yet to be revealed …

Distribution of Odontidium mesodon (i.) and Platessa oblongella (j., including P. saxonica). The solid lines represent relative sampling effort (i.e. the proportion of samples in the dataset collected in a particular month) and the vertical bars represent samples where the relative abundance of taxon in question exceeded the 90^th percentile for that taxon (20% for P. oblongella/P. saxonica and 5% for O. mesodon).

Reference

The dataset used for these analyses is that used in:

Kelly, M.G., Juggins, S., Guthrie, R., Pritchard, S., Jamieson, B.J., Rippey, B, Hirst, H & Yallop, M.L. (2008). Assessment of ecological status in UK rivers using diatoms. Freshwater Biology 53: 403-422.

Annex 1: Odontidium mesodon’s preference for still or standing water

As I included a graph showing the preference of Platessa oblongella / P. saxonica for still or standing water in “A tale of two diatoms …”, I have included a similar graph for Odontidium mesodon here.   I have not included any data from the streams that flow into Ennerdale Water’s north-west corner in this graph as this would give a distorted picture.  To date, I have only seen a single valve of O. mesodon during analyses of 14 samples from these streams but I have not yet sampled these in spring which, as the graph above shows, is the time when O. mesodon is most abundant.   Like Platessa oblongella, O. mesodon is predominately a species of running, rather than standing waters.

Differences in percentage of Odontidium mesodon in epilithic samples from Ennerdale Water and associated streams.  Data collected between 2012 and 2018.

Annex 2: notes on species-environment plots

These are based on interrogation of a database of 6500 river samples collected as part of DARES project.  Vertical lines show UK environmental standards for conditions necessary to support good ecological status: blue = high status; green = good status, orange = moderate status and red = poor status.  Note that there are no environmental standards for alkalinity and the vertical lines show a rough split of the gradient into low alkalinity (“soft water”: < 10 mg L^-1 CaCO₃), low/moderate alkalinity (³ 10, < 75 mg L^-1 CaCO₃), moderate/high alkalinity (³ 75, < 150 mg L^-1 CaCO₃) and high alkalinity (“hard water”: ³ 150 mg L^-1 CaCO₃).

pH thresholds are for clear water (see UK TAG’s Acidification Environmental Standards.  The corresponding thresholds for humic waters are lower (high/good: 5.1; good/moderate: 4.55; moderate/poor: 4.22; poor/bad: 4.03).

Phosphorus thresholds are based on UK TAG’s A Revised Approach to Setting WFD Phosphorus Standards.   Current UK phosphorus standards are site specific, using altitude and alkalinity as predictors.  This means that a range of thresholds applies, depending upon the geological preferences of the species in question.  The plots here show the position of boundaries based on the average alkalinity and altitude measurements in the DARES database.

Note, too, that phosphorus analyses use the Environment Agency’s standard measure, which is unfiltered molybdate reactive phosphorus.  This approximates to “soluble reactive phosphorus” or “phosphorus as orthophosphate” in most circumstances but the reagents will react with phosphorus attached to particles that would have been removed by membrane filtration.

Nitrate-nitrogen: There are, currently, no UK standards for nitrates in rivers.  Values plotted here are derived in the same way as those for phosphorus (see “This is not a nitrate standard”)