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July 2013 - MicrobeHunter Microscopy Magazine - 1

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ISSN 2220-4962 (Print)ISSN 2220-4970 (Online)

Volume 3, Number 7July 2013

The Magazine for theEnthusiast Microscopist

http://www.microbehunter.comMicroscopy Magazine

Lighting Techniques Crawling Ciliates Microscopy of Pollenand Bees

2 - MicrobeHunter Microscopy Magazine - July 2013

Microbehunter Microscopy MagazineThe magazine for the enthusiast microscopistMicrobeHunter Magazine is a non-commercial project.

Volume 3, Number 7, July 2013

ISSN 2220-4962 (Print)ISSN 2220-4970 (Online)

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Images and Articles by: Joshua Grosse, Charles Crookenden,Marc Bos, Salah Deeb, Mahmoud El-Begawey, Khalid El-Nesr,Emad Mahdi, Neill Tucker, Randy Oliver,John Severns (Severnjc)

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Front Cover: Citric acid in polarized lightLeft image: Neill TuckerMiddle image: Joshua GrosseRight image: Gretchen D. Jones

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4 Biology of Crawling CiliatesCrawling ciliates use cirri for attaching andmoving on surfaces

Joshua Grosse

8 Focus on BeesMicroscopes are valuable tools for diagnosingColony Collapse Disorder in bees.

Charles Crookenden

12 GalleryBdelloid rotifer images

Marc Bos

14 Direct Specifying of the Region of Interest forPreparation of Tissue Microarrays

Salah Deeb, Mahmoud El-Begawey,Khalid El-Nesr, Emad Mahdi

16 Imaging TechniquesHere are a few techniques to improve the imagesrecorded with a USB eyepiece camera.

Neill Tucker

CONTENTS

Answer to the puzzle (back cover): Bee sting.

Rotifer gallery(P. 12)

Pollen(p. 8)

Unidentifiednematode (p. 6)

Lightingmethods

(p. 16)

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Crawling ciliates are among themost conspicuous protozoans,easily found in all sorts of places

– puddles, around algae or aquaticplants, water from plant pots, and so on.Anyone who has done much microscop-ic exploring is sure to have seen some,though it seems they are not alwaysrecognized. I also think they are good examplesof many of the most interesting adapta-tions, which are unfortunately beyondthe scope of usual guides. As such, Ithought it might be appropriate to re-view some of the main facts about themand give a bit more perspective on theseremarkable types, once considered thehighest evolved of all single-celled life. The ciliates I am considering herecan be recognized by the presence ofcirri, bristles formed by compound cilia.These are much more obvious than sin-gle cilia, and are usually conspicuous atrelatively low magnification, at leastwhen the body turns out of the way.

Besides these there are also mem-branelles, compound cilia in a row lead-ing to the oral cavity. A prominentadoral zone of membranelles, as thisseries is called, is typical of three mod-ern classes of ciliates: Heterotrichea,Armophorea, and Spirotrichea. Typeswith cirri are all part of the last group. In most of these the body is flat-tened, with the cirri on the lower orventral surface, acting like tiny legswhen they crawl. The membranelles,used for swimming and feeding, usuallyrun around the front, over to one side,and then to the mouth. In fact they al-ways go on the left side, defined as seenfrom above – though note ciliates willhappily turn upside-down and crawl onsurface films or cover-slips. If the ciliate is not too small orquick, another feature you may notice isthe contractile vacuole or water-expul-sion vesicle. Spirotrichs typically haveonly one set to one side. This growsinto a clear circle as it collects excess

water, then periodically vanishes as itscontents are expelled.

Classification

Ciliates with cirri used to be classi-fied together as the hypotrichs, but arenow understood to make up two sepa-rate groups. These are easy to distin-guish but unfortunately hard to name,since different protozoologists call themdifferent things, some using “hy-potrichs” for one and some for the other.As a way to keep things clear I am goingto call them “euplotids” and “sticho-trichs”, although those terms do admitsome variation, too. Euplotids have cirri in scatteredgroups, never in rows along the sides.The shape tends to be oval and rigid,with the water-expulsion vesicle on theright and near the posterior. The pelli-cle is inflexible and some have promi-nent ridges running down the back.

Aspidisca have only inconspicuousmembranelles restricted to the posteriorhalf, but in most others they run aroundthe front and past the midline. The otherones common in freshwater and soil areEuplotes. This is a very large group thatcould maybe stand to be divided up, butso far only a few of the proposed sub-groups appear natural – notably Euplo-toides, including larger freshwaterspecies, and marine Moneuplotes. Stichotrichs have at least some cirriin long rows, and in flattened kinds thisalways includes a row along both mar-gins. The cells are much longer thanwide, with the water expulsion vesicleon the left and rarely behind the mid-line. Many are flexible, bending whenthey encounter obstacles, though one

BACKGROUND Ciliate classification

Biology of Crawling Ciliates

Crawling ciliates use cirri for attaching andmoving on surfaces

Joshua Grosse

Figure 1: Euplotes

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subfamily – the stylonychines – are no-table exceptions. In general, identifying different sti-chotrichs depends on the details of howthe cirri are arranged, which is not usu-ally clear in a casual viewing. In factmuch of their classification is develop-ing, and some large genera like Oxytri-cha are still being cut up. Even so, thereare some features to watch for.

Some stichotrichs have distinctlyelongated caudal cirri, usually three innumber, for instance in some flexibleOxytricha, Uroleptus, and rigid Tetme-mena. They tend to be most prominent,though, in the very common type Stylo-nychia. Those also often have the frontend bulge out on the left side, an other-wise rare combination; Leeuwenhoek’sanimalcule “figured like a Mussel-shell” may well have belonged here.

The mouth can also be important. Inmost the membranelles follow a ʕ pattern running a third the length of a ma-ture cell, or half in stylonychines. Theyform a straighter row along the side inGonostomum from soil, though, and arelinear in Stichotricha which also have aspindle-shaped body and live in tubes.Another unusual feature is seen inSteinia, where the oral cavity is verylarge and has a distinctive pit at the front. Most dramatic, though, a few kindsproduce pigments – something rare forciliates, which outside some het-erotrichs are usually only coloured byalgae or food. These usually take theform of cortical granules distinguish-able only under oil immersion, but infreshwater types now called Rubri-oxytricha, the cytoplasm itself has areddish or brown colour.

Feeding and Symbiosis

When people learn about most ani-mals, from dinosaurs to insects, whatusually gets stressed is how they fit inthe food chain; what different types eat,and eats them, and how they interactwith other organisms. For protozoansthese questions are often left to a fewkey examples like Paramecium, but Ithink they are important for appreciat-ing their diversity, and so have goneinto some more detail about them. For the most part spirotrichs arefilter feeders. Kinds like Aspidisca withsmall mouths feed mainly on bacteria orsmall algae, while ones with largermouths tend to be omnivores and willalso catch other protozoans. This feed-ing is not necessarily indiscriminate,though; for instance Stylonychia in cul-tures have been found to prefer eatingalgae to Euplotes, but still pursue themwhen other food is scarce. The odd stichotrich Kerona pedicu-lus is a special case. These kidney-shaped ciliates live on polyps – mostlyHydra, like the unrelated ciliateTrichodina, but also sometimes bryozo-

BACKGROUNDCiliate classification

Figure 2: Stichotricha

Figure 3: Stylonychia

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Ciliate classificationBACKGROUND

ans. There has been uncertainty in thepast if these “polyp-lice” might beharmful or beneficial, but for the mostpart they seem to live off waste materiallike lost cells, supplemented by algaeand diatoms. Symbiotic green algae, calledzoochlorellae, are not as common as insome other ciliates, but are typical of afew species like Oxytricha chlorelligeraand Euplotes daidaleos. The latter alsorely on symbiotic bacteria, and in factso do all members of the Euplotoidessubgroup to which they belong. Theseciliates have lost the ability to use stor-age carbohydrates in their absence, andso in the long term die unless they arere-established. The bacteria likewiseonly live in the ciliates; so far only onetype has been named, Polynucleobacterfrom E. aediculatus. A different sort of endosymbioticbacteria has been found in the Moneu-plotes subgroup. These are very similarto the bacteria found in mate-killer Par-amecium; they occur in certain carrierstrains, and kill sensitive cells that con-jugate with them. This mostly soundslike a sexually transmitted disease, but Ihave also seen it discussed as a possibleform of intra-species competition. Something else that might look likeintra-species competition is cannibal-ism. This is seen in species here like E.versatilis and Tetmemena pustulata(formerly in Stylonychia or as Oxytri-cha bifaria), as well as some hymenos-tome ciliates; as food is exhausted,

some cells grow several times in sizeand devour the others. This is not aquestion of different strains, though, butoccurs just the same when all cells aregenetically-identical clones, so maysimply reflect that the odds of survivalare higher for a few larger cells.

Induced Defenses

Any microscopic life is going to below on the food chain, and so for in-stance falls prey to larger filter-feederslike crustaceans. However, there arealso many predators that are more activein capturing them, including smallworms, amoebae and heliozoans, andother ciliates. Of the last, litostomesand suctorians are noted for toxicysts,small bodies that release enzymes tobreak down and paralyze potential prey. In Visscher’s early observations ofpredation by Dileptus he reported thatEuplotes were protected by their stiffpellicle, which he calls a lorica, but laterstudies have found they are not so invul-nerable. However, they have some pro-tection in the form of induced defenses– responses triggered by the presence ofpredators. Taken at their broadest induced de-fenses include simple behaviour chang-es, such as moving in patterns that aremore difficult to follow. In the Euplo-toides subgroup, though, largeramounts of certain predators also in-duce changes in size and shape. Thecells delay reproduction to develop into

larger ‘winged’ forms, with extendedsides and prominent dorsal and ventralkeels, making them more difficult toingest. This reaction is tailored to the pred-ator. Daphnia and Amoeba are just ascapable of eating winged cells, and atbest weakly induce them, althoughsome species do have an avoidance re-sponse to the latter. The shape changeis an effective defense, though, againsttypes with limited mouths like the cili-ate Lembadion or flatworm Stenosto-mum. These are sensed at a distance viadissolved proteins, while for the part-time predator Stylonychia, winged cellsare only induced by repeated contact. A similar induced shape change isknown in the stylonychine Sterkiella,and Aspidisca lynceus have been foundto develop dorsal spines, something for-merly considered characteristic of aseparate species A. turrita. The raretype Styxophrya quadricornuta (for-merly in Onychodromus) is interestingin showing both cannibalism and a de-fensive form with enlarged spines,which can be induced by its own giantclones.

Chemical Defenses

Still other defenses have been foundin marine euplotids. A number of spe-cies here are eaten by the litostomesAmphileptus marinus (formerly as ma-rine Litonotus lamella). Like the morefamous Didinium, which mainly eatsparamecia, these are highly specialistpredators; their preferred prey will at-tract them from even a few centimetresaway, while most other ciliates are ig-nored. In fact A. marinus discriminate be-tween even closely related species.They rapidly ingest Euplotes crassusand other Moneuplotes, but while E.charon attract them they are not eaten,and others like E. rariseta are avoided.Euplotidium may also be eaten, but notEuplotidium itoi, which host very pecu-liar bacteria called epixenosomes thatkeep them safe by exploding.

Figure 4: Probably Tetmemena4


BACKGROUNDCiliate classification

For the others the difference in pal-atability has to do with what com-pounds they produce. Protozoans areunderstandably difficult to investigatein this respect, and most of what isknown concerns the pigments and de-fenses of heterotrichs. However, spe-cial attention has been given to marineEuplotes and several different defensivecompounds have been characterized. These compounds are all terpenoids,a very large group of compounds thatincludes many familiar aromas, fla-vours, toxins, and even basic membranecomponents from bacteria, plants, sea-weeds, fungi, and animals. All of themare built by repeating a basic unit of fivecarbon atoms, but from this there arisean incredibly diverse range of molecu-lar structures. Different Euplotes haveeach been found to have their own pre-viously unknown type, and most alsoshow slight variation between regions,which might be an indication of crypticspecies. The common species E. crassus aresomething of an exception as strainsfrom all over the world have mostlybeen found to produce the same euplo-tins. While these do not protect themfrom A. marinus, they are apparentlystrong contact toxins against other Eu-plotes that might compete with them,impairing calcium ion pumps that havea key role in movement and maintainingthe balance of the cell. There have also been a number ofpapers investigating the toxicity of eup-lotins toward pathogens and cancer celllines, obviously with an eye towardpossible drug design. It is a very longjourney from those sorts of investiga-tions to any sort of useful treatments,and few ever make it. I mention, them,though, because such explorations arestill an important source of leads, and itis interesting to watch these ciliates andconsider them someday saving peoplethe way Penicillium and Streptomyceshave. There is certainly much more to beexplored. So far as I know, the onlyother compounds that have been exam-ined are red pigments in two marinestichotrichs, Pseudokeronopsis rubraand P. riccii. These are alkaloids, relat-ed but somewhat different and with

nothing at all to do with terpenoids.Considering this, you can see how farwe are from even knowing how muchmore diversity there might be to discov-er here.

References

van Leewenhoek, A. (1677). Observa-tions, communicated to the publisherby Mr. Anton van Leewenhoek, in aDutch letter of the 9th of Octob. 1676.Philosophical Transactions, 12, 821-831.

Visscher, J. P. (1923). Feeding reac-tions in the ciliate, Dileptus gigas, withspecial reference to the function oftrichocysts. Biological Bulletin, 45(2),113-143.

Kusch, J. (1995). Adaptation of induc-ible defense in Euplotes daidaleos (Cil-iophora) to predation risks by variouspredators. Microbial Ecology, 30(1),79-88.

Wicklow, B. J. (1997). Signal-induceddefensive phenotypic changes in ciliat-ed protists: Morphological and ecologi-cal implications for predator and prey.Journal of Eukaryotic Microbiology,44(3), 176-188.

Berger, H. (1999). Monograph of theOxytrichidae (Ciliophora, Hypotri-chia). Dordrecht, The Netherlands:Kluwer Academic Publishers.

Tuffrau, M., Fryd-Versavel, G., Tuf-frau, H., & Génermont, J. (2000). De-scription of Euplotes versatilis n. sp., amarine tropical ciliate exhibiting anunusually extensive phenotypic plastic-ity. European Journal of Prostistology,36(4), 355-366.

Wiąckowski, K., Fyda, J., & Ciećko,A. (2004). The behaviour of an omniv-orous protozoan affects the extent ofinduced morphological defense in aprotozoan prey. Freshwater Biology,49(6), 801-809.

Görtz, H.-D. (2006). Symbiotic associ-ations between ciliates and prokary-otes. The Prokaryotes, 1, 364-402.

Foissner, W., Moon-van der Staay, S.Y., van der Staay, G. W., Hackstein, J.H., Krautgartner, W.-D., & Berger, H.(2004). Reconciling classical and mo-lecular phylogenies in the sticho-trichines (Ciliophora, Spirotrichea),including new sequences from somerare species. European Journal of Pro-tistology, 40(4), 265-281.

Vannini, C., Lucchesi, S., Rosati, G(2007). Polynucleobacter: Symbioticbacteria in ciliates compensate for agenetic disorder in glycogenolysis.Symbiosis, 44, 85-91.

Achilles-Day, U., Pröschold, T., Day,J. G. (2008). Phylogenetic position ofthe freshwater ciliate Euplotes daida-leos within the family of Euplotidae,obtained from small subunit rDNAgene sequence. Denisia, 23, 411-416.

Lynn, D. H. (2008). The Ciliated Pro-tozoa: Characterization, Classifica-tion, and Guide to the Literature (3rd

ed.). Springer.

Rosati, G., Modeo, L., & Verni, F.(2008). Micro-game hunting: Predatorybehavior and defensive strategies inciliates. Microbial Ecology ResearchTrends, 65.

Guella, G., Skropeta, D., Di Giuseppe,G., Dini, F. (2010). Structures, biologi-cal activities and phylogenetic relation-ships of terpenoids from marine ciliatesof the genus Euplotes. Marine drugs,8(7), 2080-2116.

Guella, G., Frassanito, R., Mancini, I.,Sandron, T., Modeo, L., Verni, F., Di-ni, F., Petroni, G. (2010). Keronop-samides: a new class of pigments frommarine ciliates. European Journal ofOrganic Chemistry, 3, 427-434.

The author can be reached at:[email protected] ■


BACKGROUND Uses of microscopes for beekeeping

Focus on Bees

Microscopes are valuable tools for diagnosingColony Collapse Disorder in bees.

Charles Crookenden

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Much has been written aboutthe continuing disappearanceof the honey bee, the corre-

sponding demise of commercial bee-keeping and the various culprits thataccount for the decline or colony col-lapse disorder (CCD) as it is more fa-miliarly known.. Ask a dozenbeekeepers what is causing CCD andyou will receive as many responses:Pesticides, fungi, excessive antibiotics,poor husbandry, loss of habitat, cellphones or the inevitable repetition ofHistory. History? Noah Wilson-RichPh.D, the founder of Best Bees(http://www.bestbees.com/), has found

that honey bees have endured “great dieoffs” on a regular basis. This currentdecline may just be another manifesta-tion. It is a heated debate that continuesalongside a variety of initiatives tosolve the conundrum throughout USuniversities and research institutions. Meanwhile, the backyard beekeeper- the humble amateur - has quietly beenon the rise. While statistics are hard tocome by, the circumstantial evidence ismounting. In Virginia, for example, thenumber of hives has doubled over thepast decade. Local beekeeper clubs areseeing record enrollment thanks in partto the publicity surrounding Michelle

Obama’s hives in the White House gar-den. Williams Sonoma’s new Agrariancatalog focuses on beehives (and chick-en coops), while the rise of homestead-ing and the Local Food movement bothcontribute to beekeeping's increasingpopularity. All intertwine to encouragebackyard beekeepers. Kim Flottum, managing editor ofthe magazine Bee Culture(http://www.beeculture.com/) and along-time pillar of the beekeeping com-munity has maintained a combination ofstatistics from which he extrapolatesthat “backyard beekeeping has grown30-32% over the past seven years”. Heestimates that over half that growth hasbeen in the past three years. “Most ofthe bees are West of the Mississippiwhere the commercial beekeepers arebased. Most of the beekeepers are Eastof the Mississippi.” In other words, therecent increase in the number of bee-keepers is concentrated in the majorurban areas in the East, not least due tothe relaxation of city ordnances overbeekeeping. This confluence of urban beekeep-ers and the search for explanations intoCCD has led to another trend. Both thenewer urban beekeepers and the moretraditional, rural beekeepers are increas-ingly turning to more sophisticated ho-listic approaches to colonymanagement, to science and in particu-lar, to the practical application of themicroscope. The newer urban beekeep-er is more comfortable with such tech-nology while more traditionalbeekeepers are turning to microscopesdue to necessity. There is a growingawareness of the need for more accurate

Figure 1: Agave pollen

(Image by Gretchen D. Jones)

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BACKGROUNDUses of microscopes for beekeeping

and earlier diagnosis, of the benefits ofimmediate on site analysis and subse-quently of swifter, targeted treatment ofpotentially, disastrous, hive infections. Part of this growing acceptance ofmicroscopes among beekeepers is thedawning realization that microscopesare not as daunting as their High Schoolmemories suggest! In part, it is becausethe cost of microscopes has fallen. Thecost is also defrayed by savings on lab-oratory analysis fees and perhaps moreimportantly, by the savings implicit inearlier treatment that decreases the riskof CCD. Beekeeper clubs across thecountry are also investing in micro-scopes so that more beekeepers haveaccess to what previously, was not intheir individual budgets. Many beekeepers, however, still re-ly on experience and perception alone.With no diagnostic tools beyond theireyeballs, there is a tendency to diagnosehive infections at a more advanced stageof infection. As a result, many beekeep-ers apply heavier treatments than is nec-essary with lower rates of success andhigher risks of long-term resistance tothe drugs. Worse, many beekeepersdose their hives with antibiotics with noidea of the nature and scale of the prob-lem at all. Randy Oliver(http://scientificbeekeeping.com/), aleading guru at the forefront of the jux-taposition of beekeeping and science,started an article in 2012 with, “It isgreatly surprising to me […] how few(beekeepers) make the effort to monitorthe levels of parasites in their colonies!Even more surprising is that, despite theconsiderable expense, many blindlytreat their colonies without any idea asto whether their bees are actually infect-ed!” At a 2011 Eastern Apiculture Soci-ety conference, the author encountereda surprising number of beekeepers whoconfessed to this type of blind dosingevery spring, “just in case” their hiveswere infected. When asked if they dothe same with their children, theylooked puzzled!

So, to what end are beekeepers us-ing microscopes? First and foremost, acompound microscope is a highly effec-tive diagnostic tool for infections. Forexample, the fungal infection of Nose-ma ceranae has proved particularlydeadly over the past few years. It hasbeen a prime suspect in the search for anexplanation for CCD. Traditionally,beekeepers have responded to Nosemainfections without an accurate feel forthe stage or degree of infection. Nowwith a gut sample that is easily preparedon site in the beeyards, they can self-diagnose using a rechargeable LEDcompound microscope at 400x magnifi-cation. The results are immediate. TheNosema ceranae spores look like horseracing ovals and stand out like beacons.With the addition of a simple hemocy-tometer, the beekeeper can gain an ac-curate spore count and, therefore, amore accurate picture of the degree ofinfection. Within five minutes, the bee-keeper has gone from pure guessworkto an informed opinion.

Pollen analysis is another commonuse for a compound microscope. Me-lissopalynology or the study of pollen inhoney, is not just for the laboratoryexperts. A simple compound micro-scope can help identify the dominantpollen in any beekeeper’s honey, al-though with 250,000 different plantsused by the honey bee in the US, mostbeekeepers may need some point ofreference for accurate identification.Pollen analysis helps ensure correct la-beling while it is also commonly used inforensic analysis, archeology and purehoney research. Low power stereo microscopes arealso used for colony management. Forexample, almost all beehives sufferfrom mite infections. While eyeballingworks well for seeing if mites are pres-ent, a stereo microscope is useful todetermine the mite's identity and there-fore, what treatment is required. Otherapplications include basic anatomy ofthe honey bee, training and for the moreadvanced practitioner, artificial insemi-nation of the Queen Bee. More recently,

Figure 2: Asteraceae pollen,Porophyllum scoparium.

(Image by Gretchen D. Jones)

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Figure 3: Honey bee mid- and hindgut

Figure 4: Bee hair, Nosema ceranaespores (white ovals) and pollen(larger)

(Images by Randy Oliver)

BACKGROUND Uses of microscopes for beekeeping

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Dino-Lite digital microscopes(http://www.microscope.com/digital-microscopes/dino-lite/) have proveduseful tools given their hand-held na-ture, greater portability and wider rangeof magnification than the typical stereomicroscope. Microscopes are but one tool in thebeekeeper’s armory to combat the manyvicissitudes that face bee hives. Theyare a small tool within the context of thework that needs to be done in order toresolve the plight of the honey bee inNorth America. However, they do offersome hope not least because they aresimple to use, affordable and widelyavailable for even the newest beekeep-er. They are an enabling technology thatcan lead to improved colony manage-ment - which in itself may lie the solu-tion to the demise of the honey bee.

Author’s Biography

Twitter: @microscopesBlog: In The Loupe(http://www.microscope.com/blog/)

Charles Crookenden is president ofMicroscope.com, an internet retailer ofmicroscopes(http://www.microscope.com), based inrural Virginia. Microscope.com spon-sors a training course at the annual EASbeekeepers’ conference.

Image Credits

Nosema ceranae images, Gut sampleof beebread, Mid- and hind gut sample:Courtesy of Randy Oliver(www.scientificbeekeeping.com):

Pollen samples (Aster and Agave):Courtesy of Gretchen D. Jones, Ph.D.,United States Department of Agricul-ture, Agriculture Research Service, Ar-ea-wide Pest Management ResearchUnit ■

Figure 5: Gut sample of beebread

Figure 6: Nosema ceranae spores

(Images by Randy Oliver)

BACKGROUNDUses of microscopes for beekeeping

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12 - MicrobeHunter Microscopy Magazine - March 201212 - MicrobeHunter Microscopy Magazine - July 2013 - Send images to [email protected]

GALLERY

Meiji MX5000L, Abbe Condenser, S ApoPlan 20X NA0.65, 2.5X photo eyepiece,BrightfieldCanon 40D 100 ASA , cropped

Images by Marc Bos

Bdelloidea

Bdelloidea is a class of rotifersfound in fresh water and moist soil.Bdelloids typically have a well-de-veloped corona, divided into twoparts, on a retractable head. Theymay move by swimming or crawl-ing. The latter commonly involvestaking alternate steps with the headand tail, as do certain leeches,which gives the group their name(Greek βδελλα or bdella, meaningleech). Bdelloids have been of interestto those interested in the evolution-ary role of sexual reproduction, be-cause it has disappeared entirelyfrom the group: males are not pres-ent within the species, and femalesreproduce exclusively by partheno-genesis. Each individual has pairedgonads. Despite the fact that theyhave been asexual for millions ofyears, they have diversified intomore than 300 species and arefairly similar to other sexually repro-ducing rotifer species. Bdelloids respond to environ-mental stresses by entering a stateof dormancy known as anhydrobio-sis. This dormancy form enablesthe organism to rapidly dehydrateitself. The Bdelloid will remain inthis cysted state until optimal envi-ronmental conditions re-occur atwhich point they will rehydrate andbecome active within hours. Dia-pause is the ability of the organismto produce offspring in a dormantand unhatched state[citation need-ed]. Hatching of the young will onlyoccur when conditions are at theirmost favourable. These forms ofdormancy are also known as cryp-tobiosis or quiescence.

MicrobeHunter Microscopy Magazine - March 2012 - 13Send images to [email protected] - July 2013 - MicrobeHunter Microscopy Magazine - 13

GALLERY

When these unusual creatures spring from hiberna-tion, they undergo a fascinating and possibly uniquegenetic process. A study conducted by Matthew Mesel-son of Harvard University suggests that when bdelloidrotifers recover from suspended animation, they incor-porate foreign DNA when patching up their own rup-tured cell membranes. Any DNA in proximity to theorganisms can be included in the new genome, includ-ing semi-digested food. This may be interpreted as anintermediate between true asexual and sexual repro-duction.

Bdelloid rotifers have recentlybeen shown to be extraordinarilyresistant to damage from ionizingradiation. The same DNA-preserv-ing adaptations used to survive dor-mancy are thought to work in thiscase, and may have also helped theorganisms to thrive despite their to-tally asexual mode of reproduction.

Source:

"Bdelloidea." Wikipedia. WikimediaFoundation, 08 May 2013. Web. 13Aug. 2013.


TECHNIQUES Marking slides for microarray analysis

Summary

Arrayers are instruments used toprepare tissue microarrays which allowthe assessment of hundreds of tissuespecimens on a single glass slide. Tis-sue microarrays consist of the manufac-ture of a donor, from which tissue-coresare collected, and recipient blocks. Thepresent work describes a method forspecifying and locating the region ofinterest from which the tissue-cores aretaken to be inserted in the recipientblocks. The device is a modified micro-scope that was used to mark the re-quired region of interest on theexamined glass-slide of the specimen.The marked slide is then matched withthe surface of the donor block to locatethe region of interest from which tissue-core(s) are to be taken. The device canbe adopted by all laboratories and re-searchers with minimal financial invest-ment.

Introduction

Arrayers are instruments used toconstruct tissue microarray slides. Tis-sue microarrays are a recently devel-oped technology, which allows theassessment of hundreds of tissue speci-mens on a single glass slide. The tech-nique was introduced by Kononen et al.(1998). In oncology, microarray tech-nology was used to characterize andanalyze the frequency of a molecularalterations in tumors of multiple histo-logical types (“multitumour microar-rays”, Bhargava et al., 2005) and has ledto the identification of hundreds ofgenes with potential roles in cancer orother diseases (Simon et al., 2005). Thetechnique, moreover, was used to eval-uate the prognostic markers in tumors

from which clinical follow-up data areavailable (“prognostic microarrays”,Hans et al., 2005; Rakha et al., 2005;Korsching et al., 2005; Hadjd et al.,2005), to test the potential diagnosticmarkers of tumours (“diagnostic mi-croarrays”, Ruiz-Ballesteros et al.,2005), and to study the progression oftumours in samples of different stageswithin a given organ (“progression mi-croarrays”, Fillies et al., 2005; Cho etal., 2006; Resau et al., 2006) withoutmajor changes in protocols used in con-ventional histological methods. All thehistochemical and molecular detectionmethods that can be used with regularsections can also be used with tissuemicroarrays. Tissue microarrays werealso used to prepare reference slides for

Direct Specifying of the Region of Interest forPreparation of Tissue Microarrays

Salah Deeb, Mahmoud El-Begawey,Khalid El-Nesr, Emad Mahdi

Department of Pathology, Faculty of Veterinary Medicine Beni-Suef,University of Beni-Suef, Egypt

Figure 1: Student microscope usedFigure 2: Removal of microscope tube, eyepiece and objectivesFigure 3: Insertion of a flow-marker pen to mark the region of interest.

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TECHNIQUESMarking slides for microarray analysis

educational purposes and for research(Deeb et al., 2012). Array instruments of different typesare already available on the market.These arrayers are either manual(Beecher Instruments, Gendiscovery-Biotechnology, Chemicon Internation-al), modified manual arrayers to whicha robot is attached (Biegler GmbH),semiautomatic (MTABooster), or fullautomatic type(Beecher Instruments).In all techniques, recipient blocks areprepared to insert core of a dimension offew millimeters located at an area ofinterest cut from a donor blocks. A region of interest (ROI) is a portionof an image that you want to filter orperform some other operation on and intissue microarrays it depends mainly onsampling. The aim of the present work was tomodify the microscope to facilitatespecifying and locating the area of inter-est from which cores are taken.

Material and Methods

Device: Our device consisted of amedical student microscope (Fig. 1) orany research microscope. The eyepiece,microscope tube and the objectives.The condenser and diaphragm were alsoremoved (Fig. 2). The microscope armwith coarse and fine focusing knobs,mechanical stage with the micrometerand the microscope base comprise themain components of our device. Specifying of the region of interest:To determine the region of interest, puta stained slide of the specimen andexamine it by the microscope to deter-mine the area of interest before de-as-sembling. The region of interest ismoved to the center of the field. Re-move the microscope tube and one ofthe objective lenses (4X lens) and inserta marker pen in the empty hole of thequadruple revolving nosepiece which isnow devoid of one lens to touch thesurface of the examined slide (Fig. 3) tomark the interested area. Locating the region of interest at thethe donor block: A flow-master pin isinserted in the center of the revolvingnosepiece (turret) sufficiently deep totouch the surface of the glass-slide (con-taining the section. With a gentle round-

ing of the pin while it is in place, an inkdot is marked at the region of interest.Match the surface of marked slide withthat of the surface of the donor block.Using a puncture and stylet of requireddiameters (0.6 mm, 1.0 mm, 2.0 mm ) atissue-core with its embedding paraffinis extracted. The method used for prep-aration of tissue microarrays was princi-pally the same as mentioned elsewhere(Deeb et al., 2006).

Results and Discussion

Using our device, you can specifyROI directly on the slide by using amicroscope to mark the required area,and by matching with the paraffin donorblock samples will correspond to thesame area and size as the image youwant to process. In histology, histopathology and im-munohistochemistry, the regions can begeographic in nature, such as polygonsthat involve more than one site withdifferent structures to get them in oneand the same microscopic field. Usingour device, the region of interest hasbeen simply and exactly specified. Tis-sue cores extracted from these regionswere used to make tissue microarrayswith different formats including highdensity (120 specimens per slide, 0.6mm in diameter), low-density microar-rays (60 speciemens per slide, 1.0 mmin diameter) and a trial-size arrays (10-20 specimens per slide, 2.0 mm in diam-eter). Cores specified and located by ourmethod were proved to be representa-tive to the microscopic field determinedearly. This new tool enables researchersto perform cost and time efficient mi-croarrays and throughput microscopicanalysis using various histologicalprobes of normal and abnormal tissue. Monitoring of the region(s) of interestis of special interest in pathologicalstudies (tumors, lesions of human andanimal diseases) and constitute the basisfor determining lesions, their reaction,and relation to the surroundings to reachto reliable diagnosis. Our device can be adopted by smallall laboratories and researchers withminimal financial investment.

References

Bhargava, R., O.Oppenheimer, W.Gerald, S.C.Ihanwar and B.Chen (2005) Identification ofMYCN gene amplification in neuroblastoma us-ing hromogenic in situ hybridization (CISH): Analternative and practical method. Diagn. Med.Pathol., 14: 72-76

Cho,Y. G., B. J. Choi, J. W. Song, S. Y. Kim, S.W. Nam, S. H. Lee, N. J. Yoo, J. Y. Lee and W.S. Park (2006) Abberant expression of krUppel-like factor 6 protein in colorectal carcinoma.Wrld J.Gastroenterol., 14: 2250-2253.

Deeb, S., Kh El-Nesr, M. ahdi, A. Shalaby andM. El-Begawey (2012) Technology and applica-tions of tissue microarrays (a review). Beni SueifJ.Appl.Sci., I(1): 73-85

Fillies, T. H. Buerger, c. Gartner, C. August, B.Brandt, U. Joos and R. Werkmeister (2005)Catenin expression in T ½ carcinomas of thefloorof the mouth. Intern J. OralMaxillofac.Surg., 34: 107-111.

Hadjd, Z. , I.Spendlove, S. E. Pinder, I. O. Ellisand L. G. Durrant (2005) Total loss of MHCclass I is an independant indicator of good prog-nosis in breast cancer. Intern. J. Cancer, 117:248-255.

Kononen, J., L. Bubendorf, A. Kallioniemi(1998) Tissue microarrays for high throughputmolecular profiling of tumor specimens. Nat.Med., 4: 844-847

Korsching, E., J. Packeisen, C. Liedtke, D. Hun-germann, P. Wulfing, P. J. Van Diest, B. Brandt,W. Boecker and A. Buerger (2005) The origin ofvimentin expression in invasive breast cancer:epithelial-mesenchymal transition, myoepithelialhistogenesis or histogenesis from progenitor cellswith bilinear differentiation potential. J. Pathol.,206: 451-457

Rakha, E. A., D. Abd-El-Rehim, S. E. Pinder, S.A. Lewis and I. O. Ellis (2005) E-cadherin ex-pression in invasive non-lobular carcinoma of thebreast and its prognostic significance. Histopa-thology, 46: 685-693.

Ruiz-Ballesteros, M., A. Mollejo, A. Rodriguez,F. I. Camacho, P. Algara, N. Martinez, M. Pol-len, A. Sanchez-Aguilera, J. Menarguez, E. Cam-po, P. Martinez, M. Mateo and M. A. Piris(2005) Splenic marginal zone lymphoma: Pro-posal of new diagnostic and prognostic markersidentified after tissue and cDNA microarrayanalysis. Blood, 106: 1831-1838.

Resau, J. H., R. Miller, J. B. Kaneene, V. Yuz-basiyan-Gurkan, J. D. Webster and M. Kiupel(2006) The role of c-KIT in tumorigenesis: Eval-uation in canine cutaneous mast cell tumor.Neoplasia, 8: 104-111.

Simon, R., M. Miralcher and G. Sauter (2005)Tissue microarrays. Methods Mol. Med., 114:257-268

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During my investigations of themicroscopic world I often trylittle sideline experiments, to

either get a better image of something orprepare a slide of it. Here are a coupleof techniques I have found useful toimprove images recorded with a USBeyepiece camera.

Lighting and Imaging

Although my microscope has a verygood LED based lighting system and

presents excellent views of thin sec-tions, thicker specimens do not alwayslet sufficient light through to record agood image with the camera. If the lightsource is turned up high enough to pen-etrate parts of the sample, the darkerareas are washed out by stray light inthe optics. Turning off the below-stagelight and illuminating from the top withan LED torch/desk-lamp (Figure 1) cangive quite nice dark-field type images.However, this can often result in strongdazzling reflections from shiny parts of

a specimen, a bit like when you look atthe sea on a sunny day. While thehuman eye actually compensates verywell for less than ideal illumination con-ditions, digital cameras fair less well,especially USB eyepiece cameras suchas mine. There is not a lot of controlover the exposure and its AutomaticWhite Balance (AWB) feature (Figure2) can give some bizarre results forspecimens of predominantly one colour,at best they end up rather black and

Imaging Techniques

Here are a few techniques to improve the imagesrecorded with a USB eyepiece camera.

Neill Tucker

Figure 1: Microscope & desk-lamp

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TECHNIQUES Lighting and white balance control


Figure 2: Figure 2 USB camera con-trol screen. Here white balance andother parameters can be controlled.

Figure 3: Default exposure settingsand the Auto White Balance (AWB)on.

Figure 4: Calibrated and AWB off.

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TECHNIQUESLighting and white balance control

2


white, at worst they can end up a com-pletely different colour! Dealing first with the white balanceissue. I have used a broad-leafed bladeof grass at 40x as an example; the imagein Figure 3 is with the default exposuresettings and the AWB on. As you cansee the image has some green colouringbut not really what I was seeing downthe eyepiece. I don’t know if other USB camerashave the same feature, but mine doeshave an AWB on/off option; if yourshas this option you may also be able touse the following procedure: With theAWB option ‘on’, turn down the lightsource to minimum, remove the slideand slowly increase the illumination toa comfortable viewing intensity. Theninsert the eyepiece camera and let itadjust, the screen should be predomi-nantly white/grey. Finally set the AWBoption to ‘off’ and place the slide backunder the microscope. You will proba-bly need to increase the illuminationlevel again, but the image colour shouldnow be much closer to what you seedown the eyepiece, see Figure 4. Other software may be different, butI found that this ‘calibrating’ of thewhite balance does seem to give betterresults than just turning the AWB offand adjusting the colour temperature orsaturation slider. Although the slidersdo vary the colour, they are not as effec-tive. Many digital cameras have userdefined white balance options that in-volve taking a picture of a piece ofwhite paper under the light you will beusing for the photo, it might be worthchecking to see if yours has this func-tion.

Exposure

The next issue, as I mentioned, waswith exposure. Looking at the edge ofthe grass with only sub-stage lightingresults in high contrast silhouette andvery little colour, see Figure 5. Usingonly above–stage lighting gave a bettercolour, but localised reflections resultedin overexposed patches, see Figure 6.

Figures 5 - 8: Comparison of differentlighting methods

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6

7

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TECHNIQUES Lighting and white balance control

Below-stage light only

Above-stage light only

Above & below-stage light

Below & diffuse above-stage lighting


However, by using a combination ofthe two lighting methods a very agree-able image can be produced for directviewing or recording with the camera,Figure 7. If your above-stage light hasno intensity adjustment or it is tooharsh, try covering the lamp with somegrease-proof paper to diffuse the light,this can further improve the image, Fig-ure 8. The lighting arrangement used forFigure 8 is shown in Figure 9. Note:Only use the paper cover with LEDlamps, other types may set light to thepaper! You may find that your above andsub-stage light sources have differentcolour temperatures, LED lights tend tobe ‘hot’ and near the blue end of thespectrum while tungsten lamps are‘cooler’ and tend to make things lookmore orange. Even LED lamps havesome variation; in Figures 7 & 8 thewhite balance calibration was for the‘cooler’ above-stage lamp, making thebackground from the below-stage lightappear pale blue. This could be correct-

ed to some extent using the white bal-ance slider; however, I found thesky-blue background quite pleasing tothe eye and the specimen colour reason-ably accurate, without further adjust-ment. To set the white balance for theabove-stage illuminations, put a pieceof white paper on the stage and use onlythe above-stage lamp while letting theAWB adjust during the ‘calibration’procedure mentioned above; for bestresults it is better if the paper is not infocus. I have used above-stage lightingat up to 100x but much higher than thisand the working distance of the objec-tive is too close and its aperture toosmall to let sufficient light in. The final thing to note is the vignett-ing (darkening around the edge of theexample images) which is due to thelack of optics to match the USB camerato the microscope. More expensivemodels or digital SLR camera adaptersgenerally have additional lenses to re-duce this. If it is bothersome then some

correction can be done in Photoshopalthough it is sometimes easier to justcrop the image. Although my experiences are pri-marily with a USB eyepiece camera,hopefully these techniques of adjustingwhite balance and optimising illumina-tion will find application with othermicroscope/camera arrangements aswell.

Equipment used

Camera: APEX minigrab USB camera with Minisee software supplied.

Microscope: APEX Scholar trinocular

Desk-lamps: IKEA Jansjö

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Figure 9: Microscope above-stagediffuse lighting

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TECHNIQUESLighting and white balance control


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