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MicrobeHunter Magazine - April 2011 - 1

MicrobeHunterMicrobeHunter

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

Volume 1, Number 4April 2011

The Magazine for theEnthusiast Microscopist

http://www.microbehunter.comMicroscopy Magazine

DIY Centrifuge Restoration Photomicrography

2 - MicrobeHunter Magazine - April 2011

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

Volume 1, Number 4, April 2011 (revision 2)

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

Download: Microbehunter Microscopy Magazine can be down-loaded at: http://www.microbehunter.com

Print version: The printed version can be ordered at:http://microbehunter.magcloud.com

Publisher and editor:Oliver Kim, Ziegeleistr. 10-3, A-4490 St.Florian, AustriaEmail: [email protected]: http://www.microbehunter.comTel.: +43 680 2115051

Text contributions by, alphabetically:Robert F. Hancock,G. Joseph WilhelmVíctor Rafael Zárate-RamírezOliver Kim

Image contributions by:Robert F. Hancock,G. Joseph WilhelmVíctor Rafael Zárate-RamírezLaura Paulina Barrera MuñozLeslie Hernández RamírezClaudia Peña FabiánEmmanuel Alejandro Ramírez MoralesOliver Kim

Copyright: By submitting articles and pictures, the authorshave confirmed that they are the full copyright owners of the ma-terial. Creative commons and public domain images are indicat-ed with a small text next to the image. The copyright of all otherimages is with the author of the article. You are not allowed todistribute this magazine by email, file sharing sites, web sites orby any other means.

Editorial: Article and image submissions are welcome andshould be sent to: [email protected] submission guidelines, consult the website at:http://www.microbehunter.com/submission

Disclaimer: Articles that are published in Microbehunter Micros-copy Magazine and the blog do not necessarily reflect the posi-tion or opinion of the publisher. The publication of these articlesdoes not constitute an endorsement of views they may express.Advice provided in Microbehunter Microscopy Magazine is pro-vided as a service and neither the authors nor the publisher canbe held liable and responsible for any errors, omissions or inac-curacies, or for any consequences (health, hardware, etc.) aris-ing from the use of information of this magazine and the blog (oranything else). Conduct all lab work and (microscopy) hardwaremodifications at your own risk and always follow the instructionsof the manufacturers.

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ABOUTANNOUNCEMENT

Write for Microbehunter!Please contribute both articles and pictures. Share your expe-riences, problems and microscopic adventures. If you are aresearcher using microscopes, tell the readers what your re-search is about. Please contribute, even if you consider your-self inexperienced. If you are a struggling beginner, tell ussomething about the problems that you encountered. If youare an active enthusiast microscopist then share your proj-ects, experiences and observations. Are you a teacher or lec-turer? Share your microscopic experiences from school oruniversity. This magazine is made by an enthusiast microsco-pist for other enthusiasts. Let‘s work together to make thisproject a successful one.Please send all contributions to:[email protected]

You must own the copyright of the contributions and you re-tain the copyright of all submitted articles and pictures. Whilewe are not able to pay you for your efforts, we will, of course,give you full credit for your contributions.

Guest Bloggers! Yes, guest blogging is also a possibility.Write microscopy-related blog posts, send them to me and Iwill publish them on the web site. Naturally, I’ll put a link toyour blog. Condition: it must be original content and you mustbe the copyright holder of the text (obviously). When submit-ting articles, please indicate if you want to have them pub-lished on the blog or in the magazine (or both).

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Front Cover:large image: Oliver Kim (Aristolochia stem)Left image: Robert F. Hancock;Middle: Joseph Wilhelm;Right: Maria Guadalupe Hernández Luna

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TABLE OF CONTENTS

EDITORIAL

We're now into thefourth month of publi-cation and it even sur-

prises myself on how manydifferent approaches there are toamateur microscopy. In this issueyou can read about the construc-tion of centrifuge made of VCRcomponents (page 4 ) as well asthe construction of a refractome-ter (page 20), which can be used

to measure the refractive index ofmounting media. Other amateursare heavily engaged into metalworking, which becomes usefulwhen restoring antique micro-scopes (page 8). Placing a stron-ger emphasis on the observationpart of microscopy, there is anarticle with contributions fromhigh school students from Mexi-co (page 24 and 28), which docu-

ments beautifully how stereomicroscopes can be used in aneducational setting. One neverstops learning: or did you knowthat some wasps lay eggs intoliving aphids in order to repro-duce? In any case, I hope that youenjoy reading this month's issueas much as I did.

3 Editorial

4 A Simple Centrifuge for MicroscopistsCommercial centrifuges can be expensive,

why not try to make your own?Robert F. Hancock

8 The Novitiate’s Odyssey: Episode ThreeExperiences and lessons of restoring antique

microscopes.Joseph G. Wilhelm

16 Introducing: The HemocytometerHaemocytometers are specialized specimen

slides that are designed to quantify the cell density.

Oliver Kim

20 A Laser RefractometerIt is possible to make one yourself to measure

the refractive index of different mounting media.

Robert F. Hancock

24 Photomicrography Course in MexicoFrom Mexico, sharing our love of microscopy

to young scientist aspirants.Víctor Rafael Zárate-Ramírez, M.Sc.

28 Parasitoid wasps: A Photomicrographic Survey of their Life Cycle in the Laboratory

In this contribution we describe the life cycle of Aphidius colemani, a parasitoid wasp.

Víctor Rafael Zárate-Ramírez et al.

8

16

24

Answer to the puzzle (back cover):Banana (squeezed between coverglass and slide, stained with ink)

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A centrifuge is a very usefulpiece of equipment for isolat-ing aquatic organisms. Centri-

fuging a sample of water containingmicroorganisms will drive them to thebottom of the container, where theybecome more concentrated and easier tomanipulate. A high speed laboratorymodel costing several hundreds of dol-lars isn't necessary for the purpose,something much simpler will do the jobquite adequately. At the most basic, youcan just tie a piece of cord securely tothe container and whirl it around yourhead. You can easily generate a centrif-ugal force of 10G with this method (butdo it outside in an open area, wherenothing will get damaged if it comesloose!) A safer and more convenient solu-tion is a motor driven apparatus, such asthe one I built using parts from an oldvideo recorder. It cost me only a coupleof dollars for the plastic bowl housingthe rotor head and some PVC pipe fit-tings, all the other parts were obtainedfrom my “junk collection” of defunctVCRs and other “dead” electronic gad-gets. If you don't have a dead VCRlying around, just ask a few friends andyou are bound to find someone who willgive you one. The parts required are thevideo recorder spinning head mecha-nism, plus the video cassette loadingmotor with its drive pulleys. Some new-er VCR models may not have beltdrives, a good alternative source forthese components is an old inkjet print-er, but these usually have stepper mo-

In many cases it can beuseful to enrichmicroorganisms that can befound in a water sample.Commercial centrifuges canbe expensive, why not try tomake your own?

Robert F. Hancock

Fig. 1 (top): VCR rotor top viewFig. 2 (bottom): VCR rotor bottom view

DIY


Fig. 3 (top): Rotor with flywheel re-movedFig. 4 (bottom): Disassembled rotor

tors which require a special drivercircuit to power them. If you can find anordinary DC motor which will fit thepulleys it is much simpler than trying toget the stepper motor working. You willalso need a 12 Volt DC plug pack froman old portable phone charger or similardevice. It should be capable of supply-ing at least 400 milliamps, otherwise itwill probably burn out before long. Ifyou need to, you can buy a new one forabout $15, but these are very commonitems found in garage sales for only oneor two dollars.

The VCR parts were extracted alongwith their mounting brackets andscrews, keeping the PCB with the cas-sette motor drive connections intact.The wiring to the video head rotor is notneeded and can be disconnected. Figs 1and 2 show the video head as removedfrom above and below, Figs 3 and 4show the disassembled rotor compo-nents, and Figs 6 and 7 the reassembledconfiguration. The first step is to re-move any superfluous bits from the vid-eo head rotor, including the PCB andthe stator windings underneath (Fig 3).

You need to take off the flywheel withthe magnetic ring to get at the inside ofthe rotor. At this stage, prepare an oldCD by cutting out the centre section tofit over the bottom section of the rotor.Drill 3 small holes in the CD and thebottom rotor section to take small selftapping screws and fix the CD to thebottom rotor section as shown in Fig 5. The two halves of the rotor are sep-arated and the top section refitted upsidedown, as shown in Figs 6. The magneticsensor heads and their wires can also bediscarded. Save the screws from theunderside (which is now the upper side)of the top rotor section. Replace theflywheel also in the reverse orientationto its original position. You should nowhave an assembly which looks like Fig7, seen with the assembly turned upsidedown. Depending on the model of VCR,you should have a mounting frame at-tached to the video head rotor with 3 or4 screw fixing points. You will need tomake up a base with some brackets orposts to allow the rotor to be mounted ina horizontal position. In the video re-corder, the rotors are mounted at anangle to the horizontal to allow the tape


Fig 5 (top): CD attached to rotorFig 6 (bottom): Reassembled rotorand CD

to be scanned diagonally, so the mount-ing points will usually need to be atvarying heights to get the rotor levelwhen it is used as a centrifuge. Try toget it as accurately level as possible,otherwise your centrifuge will wobblewildly when it is running. I used mdfboard for the base and two posts, and ametal bracket for the third mounting.You can use some washers under themounting frames to adjust the level ac-curately with a spirit level / bubble level. When the rotor assembly is mountedon the base board, the drive motor andits pulley will need to be located so that

the drive belt runs around the detach-able collet just above the reversed fly-wheel. The driving pulley and colletmust be aligned in the same plane, oth-erwise the belt will be thrown off whenthe motor starts. Since the drive motorand pulley configuration will varygreatly between different models ofVCR, I will not provide any specificdetails for this step, you will have tofigure it out to suit whatever you areusing. Fig 8 below shows how I ar-ranged mine. By the way, if you don'thave a drive belt of suitable length, astrong rubber band will also work.

The next step is to cut a hole in thebase of the centrifuge bowl to fit overthe rotor, and then fix it to the CDunderneath with some small screws.Figs 9 & 10 show the bowl fitted inplace. Make sure the bowl is accuratelycentred so that the spinning tubes cannot touch the sides of the bowl. ( Ofcourse, you need to get a bowl of theright diameter to accommodate yourcentrifuge tubes in the first place. I useda plastic microwave cooking cover ) To complete the centrifuge you willneed to add the trunnions and rings tocarry your centrifuge tubes. The tubes Iuse are 100 mm x 12 mm diameter, so Imade two tube carriers from black plas-tic irrigation tubing and fitted them withtrunnions as illustrated in Fig 11. Thetrunnions are formed from a threadedPVC collar and two self tapping screws.A piece of foam rubber is pushed intothe bottom of the tube carriers to cush-ion the glass tubes, and another screw isinserted across the bottom to hold thetubes in place. The aluminium bracketscrewed to the top of the rotor has holesfor the ends of the trunnion screws, asseen in Fig 12. To use the centrifuge, I fill the cen-trifuge tubes to about 15 mm from thetop with the liquid, making the levels ineach tube as near to equal as possible.They are inserted into the carrier tubesand a piece of thin mdf board is placedon top of the bowl as a lid, which helpsto reduce the wind resistance. The cen-trifuge is run for about 5 minutes at 250rpm, which generates a force of about 6G. It will need to be clamped to thework bench so that it doesn't run aroundand develop uncontrollable vibrations. Should anyone wish to have a vari-able speed version, the simplest methodis to buy a 12V LED light dimmercontrol. These are available on eBay forunder $10. I have tried one of these andit works perfectly. ■


17

Fig. 12: Completed centrifuge

Fig. 7: Reassembled rotor, bottom view Fig. 8: Drive motor, pulley and belt.

Fig. 9: Top view of centrifuge bowl Fig. 10: Side view, bowl in place.

Fig. 11: Tube carriers and trunnions


Greetings once again from theland where the average fellowhas more fishing rods than

pairs of pants and if you put twenty ofthe local girls in one room you havealmost a full set of teeth. By the timeyou read this our holiday season willhave come and gone. Our annual lateOctober bacchanalian debacle of de-bauchery is over (the Key West versionof Mardi Gras called “Fantasy Fest”)where morality and good judgment takethe back seat with Johnnie Walker andAnhauser Busch. The 50,000 peoplewho parade our streets in body paintsans most of their habiliments are goneand life is back to as normal as it willever get here. Halloween, Thanksgiv-ing, my wife’s birthday, Christmas andNew Year’s Day closely followed thiscelebrious activity. Whew! Being fullon into the winter season here the tem-perature has plummeted (It got so coldlast night I had to turn off the air condi-tioner), the snowbirds have arrived andI now find some time to resume my pento paper efforts. Update: There has been a noticeablecourse of recurrent personal illumina-tion, every week or so, that I havelearned all there is to know of microsco-py. The inevitable subsequent revela-tion that I have had inadequate tutelageleads me to suspicion the generally ac-cepted adage that “You can’t teach anew dog old tricks.” Also, my inabilityto effectively control the eBay addiction

has led to the acquisition of a legion ofmicroscopically relevant accessories aswell as a dramatic increase in my con-course of cameras with associateddevices/adaptors allowing interfacewith my microscopes. Accordingly re-inforced, I venture now to illumine thepursuance of a binocular microscopesuitably compatible with my idiosyn-cratic tendencies and the procurement

of same, which will be based upon thesound principles of déjà vu, reincarna-tion and the fact that inanimate objectsspeak to me. Allow me to explain (Please be ad-vised to reread paragraph five of epi-sode one and re-apply any of theaforementioned descriptors), but first

Fig. 1: The Zeiss GFL

This article was originally published in the January 2010 issue of Micscape Magazine and is nowrepublished here with the permission of the author and of Micscape Magazine.

Experiences and lessonsof restoring antique micro-scopes.

“Tubby or not tubby; thatis the question, pilgrim.”(John Wayne reciting Shake-speare, Hamlet 3/1 - Key West)

G. Joseph Wilhelm,Florida Keys, USA

COLLECTIBLES


turn off the Twilight Zone theme music,it’s in your head, I can hear it. As far back as my memory servesme I have always found the worldaround me quite fascinating in general,but there were instances that evoked areaction far above the norm of juvenileinterest and curiosity. These occurrenc-es were quite commonplace and sum-moned forth a considerable depth ofemotion. It wasn’t until later in life Ibegan to notice a pattern and was ableto identify the feeling. As a young lad,visiting the Cincinnati Art Museum, Ipeered into one of the late 19th century

restored room displays and was imme-diately transfixed by the roll top deskthere. This desk elicited strong feelingsof familiarity, comfort, warmth andwhat I can only describe as a tactilesensation of homesickness. The samescenario was oft repeated with differentobjects as the initiating stimulus andvarying types and degrees of senti-ments, sometimes including anxiety.The familiarity was always present.These occurrences became so routine asto be without noteworthy consequenceto me. I assumed everyone experiencedthem.

Similar above related instances havetaken place throughout my life andseem to be associated with an era be-tween the early 1880s and late 1940s.This, along with the conviction that Icouldn’t have reached my current stateof comfortable maladjustment in a sin-gle lifetime gives forth the presumptionthat I may have been here before andwhile I cannot associate it with an indi-vidual I can certainly place it in a partic-ular timeframe. This also seems toexplain my affinity with objects fromthis period that compromise 99% of mycollections. How this relates to a micro-scope selection will be revealed shortly. The Spencer stereoscope restorationrelated in the previous episode has re-warded me with some magnificentlywondrous hours of entertainment andsatisfaction. This has prepared me forthe next step into serious microscopical-ly enabled observation. I now was desirous of a quality bin-ocular instrument as modern and con-sistent with my collections as possiblewhich meant I would be looking atthose stands I considered as a “bridge”between my vintage scopes and theharsh angular design of the modern in-struments of the 20th century’s lateryears. The “top four” manufacturerswith the following requirements wereconsidered: 1. It had to be black. This may seeman irrational parameter at first, but irra-tional fits me very well. All of the grey/gray (which is really correct?) andwhite microscopes fall outside of mycollection era comfort zone.

2. It should have the 160mm tubelength because most of the (affordable)after market objectives were within thisspecification.

3. The design had to be aestheticallypleasing as per the above-cited era.4. Parts/accessories as used itemsshould be readily available as upgrades.

Fig. 2 (top): The arm as received,nothing seized and all functionsworking very smoothly.

Fig. 3 (bottom): The base. To getthe proper thickness I had to lami-nate together a piece of ¾” and ½”thick by 10” wide bar stock.


5. The binocular head needed to be on arotating 360 deg dovetail mount so as toview from behind or in front of the arm. I basically used eBay to research theavailability, price etc. of theLeitz/Leica, Olympus, Nikon and Zeissstands that fit my paramount specifica-tions. Leitz almost had me with theearly Dialux, Laborlux and the Ortholuxas depicted in Gregor and NormandOverney’s excellent article, but the170mm tube length design, while stillable to accept the 160mm objectivesdictated that the correct match up ofobjective/eyepiece could be a problem.

The Olympus BH stands were close butblack ones and parts seemed scarce andeven more so for the early Nikons. And then, browsing the MolecularExpressions microscope museum, I wasstricken, “But soft, what light through yonderwindow breaks? It is the east and theGFL65 8-56 4 is the sun.” (Romeo andZeiss, Act 2 Scene II, Capulet’s labora-tory.) I was in love! The Zeiss GFL waseverything I was looking for, beauty,style, grace, versatility, power steeringand disc brakes, functionality and avail-

ability. And yes, it spoke to me with afamiliarity of its wonderfully executedart deco spherical design and a comfortfrom what I instinctively knew was pre-cise engineering. This definitely wasthe binocular scope I had to have. Entering “Zeiss microscope” ineBay netted four pages of listings andthere were several GFL scopes there,but there was a problem, they all suf-fered severe cases of dismemberment.Like a Corvette in a chop shop, thesescopes were obviously more valuablesold off for their individual parts than asa whole. Even the most complete scopestill lacked a substage condenser, ocu-lars, lenses, illuminator, and was stillcommanding a price of over $1200.00!Did I really want to get into this? Of course I did. I was determined toplay Dr. Frankenstein and assemble aliving, breathing GFL from parts, thefinal assemblage customized to myspecifications, and do it for less than thecost of a complete scope. My goal wasunder $800.00. The process began with the pur-chase of the GFL arm with quadrupleobjective turret and substage condenserholder (see fig 2) Previous arms likethis with a base but no condenser holderwere going for about $150. Somehow Igot this one for $79 and from the sameseller I got the binocular head for $75.Still needed were the base, condenser,stage, illuminator assembly, eyepiecesand objectives. The 1.3 NA Zeiss condensers weregoing for about $70 to $100. I saw a $39“buy it now” offering but the descrip-tion said it had a small scratch. After aday I inquired about how bad thescratch on the lens was and was in-formed the scratch was not on the lensbut on the chrome on the flip out mount.I now had a condenser and the lens wasperfect. The base was the next logical step soI could stand the durn thing up. It alsoseemed the most difficult. There was anextensive truancy on eBay of this fun-damental foundation for the GFL, atleast one that was not attached to anoth-er arm or an almost complete micro-

Fig. 4 and 5: The arm interface. Itook a 3” diameter “plug” cut witha hole saw from a 1” thick piece ofaluminum plate.


scope. In fact, all of the other remainingitems I required (rotating, centerablecircular stage, light source and objec-tives) were selling at such premiumprices I stopped to re-think this endeav-or. I consulted Captain Morgan andlapsed into deep contemplation andthoughtful analysis and after three orfour seconds had my solution. Exceptfor the objectives, I could manufactureeverything I needed in my shop. Thiswould be an excellent opportunity toexpand the lighting capabilities and ver-satility of the scope in general. The final

product however, must be commensu-rate with the Zeiss level of quality. Thatwas a tall order. The base is complete and the stageabout 90% done. For those who may beinterested, the rest of the article goesinto some detailed description and pho-tos on how it was accomplished.

The Base

The original Zeiss base is a cast,hollow round section of a sphere withthe light source built in. The arm attach-es with four small screws and does not

allow for any inclination of the entirescope. We are about to change all that. The scrap bin at my work site pro-vided all the required material, which is6061-T6 aluminum. We use this stufffor a lot of fabrication, has a structuralstrength approaching mild steel and hasexcellent machining properties. Mybase was going to be solid aluminumand a larger diameter than the originalthus giving more weight and stability.The arm would be attached via a fairlyquick release interface that would alsogive more clearance below the substagecondenser and rack mechanism for in-sertion of as yet to be determined filters,mirrors, prisms, diffusers etc. To get the proper thickness I had tolaminate together a piece of ¾” and ½”thick by 10” wide bar stock. The NavyResearch Lab provided me with an ep-oxy that was stronger than the alumi-num and had the same coefficient ofexpansion. This was trimmed with asaw to a roughly circular shape (see Fig3), a center hole drilled for a bolt andthen chucked in my lathe. Outer diame-ter was turned round and both sides ofthe disc were faced so as to be paralleland the final touch was to round thebase upper edge to approximate theZeiss design. This whole process tookabout 8 hours over two days. (I wasworking at the size limit for my smalllathe and had to make numerous lightcuts.) Next was the arm interface. I took a3” diameter “plug” cut with a hole sawfrom a 1” thick piece of aluminumplate, center bored, threaded and coun-ter sunk a hole for a flat head screw, andturned it down to just slightly largerthan the arm base mounting surface.The tricky part here was to accuratelytransfer the arm attachment screw holepattern to the interface, drill and counterbore the hole from the bottom of theinterface to match the shell thickness ofthe original base so I could use theoriginal screws. The last was to lock inthe large flathead screw with a drilledand tapped hole for a setscrew flushwith the top of the interface.

Fig. 6 and 7: The arm interfacemounted to the base.


Fig. 10: Transferring the mounting hole pattern of the stageholder to paper.

Fig. 11: Finding the center of each hole.

Fig. 8: full size cross section drawing of the stage Fig. 9: Transferring the mounting hole pattern of the stageholder to paper

Fig. 12: Finding the center of each hole. Fig. 13: Finding the center of each hole.


The interface was con-nected to the base by simplydrilling a hole thru and coun-ter boring the bottom to ac-cept the nut and washer.Three rubber feet were thenscrewed in to the bottom ofthe base. The whole thingdissembled and painted withblack enamel and left to bakeon in the hot Florida Keyssun. This whole business tooktwo weekends to completebut thankfully, due to mypersonality, there are hugegaps in my social calendarand I can use the free time tomy advantage.

The stage

This was going to be a bitmore complicated. I had noZeiss stage to copy from andthe mounting hole patternhad to be exactly positionedin relation to the center of thestage, which in turn had tocoincide with the centeredposition of the condenser.Taking gross measurementsfrom the scope I made a fullsize cross section drawing ofwhat I thought the stageshould look like and woulduse it to take my measure-ments from for the machin-ing process.

Fig. 15: The inner bearing was cut from aluminum pipe.Fig. 14: Turning the stage base.

Fig. 16: the inner bearing was cut from aluminum pipe. Fig. 17: The holes for mounting the stage are clearly visible.

Fig 18: The parts of the stage.


I visually centered the condenserand took measurements around itsmount to fine tune it, then did a “rub-bing” with pencil and paper to act as amarking template. Using my SF Stereoscope I was able to then accurately findand mark the center of each mountinghole and condenser. Laying the paper on the aluminum,I center punched the pattern for theholes. Accuracy was within .05 in.Good enough for my purposes and Ibegan the turning of the stage base, themost crucial part. The stage base wasmachined from aluminum thick enoughto accept the centering screws and theadjustable rotation brake/lock. After a test fit of the stage base, theinner bearing was cut from some sched-ule 80 aluminum pipe and then the ro-tating stage itself. The bearing to stagefit is crucial for proper operation and thefinal machining of the mating surfacesfor the proper sliding fit had to be donewith 1200 grit abrasive paper. The finalresult was a perfect fit with absolutely

no play or wobble and an ever so slightresistance. A light coat of white lithiumgrease was the final touch. The adjusting screws were the nexthurdle. Backlash would be the mainobstacle to overcome in a set up likethis. Long thread engagement and agood class fit of the thread is the solu-tion for most high use applications butsince that requires more precise toolingthan I had available and the fact thatthese would see very little use after thestage was properly centered there was amore practical solution. First I marked and drilled thru thestage base at the three spots for thecentering screws and a fourth for thelocking screw. These holes were aslarge as I could comfortably make itwithout affecting the structural integri-ty. Then some brass rod was turneddown until it fit snugly into these holes.Sections of this rod was then drilled thrulengthwise and tapped. From these I cut to length whatwould become the inserts and adjusting

screw barrel shafts. Then a 4-40 screwwas placed in one end of the barrelshaft, the excess cut off and the endturned round and polished. A knob ofZeiss-compatable design from somediscarded electronic equipment was af-fixed to the other end. Now the threadedinsert was screwed over the exposedthread and the class fit was adjusted bygently tapping around the malleablebrass insert with a small hammer untilall play was removed but the screw stillturned smoothly without excess force.The insert was then unscrewed, placedin a hole and locked in place with asetscrew whose threads just gripped theinsert from the side. The front spring loaded centeringpin is shown disassembled below. Thespring and pin came from a discardedVCR (I take all these throw away itemsand disassemble them for parts). The bearing piece was then drilledthru to allow the brake/lock shaft toscrew against a small, oiled leather

Fig 19-21: Adjusting screws and centering pin.


“brake shoe” to adjust rotation resis-tance. With the thus far completed scopeand stage attached. I did a preliminarycentering check with a 20x objectiveand all seemed well but need to do alittle fine-tuning with the 40X objective.Some other minor tweaking for con-denser clearance, a degree scale, andmechanical stage attachment remain.(Speaking of a degree scale, does any-one out there have an already madeCAD drawing or similarly done render-ing of a degree scale they would bewilling to share?) The whole stage fabrication to thispoint has been three weekends of work(I coulda made a stationary stage in oneday but nooooooooooo, I had to haveone with all the bells and whistles). Sofar the total cash outlay for the arm,condenser and binocular head is $193.

Illumination

As a simultaneous effort to theabove (just to prove to my wife that Ican multitask) I dedicated some of myvast mental capability to addressing theremaining lighting requirement. Mostof the GFL stands I have seen have thelight built in to the base but some havea small illuminator that sits on top of thebase. The now solid aluminum baseprecluded my applying the former and Idid not want to give up the substagespace to the latter. In either case therewas not enough flexibility to accom-

plish all the different applications I hadin mind for photon manipulation. An external source directed thru thecondenser via prism or first surface mir-ror seemed to be the answer. My perusalof the external light source offers online left me unmoved as they were ei-ther hundreds of dollars, not powerfulenough, not complete, did not havecomplete range of adjustments (center-ing, focus, iris), hard to get bulbs, need-ed odd voltage power sources whichwere also expensive etc. etc. etc. Salvation (at least I hope so) arrivedin the form of Mr. Frithjof A.S. Sterren-burg of the Netherlands and his articleon making a “Heavy Calibre” micro-scope lamp from a 35mm slide projector(Micscape Oct. 2002). His article madea lot of sense. These things are plentiful,inexpensive and I was able to find ablack vintage projector in mint workingcondition with an Art Deco design thatcomplimented the GFL perfectly for$9.99 plus $12.00 shipping. I was theonly bidder. Below is a picture of theArgus 100 watt projector I bought. By affixing this light and the scopeto a common base I could lock in thealignment and there were enormouspossibilities for combinations andplacement of neutral density and colorfilters, frosted glass and an Iris dia-phragm. This was starting to become a whole“system” so to speak, for microscopy.Vanity has enticed me to give it a nameof its own such as the Ortholux, Axio-

phot, and Amplival have. What to nameit? Since Captain Morgan left the build-ing I consulted with Jack Daniels toponder the question. It had no built incamera, so I couldn’t use “Phot” any-where, and besides, the name was al-ready taken. Since it had a powerfullight source I considered “The Illumina-tor” but that sounded too much like anArnold Schwarzenegger character.Hmmm…the two main componentswere the Zeiss GFL and the Argus lightsource, so let’s see… if we combineZeiss and Argus by taking the first twoletters of Zeiss and the last two of Arguswe get… “ZEUS”!! Yes! That was it.Zeus, an ancient name, a powerfulname, a Greek god, said to be ruler ofOlympus so I figured he had it all overLeitz and Nikon too. “The Zeus wholemicroscope system.” A wonderful nameand much more dignified than combin-ing them the other way around whichwould have resulted in the word “Arss”. Unlike Mary Shelly’s tortured Doc-tor I am not yet ready to scream to theheavens of my creation “IT’S ALIVE!IT’S ALIVE!” Much work remains. Ihope to have Zeus finished in anothermonth or so providing I am not overtak-en by circumstances. A new set of fouraftermarket DIN Plan Achromatic lens-es is on the way. So the total at thispoint with the lenses and 10XWF eye-pieces is $465.00. Still needed are theND, Polarized and UV filters, mechani-cal stage plus the iris diaphragm withmount and the prism with holder.

So as another Episode concludesand a new year is upon us I would liketo take this opportunity to thank theeditor’s of Micscape for their patienceand forbearance in allowing me to sharemy sometimes-outlandish pedagogicpoints of view. My wife, ChaunceyTubbs and myself send everyone bestwishes for a fruitful and enlightening2010. For Christmas my wife presentedme with a copy of “Robert’s Rules ofOrder” to enhance our evening conver-sations and possibly make me morereceptive and democratic in allowingher to complete her “recitations of re-spectful criticism” before rudely inter-rupting. With this charitable attitude Iencourage you to send me yourthoughts or questions [email protected] trails. Comments can be ad-dressed to me. (remove_nospam, ed.) ■

Fig. 22: Argus projector as an illu-mination source.


The hemocytometer (or haemo-cytometer or counting chamber)is a specimen slide which is

used to determine the concentration ofcells in a liquid sample. It is frequentlyused to determine the concentration ofblood cells (hence the name “hemo-”)but also the concentration of sperm cellsin a sample. I have used hemocytome-ters to determine the concentration ofbacteria in liquid growth medium. The cover glass, which is placed onthe sample, does not simply float on theliquid, but is held in place at a specifiedheight (usually 0.1mm). Additionally, agrid is etched into the glass of the hemo-cytometer. This grid, an arrangement ofsquares of different sizes, allows for aneasy counting of cells. This way it ispossible to determine the number ofcells in a specified volume.

Construction

A hemocytometer requires twoparts, the hemocytometer slide itselfand special cover glasses. The slide isquite a bit thicker than a normal slideand contains a grid etched into the sur-face. The cover glass is larger and thick-er than the conventional ones, whichhave a thickness of only about 0.15mm.The cover glass rests on two “stages”which elevate the cover glass 0.1mmabove the grid. This way there is a de-fined volume of liquid above eachsquare on the grid. The hemocytometerslide also has an overflow area. Excess

sample fluid therefore does not result inthe cover glass floating on the liquid.The cover glass is thicker in order toprevent surface tension of the samplefluid to bend the glass and therebychanging the defined distance to thegrid.

Preparing the sample

The fluid containing the cells mustbe appropriately prepared before apply-ing it to the hemocytometer. The fluidcontaining the cells should be a homog-enous suspension. Cells that stick to-gether in clumps are difficult to countand they are not evenly distributed. Anuneven distribution may result in sam-pling error. The concentration of the cellsshould neither be too high or too low. Ifthe concentration is too high, then thecells overlap and are difficult to count.A low concentration of only a few cellsper square results in a higher statisticalerror and it is then necessary to countmore squares (which takes time). Sus-

pensions that have a too high concentra-tion should be diluted 1:10, 1:100 and1:1000. A 1:10 dilution can be made bytaking 1 part of the sample and mixingit with 9 parts water (or better saline ofcorrect concentration to prevent burst-ing of the cells). In some cases, a dilution factor of1:11 (for white blood cells) and 1:101(for red blood cells) is suggested. Thesedilution factors are simpler to workwith, because you simply add 10 or 100parts of liquid to the sample. Whateverthe dilution factor used, it must later beconsidered when calculating the finalconcentration.

Counting the cells

Cells that are on the line of a gridrequire special attention. Cells thattouch the top and right lines of a squareshould not be counted, cells on the bot-tom and left side should be counted. The lower the concentration, themore squares should be counted. Other-wise one introduces statistical errors.

Haemocytometers arespecialized specimen slidesthat are designed toquantify the cell density.This article gives a briefintroduction.

By Oliver Kim

There are different types of counting chambers available with different grids. Thisone is the “Neubauer Improved”.

BACKGROUND


How many squares? To find out onecould calculate the cell concentrationper ml based on the numbers obtainedfrom 2 different squares. If the finalresult is very different, then this can bean indication of sampling error.

Calculating the cell density

Here it is necessary to do some sim-ple math. The following numbers areneeded: number of cells counted in thesquares, area of the square, height of thesample, dilution factor. The objective isto find the number of cells in 1ml oforiginal solution. Step 1 – Averaging: If one did notcount all of the cells in a large square(1mm x 1mm) then it is necessary toaverage the results first before proceed-ing. For the purpose of this example, Iuse an average cell count of 123.456cells in a 0.25mm x 0.25mm square. Step 2 – Computing the volume: Itis necessary to determine the volumerepresented by the square. The widthand height of the square (e.g. 0.25mm x

Yeast cells in the counting chamber. Each square holds a defined volume of liquid.

The squares hold a different volume. The size of the square must be multipliedwith the cover glass to slide distance (usually 0.1 mm).


0.25mm) must be multiplied by theheight of the sample (often printed onthe hemocytometer, in this example it is0.1mm):

volume = 0.25mm x 0.25mm x 0.1mm= 0.00625mm³ = 0.00625ml

Step 3 – Calculating the number ofcells in 1 ml: if there are 123.456 cellsin 0.00625ml, then how many cells arethere in 1ml (=1000ul)? We do simpledirect proportion:

123.456cells/0.00625ul = X/1000ul (123.456cells*1000ml)/0.00625ml = X

X = 19 752 960 cells

Step 4 – Correcting for dilution: Ifthe sample was diluted before counting,then this must be taking into consider-ation as well. We assume that the sam-ple was diluted 1:10. The final result istherefore:19 752 960 cells x 10 = 197 529 600cells / ml. That a lot of cells.

Things to watch out for

Type of counting chambers: Thereare different types of counting cham-bers available, with different grid sizes.One counting chamber also has grids ofdifferent sizes. Take care that that youknow the grid size and height (read theinstruction manual) otherwise you’llmake calculation errors. Use the provided cover glasses:They are thicker than the standard coverglasses. They are therefore less flexibleand the surface tension of the fluid willnot deform them. This way the height ofthe fluid is standardized. Moving cells: Moving cells (such assperm cells) are difficult to count. Thesecells must first be immobilized. Objective: The hemocytometer ismuch thicker than a regular slide. Becareful that you do not crash the objec-tive into the hemocytometer when fo-cusing. ■

Disclaimer: This article is intended purely foreducational purposes. Do not use this infor-mation for medical diagnosis. No guaranteeis given for the correctness or completenessof the information published in this article.Consult an official manual or instruction sheetto obtain information.

Yeast suspension applied to thehemocytomer. Notice the liquid inthe overflow area (arrow).

The surface tension of the samplepulls the cover glass to the slide.The sides of the counting chamberare higher (0.1mm) creating a de-fined volume over the countinggrid.

Cells that are located on the top oron the right of a square should notbe counted. Cells that are on theleft or on the bottom should becounted to a square.


In microscopy, it is often desirableto know the refractive index (RI) ofliquid media. In a well equipped

laboratory, the standard instrument forRI measurement is an Abbe refractome-ter, but the cost of these is well beyondthe budget constraints of most hobby-ists. Fortunately there is a simple lowcost alternative, for transparent liquidsat least. The apparatus basically consistsof a hollow equilateral triangular glassprism which is filled with the test liquid.A narrow beam of light from a laserpointer is directed through the apex ofthe prism, and the angle by which thelight beam is deviated is measured on aprotractor scale. This is the principle ofthe prism refractometer. It can be shownthat when the deviated angle is a mini-mum value, the refractive index of themedium inside the prism can be calcu-lated from

RI = 2 sin 1∕2 ( d + 60 )

where d is the minimum deviation angleof the light beam. The derivation of thisformula can be found in most physicstext books (for example, “Physics Part I& II”, Resnik & Halliday (1966),p.1018). Fig 1 below illustrates the pathof the beam through the prism. Theminimum deviation occurs when thebeam passes through the prism parallelto the base. The construction of a prism refracto-meter is relatively simple. The prismcell can be made from 25mm squarepieces of microscope slide which arecemented together at the edges andmounted on another slide. Silicone seal-ant is suitable for use with most liquids,but other clear sealants can be used,provided that they are inert to the testliquid. To ensure that the angles of thecell are as near as possible to 60 de-grees, an equilateral triangular templatecan be made from stiff card and the cellassembled around it, using long model-ing pins to hold the sides in place whilethe sealant cures. Care needs to be takento keep the sealant confined to the jointsurfaces only. If any is accidentallysmeared on the faces of the cell, it canbe carefully removed with a razor bladeafter curing. The stages of the construc-tion of a prism cell are shown in Fig 2.

Construction

Once the cell has been made, itneeds to be mounted on a rotating table.An easy means of doing this is to obtainthe base section of a blank CD-R con-tainer and one of the clear plastic spac-ers which came in the CD pack. Thecentral spindle is cut off about 2.5mmabove the base and the clear spacer discis slipped over this short axle. The slidewith the glass prism is then fixed to thespacer disc with double sided foamtape, so that it just clears the centralspindle. The prism cell should then ro-tate freely about the spindle when theplastic spacer is turned. If the spindle istoo high, file a small amount off untilthe prism cell and slide do not touch it(Fig. 3). Next, you will need some rigid clearacrylic packaging material approxi-mately 0.5mm in thickness. From this,the index arm is cut using the pattern inFig 4. If the CD container you have isdifferent, you may have to make somealterations to the pattern. The verticalindicator card can be glued to a short“L” section as illustrated. Another vertical index tab is re-quired for the zero mark of the refracto-meter. This can be made in the sameway as the moveable index arm, but itdoes not need the central ring as it willbe fixed in position. A pattern for thispart is shown in Fig 5.

A refractometer measures therefractive index of differentmedia. It is possible to makeone yourself to measure therefractive index of differentmounting media.

Robert F. Hancock

DIY

Fig. 1: Path of the light beamthrough the prism filled with thetest liquid.


Fig. 2: Prism cell construction.

A protractor scale now needs to bemade, which is placed underneath theindex arm and the clear disc holding theprism, and glued to the base of the CD-R container. A photocopy of a 360 de-gree protractor was used in the exampleillustrated, but only one quadrant of thescale is actually needed. It is a good ideato clear laminate the protractor scale ifyou can, this will preserve the markingsindefinitely. A 40 mm diameter hole iscut in the centre to fit over the base ofthe CD-R container. The zero index tabis glued in position to coincide with the

zero mark on the protractor scale. En-sure that the vertical index line is exact-ly aligned with the centre of theprotractor and the zero degree mark.The index arm is placed on top of theprotractor scale and underneath theclear spacer disc carrying the prism cell.It should rotate freely without causingthe spacer disc to move with it. Thecomplete rotating table assembly is at-tached to a base board. The componentsare assembled as shown in Fig 6. The next step is to mount the laserpointer (only a 1mW pointer should beused – higher powered lasers are dan-

gerous to your vision) so that the beampasses exactly above the centre of therotating table, and is aligned on thevertical mark of the zero index tab. A75mm length of rigid PVC tubing andan aluminium bracket is used to mountthe pointer on the base board. Theheight of the laser pointer must be suchthat the laser beam passes through thecentral section of the prism on the rotat-ing table. The tube is drilled with twosets of 3 small holes spaced at 120° totake the machine thread screws, whichwill hold the laser pointer in position.The screws should be a tight fit in the

Fig. 3: The rotating table.


holes, so that they cut a thread into thePVC when they are inserted. Anotherhole is drilled above the pushbuttonswitch and a screw is inserted in this sothat the laser beam can be turned on oroff by turning the screw. The position ofthe pointer is then adjusted with thescrews to align the beam with the zeromark. Be careful to protect your eyesfrom the laser beam when it is switchedon. Fig. 7 shows the completed refracto-meter in use. Once everything is correctlyaligned, the prism can be filled with thetest liquid, using a pasteur pipette.About 5 ml of sample is required. If theliquid is volatile, place a 25 mm squareof microscope slide glass on top to re-duce evaporation. The prism and theclear plastic disc are then carefully ro-tated until a position is found where thedeviation angle of the laser beam has aminimum value. A magnifying lens willbe found useful here to read the angleoff the protractor scale, again takingcare not to look into the laser beam. It isa good idea to switch off the laser whileyou are inspecting the protractor scale.The prism should then be rotated by120° so that the other two corners are atthe apex, and the minimum angle read-ings taken for each apex. If your prismhas been accurately made with 60° an-gles, you should obtain the same mini-mum deviation angle for each of the 3vertices of the prism. If they are slightlydifferent, then just use the average valueof the 3 readings, this will not introduce

any significant error. The average valueof d is substituted into the formula

RI = 2 sin 1∕2 ( d + 60 )

to calculate the refractive index, or theRI value can be read from the graph(Fig. 9).

Sources of Errors in R.IMeasurements

Some comparisons were made be-tween R.I measurements made on thisapparatus and a laboratory grade Abberefractometer reading to +/- 0.0002. Theresults are tabulated in Fig. 10. As can be seen, the average error isabout 0.5 % . This may be adequate formost amateur microscopy purposes.The sources of error can be identified inthree main categories:· Measurement of the Minimum De-

viation Angle· Wavelength and Temperature Errors· Dimensional Irregularities in the

Prism Cell

The protractor scale can only beread to +/- 0.5 degrees, which corre-sponds to an uncertainty of around +/-0.005 in the refractive index value. Theaccuracy of the angle measurementcould be improved by projecting thelaser beam at right angles onto a wall ata distance of a few metres and measur-ing the minimum lateral displacementof the beam in the horizontal plane. The

minimum deviation angle can then becalculated as d = arctan ( h/ l ), where h= lateral displacement and l = distancefrom the centre of the prism cell to thewall. Assuming l = 2000 mm and d =300, and thus h = 1155 mm, with thelaser pointer that I used, the diameter ofthe laser beam spot on the wall is about3 mm. This corresponds to an uncer-tainty in d of about 1.5 arc minute, or+/- 0.0003 in the refractive index value,which is approaching the accuracy oflaboratory instruments (see Fig. 11).

Wavelength and TemperatureErrors

Refractive Index decreases with in-creasing wavelength of the light source.The standard wavelength for R.I mea-surements is 589.3 nm, the wavelengthof the sodium D line, whereas cheap redlaser pointers produce a wavelength of670 nm. For most organic liquids, thisprobably introduces an error of about0.002 in the R.I value, the value at theNa D line being higher. If you can ob-tain a yellow-orange laser pointer, thiswill give much more accurate results, asthese lasers have a wavelength of 583nm. Similarly, the R.I decreases withincreasing temperature, for most sub-stances this amounts to about – 0.0003per degree Celsius. For optimum accu-racy, the temperature of the sampleneeds to be controlled.

Fig. 4 (left): Index arm table.

Fig. 5 (bottom): Index arm patternzero mark


Dimensional Irregularities in thePrism Cell

Variations in the thickness and per-pendicularity of the glass walls and theprism angles will cause errors in the R.Ivalues obtained. A difference of +/- 5degrees from the correct prism anglesof 600 will produce an error of about+/- 0.001 in the R.I value determinedby averaging the three d values. Hollowglass and plastic prisms can be obtainedcommercially from scientific equip-ment suppliers, and these are probablymore accurate than home made prisms.

Calibration of the Refractometerwith Distilled Water

The best method of checking therefractometer's accuracy is to measurethe R.I of a sample of distilled (or de-ionised) water. At 670 nm and 200Celsius, the refractive index of water is1.3317.

R.I Value of Mixtures

In microscopy, it is often requiredto produce a series of liquids havingknown R.I values. It is only necessaryto obtain two liquids having known R.Ivalues which span the range of interest,and a series of intermediate R.I valuescan be matched by blending the twocomponents. To a fairly good approxi-mation, the R.I value of a mixture willbe proportional to the volume concen-tration of the two components., provid-ing the two liquids do not reactchemically. Thus, given liquid A withR.I = a, and liquid B with R.I = b, thepredicted R.I value of any mixture isgiven by R.I = Aa + Bb, where A =volume fraction of liquid A and B =volume fraction of liquid B. Note also

Fig. 7 (top): Prism & table assembly

Fig. 8 (middle): Completedrefractometer in use

Fig. 9 (bottom): Once you have de-termined the minimum deviation an-gle, you can either directly calculatethe refractive index or use thisgraph to read it off.


that B = 1- A. Figure 12 shows thisrelation ship.

For example, mixing methyl salicy-late (R.I = 1.5365) with Eucalyptus Oil(R.I = 1.4572) in the volume ratio of70:30 is expected to produce a mixturewith R.I = 0.7 x 1.5365 + 0.3 x 1.4572= 1.5127. An experimental mixture ofthese proportions was measured on anAbbe refractometer and the resultingR.I was 1.5128. This simple calculation(known as the Arago – Biot equation)assumes ideal behaviour of the two liq-

uids, but generally the predicted R.Ivalue will be within +/- 0.1 % of theactual value. Small differences betweenthe predicted and actual values mayoccur, these are caused by minor vol-ume contractions or dilations which oc-cur when dissimilar liquids are mixed.There are more complex equationsavailable to predict R.I values takingthese interactions into account, for ex-ample the Lorentz – Lorenz equation(see Proc. Indian Acad. Sci. (Chem.Sci.), Vol. 115, No. 2, April 2003, pp

147–154). On the graph (Fig. 12), thepredicted R.I values from the Lorentz –Lorenz equation are also shown, togeth-er with the differences from the Arago -Biot predictions (magnified 1000x). Ascan be seen from the graph, the differ-ences are much less than 0.1 % of theactual values of R.I. These equationscan also be applied to ternary, quaterna-ry, etc mixtures, ie those having three ormore components. ■

Fig. 10 (top): Comparing theme�asured R.I. with the theoreticalvalues. The average error is about0.5%.

Fig. 11 (middle): The minimum de-viation angle can then be calculat-ed as d = arctan (h/l).

Fig. 12 (bottom): R.I. Values of mix-tures.


In February of this year we imple-mented a digital photomicrographycourse in the CCH-Oriente (Cole-

gio de Ciencias y Humanidades – Plan-tel Oriente), one of the two branches ofthe high school of the UNAM (Univer-sidad Nacional Autónoma de México).The main objective of this course was tointroduce the students into the differentset-ups for photomicrography used in

faculties and the investigation centers ofthe medical-biological departments ofthe most important University in Méxi-co. In the eastern side of Mexico City,with more than ten thousands of stu-dents, the CCH-Oriente is a referencecenter for science and humanities edu-cation for high schools, because ourstudents have a complete curricular pro-gram in a context of a constructivisteducational model. The curriculum alsoincludes, a wide variety of extracurricu-lar training courses with a propedeuticapproach. Professors and other faculty stafffrequently criticized that high schoolstudents show a deficiency of abilitiesin the use of the microscopic and related

techniques, especially in careers likeBiology, Medicine and Veterinary,among others. SILADIN (Sistema de Laboratóriospara el Desarrollo y la Innovación), theSystem of Laboratories for Develop-ment and Innovation, is present in allschools of the CCH’s, and has a wellequipped academic facilities for profes-sors who want to work in advancedinvestigations with the participation ofstudents. The microscopy equipmentincludes Olympus epifluorescence, CarlZeiss compound microscopes (withbright filed, dark field and phase con-trast), and stereoscopic microscopes. Inthese facilities we implemented a 20hours propedeutic training course forstudents in the last year of high school

From Mexico, sharing ourlove of microscopy to youngscientist aspirants.

Víctor RafaelZárate-Ramírez, M.Sc.

Hemipteran Insect, Planoccocus citri, Tucs-en 1.3 MP, stacked from 10 images withCombineZP, postprocessing with Gimp.

Image: Víctor Rafael Zárate-Ramírez

OBSERVATION


Protozoan, Spirostomum spp., Tucsen 5.0MP, bright field, postprocessing with GimpImage: Karla Mariana Peña Gutierez

Rotiferan, Philodina roseola, Canon EosRebel T1i, phase contrast, postprocessingwith GimpImage: Jesús Vázquez Ramos

Hemipteran insect, Aphis gossypii, a “mummy” witha parasitoid wasp inside, Canon EOS Rebel T1i,postprocessing with Gimp

Image: Rosa Jazmín Sánchez Arguello

mainly, and with plans for a scientificcareer. The course was divided into tensessions. The first session was an introductioninto compound (Axiostar Plus) and ste-reoscopic (Stemi DV4) microscopeswith an emphasis on phase contrast,bright field and dark field. Top andbottom illumination was also addressed,according to the nature of the sample inthe case of the dissection microscope. In this context the cameras to beused in the course were presented aswell: Two dedicated cameras with Tuc-sen chip (1.3 and 5.0 MP), a digitalreflex Canon Rebel EOS T1i (15 MP)with T-mount, threaded 10X lens and23 and 30 mm microscope adapters, aswell as a Nikon Coolpix S203 (10 MP)with a Celestron 93626 digital cameraadapter, for afocal microphotography.All this (with some variations) was usedas standard equipment in mostly of fac-ulties and research centers of medical-biological area of the UNAM.


The second session was an introduc-tion to the software: Micam for an alter-native to the native software includedwith the cameras, CombineZP for photostacking and Gimp for postprocessing.We began to take photos of the protozoaParamecium caudatum, Spirostomumspp., Astramoeba radiosa and RotiferaPhilodina roseola, all previously culti-vated in laboratory in glass dishes andfed with unicellular algae. All speci-mens were mounted in wet prepara-tions, but we also applied a little drop ofGram's iodine to reveal cilia in Parame-cium caudatum samples. For the third session, the studentscollect leaves of plants to watch stomain the lower epidermis. In session fiveto eight, each student designed and tookphotos of a personal project. Theseprojects ranged from aphids (we found“mummies” containing parasitoidwasps), to pollen, and a wide selectionin the middle. In session nine and ten, the studentspresented to the class their individualprojects. All the students were ap-proved, taking their skills with micro-scope to new levels. In this contributionwe present some of these photos takenby the students and myself.

Acknowledgments

This course would not have beenpossible without the invaluable supportof Arturo Delgado González, Directorof CCH-Oriente; Tomás NepomucenoSerrano, Technical Secretary of the SI-LADIN and Esperanza Martínez Lópezadministrator of the upstair hall in theComputation Tower. ■

Protozoan, Astramoeba radiosa, tucsen 5.0 MP, phase contrast, postprocessingwith Gimp

Image: Maria Guadalupe Hernández Luna

Web sites:

UNAM:http://www.unam.mx/

CCH-Oriente:http://www.cch-oriente.unam.mx/

SILADIN Plantel Oriente:http://siladin.cch-oriente.unam.mx/

Protozoan, unidentified free living amoeba, Nikon Coolpix S203 (afocal), phasecontrast, postprocessing with Gimp

Image: Lesli Tiare Figueroa Arriaga


Protozoan, Paramecium caudatum, asexual division and full or-ganism, Tucsen 1.3 MP, postprocessing with Gimp.

Image: Laura Paulina Barrera Muñoz

Protozoan, Paramecium caudatum, with Gram’siodine, to reveal cilia, Canon EOS Rebel T1i, phasecontrast, postprocessing with Gimp.

Image: iare Figueroa Arriaga

Hemipteran insect, Aphis gossypii, lower left a juvenile, inthe middle an adult with cornicles visible one emitting greencolor pheromones, Canon EOS Rebel T1i, postprocessingwith Gimp.

Image: Maria Guadalupe Roque Ortega

Hymenopteran insect, Aphidius colemani, parasitoid wasp,Canon Eos Rebel T1i, stacked from 8 images, postpro-cessing with Gimp.

Image: Víctor Rafael Zárate-Ramírez


Introduction

In February 2011, in CCH-Oriente,UNAM, Mexico, we implemented a mi-crophotography course for students. Aspart of their personal projects, somestudents collected a flower of pome-granate with some aphids. They identi-fied as the species as Aphis gossypii, awell know homopteran parasite of com-mercial interest in Mexico. A prelimi-nary search in primary literature and theInternet showed us the possibility of thepresence of one of their natural preda-tors, parasitoid wasps of the speciesAphidius colemani. We conducted acareful search and found a “mummy”aphid. The wasp used the aphid to repro-duce by laying an egg into it, transform-ing it into a mummy. We began with aninvestigation with the main objective ofcorroborate the information of the liter-ature, with the possibility of new find-ings. The aphid-wasp relationship is anexample of co-evolution and a learningobjective for the Biology class of somestudents.

Aphis gossypii, Glover 1987(Homoptera: Aphididae)

The aphid Aphis gossypii is a cos-mopolitan homopteran, wherever itshost plants are grown we can be certainto find it as well. The aphid generallyprefers warmer climates. It can also befound in greenhouses, where it is a com-mon pest. The aphid attacks the plant byfeeding of its green parts, such as.young shoots and leaves. The females ofthe winged morphs have a black color,the abdomen of often colored different-ly. The wingless forms can show manydifferent colors, ranging from yellowishto green, even gray. The wingless adultsare 1-2 mm in length.

Aphidius colemani, Viereck 1912(Hymenoptera: Braconidae)

Aphidius are small wasps, which re-produce by laying single eggs intoaphids. The eggs hatch and the larvaeconsume the aphids. During this process

the aphids are killed. The aphids thusturn into “mummies”. The larvae thenpupate. After metamorphosis, an adultwasp emerges through an exit holewhich they cut into the mummy. This isnot the only thing that kills the aphids.The wasps search for suitable hosts andthereby disturb the aphid colonies.Many aphids therefore fall off the plantand die. Aphidius colemani can reproduce inmany different kinds of aphids, such ascotton and green peach aphids. Otheraphids (potato or foxglove aphids) areresistant. The morphology of Aphidiuscolemani is similar to A. matricariae,but there are differences in the numberof eggs produced. The size of Aphidius colemani rang-es from 0.5 to 1cm in length. This de-pends also on the size of their aphidhosts. Males and females differ in theshape of their abdmonen. Males tend tohave a rounder abdomen, while femaleshave a pointed ovipositor to place eggs.

In this contribution wedescribe the life cycle ofAphidius colemani, aparasitoid wasp. It is anatural predator of Aphisgossypii (Glover, 1987) ahomopteran insect parasiteof plants. This high schoolinvestigation confirms datafrom literature.

Víctor Rafael Zárate-Ramírez,Laura Paulina Barrera Muñoz,Leslie Hernández Ramírez,Claudia Peña Fabián andEmmanuel Alejandro RamírezMorales

OBSERVATION

Pomegranade trees in SILADIN of CCH-Oriente, UNAM.


The color of adults is dark brown toblack.

The Beginning - Searching forMummies on Plants

We collected some flowers, bulbsand leaves of pomegranate (Punica gra-natum, Linneo 1762), found in SILA-DIN of CCH-Oriente, UNAM, in theeastern side of México City(19°23'2.31"N, 99° 3'35.39"E). On oneleaf we found one aphid mummy and onone bulb we found three more (watchyour own plants too, maybe they haveaphids and mummies too). All of themwere placed in petri dishes at room tem-perature. For three days we monitored theaphid mummies with a Carl Zeiss stereomicroscope DV4, a Celestron universaldigital camera adapter and a NikonCoolpix S203 digital compact camera(10MP), and a Olympus Ve3 with aCanon Rebel EOS T1i (15 MP) withT-mount, threaded 10X lens and 30 mmmicroscope adapter. All microscopesbelonged to LACE (Laboratorios Avan-zados de Ciencias Experimentales) Lab-oratories in SILADIN.

The sprouting - any similaritywith alien movies is only acoincidence

After three days of monitoring, wecould see how the wasp began to bite acircular pattern into the dead exoskele-ton of one of the mummies. This hap-pened in the abdomen between thecornicles. This hole was finished in nomore than 5 minutes and the waspemerged 32 seconds later. In a secondcase the wasp completed the hole in 3hours and emerged in 5 hours.

Infection’s under laboratoryconditions (hurray!!! they werefemale!)

If the first emerged wasp was fe-male, it would have been ready to layeggs in aphids. We therefore prepared apetri dish with ten aphids with differentsizes and also ten exuviae. The exuviaeare the skins of (dead) aphids. They areused as a control and should not berecognized by the wasps. We waitedseveral hours for the first emerged waspto walk to prepared part with aphids andexuviae. The wasp was female becausethey begin immediately to lay eggs inthe aphids. The exuviae was not recog-nized as valid prey and the wasps did

An aphid “mummy” on a leaf ofpomegranade. The parasitoid waspstarted to form a hole. The imagesshow the attempt for the wasp toemerge from the aphid.

Pomegranate bulb with aphids (Aphis gossypii), of different sizes and colors,even a winged female.


not try to lay eggs into them. Naturalselection was still doing its job! When a wasp finds a suitable aphidit doubles the abdomen under the frontlegs, beats the wings of an intense wayand places them backwards, with theovipositor lay their eggs into the aphid.The whole process was documentedwith video and photomicrography.

Conclusions

The colony of aphids Aphis gossypiipresents in pomegranate of the SILA-DIN in CCH-Oriente, UNAM, are dep-redated by the parasitoid wasp Aphidiuscolemani. The data taken in video andphotos support previous information,but, we found inconsistencies in thebehavior reported about the exuviae andfurther study is necessary. The studentsreinforced their skills with microscopyand photomicrography. This investigation from high schoolstudents resulted in a good learningstrategy, because they worked in everyphase, from field to laboratory. In thisway they acquired and they reinforced

themes related to co-evolution and nat-ural selection.

Acknowledgements

We appreciate the support of ArturoDelgado González, Director of CCH-Oriente, UNAM; Tomás NepomucenoSerrano, Technical Secretary of the SI-LADIN; and Ricardo Abad Moraleschief of Audiovisual Department fortechnical help in Canon mount’s.

References

Escoto Rocha J., E. V. De EriceZúñiga y L. Delgado Zaldivar. 2003.Homópteros de la Colección Ento-mológica del Departamento de Biologíade la Universidad Autónoma de Aguas-calientes. Investigación y Ciencia de laUniversidad Autónoma de Aguascali-entes, (29): 11-16.

Gaona García G., E. Ruíz Cancino yR. Peña Martínez. 2000. Los pulgones(Homoptera: Aphididae) y sus enemi-gos naturales en la naranja, Citrus sin-ensis (L.), en la zona centro de

Tamaulipas. Acta Zoologica Mexicana,81: 1-12.

Muratori F. B., D. Damiens, T.Hance y G. Boivin. 2008. Bad house-keeping: why do aphids leave their exu-viae inside the colony? BMCEvolutionary Biology, 8: 338-345.

Sánchez Lamar A, R. Cozzi, E. Cun-dari, M. Fiore, R. Ricordy, G. Gensa-bella, F. Degrassi y R. De Salvia. 2005.Extracto de frutos enteros de Punicagranatum L. como agente protector deldaño inducido por el peróxido dehidrógeno. Revista Cubana de plantasmedicinales, 10 (2).

Stary P. 1974. Aphidius colemaniVierek: its taxonomy, distribution andhots range (Hymenoptera, Aphidiidae).Acta Entomologica Bohemoslovaca,73: 156-163.

Stary, P. y G. REMAUDIERE.1983. Complements to the aphid para-sitoid fauna of Mexico (Hymenoptera,Aphidiidae). Annales de la Société En-tomologique de France, 19: 113-116.

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OBSERVATION

Top left: Aphidius colemanii, a parasitoid wasp afteremergence, ventral view, inside a petri dish.

Top center: The first obtained female of the parasitoidwasp, preparing to lay eggs inside an aphid.

Top right: Laying their eggs with the ovipositor.

Right: Trying to lay eggs in a young aphid. At the rightthere is an exuviae. Never try to lay eggs in them!


1. A second aphid “mummy”, the wasp begins to push frominside.

2. The head of the wasp its out now.

3. The wasp’s antenna is now completely out.

4. Half of the wasp’s body is now out.

5. In the struggle, how many of them die in the process?

6. Almost completely out now.


What’s this? Answer on page 3.

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