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MicrobeHunter Microscopy Magazine - November 2011 - 1

MicrobeHunterMicrobeHunter

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

Volume 1, Number 11November 2011

The Magazine for theEnthusiast Microscopist

http://www.microbehunter.comMicroscopy Magazine

Yogurt Bacteria Staining EpithelialCells

Purple Sulfur Bacteria

Essential oilsas clearing agents

Field Notes onScience andNature

ObservingYogurt Bacteria

A Wonderful Giftfor Christmas?

Sulfur Bacteriaon a Leaf

2 - MicrobeHunter Microscopy Magazine - November 2011

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

Volume 1, Number 11, November 2011

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 and image contributions by:Andrew ChickCharles GuevaraOliver KimManfred RathWilhelm ReschZachary J. Smith et al. (cc)Anthony ThomasWayne WilsonEurico Zimbres (cc)

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 or in the caption. Thecopyright of all other images is with the author of the article. Youare not allowed to distribute this magazine by email, file sharingsites, web sites or by any other means. If you want to have acopy of this magazine, either order one from Magcloud (see linkabove) or vistit www.microbehunter.com.

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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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]

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Front Cover:

Large image, left image, middle image: Oliver KimRight: Charles Guevara

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4 A Review of Essential Oils as clearing agents for natural history specimens

Certain essential oils have previously been used in micros copy as clearing agents. While they certainly smell better than xylene do they actually perform?

Andrew Chick

5 Bad PicturesWhy report back only the successful microscopy attempts?

This one resulted in quite disappointing images.Oliver Kim

6 New Techniques in MicroscopySmartphones as microscopes, scanners that scan slides

instead of traditional microscopes…. Where will thefuture take us?

Oliver Kim

8 Image GalleryPictures by Anthony Thomas and Manfred Rath.

10 Field Notes on Science and NatureKeep a scientific notebook which documents yourobservations! A book review.

Wayne Wilson

11 Polarizing Microscope and ApplicationsWhat are the areas of applications of polarizingmicroscopes?

Monty Apollo

12 A Wonderful Gift for Christmas?How well do toy microscopes perform? Wilhelm Resch

bought one to give it a test.Wilhelm Resch

14 Observing Yogurt BacteriaYogurt bacteria can be observed in a wet mount orafter heat-fixing and staining them.

Oliver Kim

18 Large phototrophic purple sulfur bacteria on a leafA magenta-colored bloom can be an indication for the

presence of purple phototrophic bacteria. Here, aggregates of these bacteria were found on a decaying leaf.

Charles Guevara

22 Staining Epithelial Cells with InkRegular fountain-pen will stain epithelial cells.

Oliver Kim

Answer to the puzzle (back cover):Tracheal system of an insect.

CONTENTS

20

cc-by-sa Eurico Zim

bres

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Numerous small animals aremounted on microscope slidesfor identification, and ar-

chiving. These are normally mounted ineither a gum based mountant such asBerlese, Hoyers or Modified Dionis(Chick, 2010), Euperal or one of theresinous mountants such as Canada Bal-sam, Gum Damar or an un-recommend-able synthetic mountant such as DPX(Brown, 1997 states that the BritishNatural History museum no longer ac-cepts specimens mounted in DPX). Themethod for mounting insects and otherinvertebrates for study in resinous me-dia is rather well known and is charac-terised by Oldroyd (1970) asmaceration of the soft tissues,bleaching/staining as necessary, dehy-dration, clearing and mounting. It iswith the clearing stage with which thispaper is concerned. Clearing is requiredto remove the dyhydration agent (usual-ly alcohol, however Acetone or Glacialacetic acid among others are recom-mended by some protocols). Histolo-gists traditionally use xylene to removedehydration agents as this is the solventoften used as a carrier of the resin in themounting media. However for the stu-dent of natural history it is deemed un-satisfactory by Oldroyd (1970) due tothe speed of evaporation not allowingadequate time for dissection. From apersonal perspective, xylene is ratherdifficult for the amateur microscopiststo obtain, and the fumes can result intremendous headaches. It is also consid-ered carcinogenic all added together itis best avoided!

Traditionally, entomologists haveused essential oils as clearing agents,

normally clove oil or cedarwood oil,however Gray (1954) lists numerousoils and how pure the alcohol they arecleared from must be. A few of thesewere tested for there suitability as clear-ing agents for insect specimens.

Methodology

For this investigation the humblelarvae of Calliphora vomitoria wasused, this was picked as it is commer-cially grown for fishermen to use asbait. The larvae were fixed using freshlyboiled water and preserved in 70% alco-hol. They were mounted in Gum Damarusing the method of Chick and Cassella(2011) as follows:

1. Maceration in 10% Sodium hydrox-ide,

2. Stain was Acid Fuchsin3. Dehydrate using 70% then 100%

IPA alcohol.4. Clear in oil5. Mount in Gum Damar

The essential oils chosen were as fol-lows:� Reagent grade clove oil (some what

of a luxury but a useful control)� Clove oil B.P.� Tea Tree oil B.P.� Eucalyptus oil B.P.� Distilled turpentine� Cedarwood oil (reagent grade)� Lavender oil� “Olbas oil” B.P. a commercial mix

of essential oils, including Eucalyp-tus and clove.

Where possible, B.P. (British pharma-copeia) grade oils were selected.

Gray (1954) notes the followingabout the chosen oils: Clove oil has arefractive index of 1.53, equal to bothglass and Canada balsam. Objects canbe transferred from a minimum 75%alcohol.

Eucalyptus oil requires a minimumof 80% alcohol as the final dehydrationstep. And it has a lower refractive indexof 1.46

Turpentine has the refractive indexof 1.47 and requires near perfect dehy-dration with 95% alcohol.

Cedarwood oil again requires nearperfect dehydration from 95% alcohol,and has a refractive index close to glassat 1.50.

Lavender oil has identical water tol-erance and optical qualities to Eucalyp-tus.

Tea Tree oil and Olbas oil were notconsidered by Gray (1954)

Results

Clove oil (reagent): Some fading ofstain over time, (over a month aftermounting), however appears to be easi-ly counter acted by using a double stainof Acid Fuschin and Methylene Blue(50/50 mix)Clove oil B.P.: Appears similar toaboveEucalyptus oil: Strips stain within 10minutesLavender oil: Strips stain within 10minutesDistilled turpentine: Strips stains with-in days, clouding of mount noticed

METHODS Essential Oils

Clearing prior to mounting in resinous media is often precluded by xylene, however certainessential oils have previously been used in microscopy as clearing agents. While theycertainly smell better than xylene do they actually perform?

Andrew Chick


Cedarwood oil: Strips stain withindays of mountingTea tree oil: Strips stains within days ofmountingOlbas oil: Strips stains within 10 min-utes.

From the above it would appear thatclove oil is the best of the tested oilswith regards to stain preservation.

Discussion

With phase contrast and anhydrousalcohol, any of the oils would be suit-able for use in microtechnique, indeed itwas suggested that clove oil leavesspecimens brittle so the other oils wouldbe preferable. However, if staining isrequired, clove oil (either reagent gradeof pharmacy grade) are the choice ofclearing oil. This combined with itstolerance for imperfect dehydration,

Gray (1954) notes that anhydrous re-agents are extremely difficult to keepwater free as they will absorb moisturefrom any air around them. Indeed Fow-ell (1946) recommends clove oil as amore forgiving clearing oil for students.It is worth noting on this point that thereare two essential oils to be avoided at allcosts these are oil of wintergreen and oilof juniper as both oil require perfectdehydration with 100% alcohol, whichmay be impossible to obtain.

In conclusion, as a general clearingagent clove oil B.P. would appear to beof most use or the amateur showing noapparent difference to the reagent gradeoil in use.

References

Brown, P.A., 1997, A review of techniques usedin the preparation, curation and conservation ofmicroscope slides at the Natural History Museum,

London, The Biology Curator, 10: Special Sup-plement, 33pp.http://natsca.info/sites/natsca.info/files/The_Biology_Curator_Issue_10_Supp.pdf

Chick, A.I.R., 2010 A modification of DionisMountant as a substitute for Berlese Mountant.Entomologists Monthly Magazine. 146 (2) 117-118

Chick, A.I.R., Cassella J.P., 2011 Gum Damaras a substitute for Canada Balsam in mountingmedia for microscopical specimens, Entomolo-gists Monthly Magazine, 147 (2) 111-114

Fowell, R.R., 1946, Biology staining schedulesfor first year students. H.K. Lewis and Co. LtdLondon

Gray, P., 1954., The Microtomists Formulary andGuide. Constable and Company, Ltd, London

Oldroyd, H., 1970 Collecting, Preserving andStudying Insects. Hutchison scientific and techni-cal. London

Contact [email protected] ■

Bad PicturesWhy report back only the success-ful microscopy attempts? This oneresulted in quite disappointing im-ages.

I have two slide duplicators in mypossession. These duplicators areused to make copies of negative

film and slide film. One duplicator canbe mounted directly on my SLR cam-era using a T2 adapter ring. The dupli-cator directly acts as a macro lens.Focusing is not possible with this sys-tem. The other duplicator attaches tothe front of of my camera zoom lens. Itis possible to focus using the zoomlens. Both systems are able to achievea high magnification.

I wondered if these two set-ups arealso suitable for microscopy, andmounted some permanent slides intothe film holder. The results were ratherdisappointing. The contrast is low andthe resolution bad. Especially the com-bination of the duplicator with an exist-ing objective resulted in significantloss of sharpness towards the corners.

What a pity. I already hoped to havefound a method to quickly make use-able overview slides at the click of abutton. I suspect that part of the prob-

lem was the inability to carefully focusthe specimen slide. Maybe scanning aslide in high resolution results in moreuseful pictures (ed.). ■

A disappointing result: Both slideduplicators delivered low-resolu-tion images of low contrast. Vi-gnetting could also be observed.

Wood

Dandelion

Pine flower

With T2adaptor

Extension ofthe objective


Smartphone Microscope

Researchers have developed a low-cost microscope which uses the cameraof a mobile phone together with a 1mmball lens mounted in front of the cameraobjective of the mobile phone. The cell-phone microscope was able to magnifyup to 350x with a nearly diffractionlimited resolution, the theoreticallymaximum resolution possible. The res-olution of the microscope was deter-mined to be 1.5 micrometers at thecenter of the image. While the imagequality rapidly deteriorates towards theedges, the quality is still sufficientlyhigh to conduct routine blood diagnos-tic tests.

The quality of the image can also beimproved digitally by stacking severalimages taken at a different focus. Fo-cusing is possible by changing the dis-tance between the slide and thespherical lens. It is also possible to usea cylindrical GRIN lens (gradient indexlens) instead of a spherical lens (figures1 and 2). This greatly improves theimage quality towards the sides, butreduces the overall image resolution.

The researchers demonstrated that itis possible to diagnose the blood diseas-es sickle cell anemia as well as irondeficiency anemia from blood smears(figure 3).Reference: Smith ZJ, Chu K, Espenson AR, Rahimza-deh M, Gryshuk A, et al. (2011) Cell-Phone-Based Plat-form for Biomedical Device Development and EducationApplications.PLoS ONE 6(3): e17150.doi:10.1371/journal.pone.0017150

URL of original Article: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0017150

Image credits: All images are under the Creative Com-mons Attribution License on http://www.plosone.org.The image credit goes to Zachary J. Smith et al., 2011.

■

Figure 1 (left): A sphereical 1mm diameter lens (to the right of the coin) producesan image which is sharp in the center but out of focus on the side.Figure 2 (right): A GRIN lens is cylindrical. It produces an image which is also sharpat the sides, at an overall lower resolution. Image: Zachary J. Smith et al.

Figure 3: The top rows show images using a regular microscope, the bottom rowuse the cell phone camera. Left: red blood cells of healthy blood; Middle: anemiadue to low blood cell count; Right: sickle cell anemia. The cell phone microscopeallows for a diagnosis of these blood illnesses. Image: Zachary J. Smith et al.

NEWS Recent Developments

Smartphones as microscopes, scanners that scan slides instead of traditional microscopes….Where will the future take us?

Oliver Kim


“iPad on Steroids”: A newGesture Controlled Microscope

Finnish researches developed a mi-croscope with a truly different ap-proach. The microscope can becontrolled with hand and finger gestureson a large touch screen. The gesturesallow for panning and zooming of digi-tized microscopic images.

A specimen slide is first scannedwith a high resolution scanner. The im-ages, which can have a size of up to200GB, are stored on an image serverand loaded as the user zooms and navi-gates. It is then possible to zoom intothe digitized image from original size to1000x magnification. Johan Lundin,one of the researchers, compares thenew method with an iPad: "The giantsize, minimum 46 inch screen lookssomewhat like an iPad on steroids".The navigation of the specimen is in-deed similar to Google Maps, whichalso permits for seamless zooming andnavigation.

The multitouch microscope haspromising applications in the area ofeducation and for scientific meetings.Several people can stand around thetouch screen and collaboratively viewand discuss the specimen.

Reference: University of Helsinki (2011, March 24).Gesture-controlled microscope developed by Finnishresearchers. ScienceDaily. Retrieved October 23, 2011,fromhttp://www.sciencedaily.com/releases/2011/03/110324103145.htmhttp://youtu.be/ihaM3DvyUHE?hd=1

■The Breaking Abbe’s Law

In 1873 Ernst Abbe formulated alaw, which limits the theoretically high-

est resolution of a microscope. Due tothe physical nature of light, it is notpossible to resolve structures that arecloser together than appox. 200 nm. Onthe 7th of September 2001, Stefan Hellfrom the Max Planck Institute for Bio-physical Chemistry received a prize forhis research that allowed him to circum-vented this law.

In STED microscopy (StimulatedEmission Depletion microscopy), aspecimen is first stained using a fluores-cent dye. In traditional confocal fluores-cence microscopy, the specimen isscanned with a laser beam and the X/Ycoordinates of the laser position arestored by a computer. All labeled struc-tures that are within the laser beam’sfocus start to light up at the same time.If two structures are too close together,then the two emitted spots of light willoverlap and it is not possible to see themas separate. Abbe’s law does thereforeapply.

Stefan Hell now managed to careful-ly control the time when the dye mole-cules emit their light. He used two laser

beams to achieve this. The first laser,the excitation beam, was quickly fol-lowed by a second laser beam, the de-excitation or STED beam. The excita-tion beam caused the dye molecules tolight up normally. The shape of theSTED laser spot was quite different,however. It was ring-shaped with a darkspot in the center. Dye molecules thatwere illuminated by the bright ringstarted to quickly emit their light, whilethe dye which was hit by the dark centerstarted to continue to shine slightly lon-ger. By carefully tuning the energy ofthe STED beam, it is possible to changethe size of the dark center. The smallerthe size of the “hole” the higher theresolution.

STED microscopy allows to theoret-ically achieve an arbitrary resolutionthis way. STED microscopy resolvesstructures that that are one molecule insize. As of 2009, the maximum resolu-tion achieved by this method was 5.8nm.

Reference: Stafford, Ned. "The million dollar micro-scope." Chemistry World. Royal Society of Chemistry,n.d. Web. 22 Nov. 2011.http://www.rsc.org/chemistryworld/Issues/2007/March/TheMillionDollarMicroscope.asp. ■

Figure 4: Comparison of various im-ages using regular microscopy (toprow) and the cell phone camera (bot-tom row). Image: Zachary J. Smith et al.

NEWS Recent Developments

Figure 5: Comparison of confocal mi-croscopy (left) with STED (center).The image on the right is a mergedimage of both techniques.

Image: ZoltanCseresnyes, UlfSchwarz, Cathe-rine M. Green


These are photosynthetic diatoms thatare common in a local drainage ditch.Olympus BH2, 20x SPlan Apochromaticobjective, 1.25x intermediate lens, 2.5xrelay lens, DIC. Nikon D90 camera.

(Anthony Thomas)

GALLERY Send images to: [email protected]

Anterior end of a young pupa of a FruitFly (Drosophila). You can still see thelarval mouthparts. Specimen in glycerineon a slide. Nikon 20x ELWD� objective ona bellows.

(Anthony Thomas)

Close-up of the Drosophila breathingtubes. Nikon 40x LWD objective on a bel-lows.

(Anthony Thomas)


Anchoring roots of ivy, stack of 25 imag-es made with Combine ZP.

(Manfred Rath)

A leaf cast of horseradish done by usingclear nail varnish. It is a mix of Rhein-berg- and oblique illumination.

(Manfred Rath)

GALLERY Send images to: [email protected]

A vitamin C crystal under polarized light.Vitamin C was dissolved in water andapplied to the slide. Crystal growth startswhen the Vitamin C has reached its satu-ration limit.

(Oliver Kim)


In a recent issue of the journal Na-ture, the book review section dis-cussed a new book titled "Field

Notes on Science and Nature", edited byMichael R. Canfield. Intrigued by thereview of this book I purchased a copy,and upon receiving it sat down and readit through in two days. The organizationof this book is unique and providesuseful information, examples, and ad-vice on keeping a scientific notebook.Each chapter in the book covers a dif-ferent aspect of scientific note keepingand each chapter is written by a differ-ent scientist who provides photographsof actual pages from their notebooks.The actual notebook pages provided inthis text are perhaps the most fascinat-ing and interesting part of the book, butthe comments of the scientists them-selves are bursting with information ofuse to anyone who keeps a scientificnotebook.

The foreword of the book is writtenby Edward O. Wilson, probably bestknown for his work with ants. A fewsamples of his notebook pages are pro-vided as well, and it is a pleasure to seethe simple yet clear descriptions he pro-vides of his observations. Once past theforeword of the book, each chapter iswritten by a different scientist, eachwell-known within their own field, andall offering differing perspectives andopinions on what is good and bad aboutthe notes they have kept. In one case ascientist goes back to notes he kept as achild and teenager in a small notebook.This gentleman also happened to be arunner, and in his earliest notebooks thebacks of pages lists various competi-tions participated in, listing the times,distances, his placement, and other de-

tails about his running meets. On thefronts of the notebook pages he wroteabout various observations he made(primarily) about birds and their nestinghabits, as well as other natural historydetails. As he progresses in his profes-sional career, we see his natural historynotes improve and expand.

Other chapters cover specific notetaking methods, such as "list keeping"and how it can be either of great value,or almost totally useless. Another chap-ter stresses the importance of includingsketches, maps, and other drawings, aswell as photographs. One of the fewthings almost all the contributors agreeupon is the importance of sketchingwhat you see. Many make a distinctionbetween the quick field notes one takeswhile observing or collecting, and themore detailed notes that are written outin greater detail when back in camp (orlab) as soon as one returns. Much moredetailed, such notes expand upon thefield observations that were made. Afew of the chapter names give you someinsight into the areas covered in thisbook: chapter 1 "The Pleasure of Ob-serving" by George B. Schaller, chapter3 "One and a Half Cheers for List Keep-ing" by Kenn Kaufman, chapter 5"Linking Researchers Across Genera-tions" by Anna K. Behrensmeyer, chap-ter 6 "The Spoken and the Unspoken"by Karen Kramer, chapter 8 "WhySketch?" by Jenny Keller, and chapter12 "Why Keep a Field Notebook?" ByErick Greene.

One of my favorite lines in the bookconcerns a scientist who states that bylooking in his journal notes from 50years ago he is able to bring back thatparticular day with great clarity. I can

think of no greater reason for keeping adetailed notebook. I strongly encourageyou to consider reading this book. Itwill teach you to take better notes, re-mind you why it is so important to takenotes, and provide notes from profes-sional scientists, including Charles Dar-win, that anyone involved with naturalhistory would realize are well withintheir own abilities to write. We may notall have the insights of a Charles Dar-win, but Darwin, without his noteswould be unknown today.

Wayne Wilson, amateur microscopist ,and professional pharmacist.McKinleyville, CA [email protected] ■

BOOK REVIEW Note taking

Keep a scientific notebook which documents your observations! A book review.

Wayne Wilson

Title:Field Notes on Science & Nature

Author:Michael R. Canfield (Editor), et al.

Publication Date: May 30, 2011Edition: 1Hardcover: 320 pagesPublisher: Harvard University PressISBN-10: 0674057570ISBN-13: 978-0674057579


A polarizing microscope is a spe-cial kind of microscope thatutilizes two polarizing lens to

acquire certain optical data from thespecimen. The polarizing microscope isused extensively in the field of opticalmineralogy which supports such appli-cations as geology, asbestos testing, andforensic science. Often those working indifferent fields will sometimes refer tothe polarizing microscope by differentnames such as geology microscope, pe-trographic microscope, pol microscope,and PLM (polarized light microscope.)

The key difference between the po-larizing microscope and a standardcompound microscope is the addition ofa fixed polarizer between the lightsource and the specimen and the addi-tion of an adjustable polarizer betweenthe objective and the eyepieces. The

2nd polarizer is called the "analyzer"and usually can insert in and out on arotating piece in the neck of the micro-scope. Other common accessories in-clude a rotating stage and insertableretardation plates made from gypsum orquartz.

With these additional elements, thethis microscope can acquire optical datafrom a specimen that no other micro-scope can. The key optical informationavailable includes refractive index, bi-refringence, sign of elongation, pleochr-oism, and angle of extinction, all ofwhich provide clues to the crystallogra-phy of the material that is being investi-gated.

The first uses of these kinds of mi-croscopes over one hundred years agowas the identification of minerals ingeology. In addition, the most common

form of lab analy-sis to test for as-bestos isperformed with apolarizing micro-scope. Because ofthe their ability toprovide opticaldata, these kindof microscopesare very com-monly used in fo-rensic sciencewhere the identi-

fication of unknown materials is a rou-tine part of the job. Some of the firstevidence disputing the claimed age ofthe so-called "Shroud of Turin" wasdetermined by analysis of pigments us-ing a polarized light microscope.

The polarizing microscope is a veryversatile and powerful instrument in theidentification of materials. It is a keytool in several scientific fields, and cansometimes be the best option over moreexpensive technologies. For example, inroutine asbestos analysis the polarizingmicroscope was determined to be moreaccurate and much more cost effectivethan the other high-tech options thatwere first investigated, such as x-raydiffraction and scanning electron mi-croscopy. It is a powerful tool withmany applications.

Nikon's MicroscopyU has quite a bitof information on how the polarizersand the science work to provide analyt-ical information:http://www.microscopyu.com/articles/polarized/polarizedintro.html

References:To learn more about microscopes please reviewhttp://www.where-to-buyazmicroscope.com

More articles written by MontyApollo can be found athttp://montyapollo.blogspot.com/

Article Source (excluding teaser lines):http://EzineArticles.com/?expert=Monty_Apollo

■

What are the areas of applications of polarizing microscopes?Monty Apollo

Figure 1: Polarizing image of a thin section of muscovite.Note the characteristic birefringence of muscovite.Rock: Nepheline syenite gneissLocation: Rio de Janeiro, BrazilImage credit: Eurico Zimbres

BEGINNER Polarization


When I search eBay with thekeyword "Microscope",amongst real microscopes, I

see these cheap nice looking instru-ments, offered at a real low price (fig-ure 1). Because I wanted to write thisarticle, I bid 99 cents on one of these. Idid win the microscope for 99 cents.

The catch was, it cost me almost$20.- to have it shipped. When I got it,I was not disappointed, it is a piece ofjunk. It is so hard to focus and hard onthe eye, that giving this as a gift to someone as an introduction to this wonderfulhobby should be a crime. I actuallypicked this one, because the ad said thatit had top quality optics, powersclaimed: 75X, 300X, 900X.

I could not get any image into focusat 900X. I did see something at 75X. Ithen took pictures of an easy object, adiatom with relatively large detail (fig-ure 3). For illumination I used a fluores-cent lamp with a ground glass in front of

it. After much effort to get it in focus,figure 3 shows what I got at 75X (Iworked hard on this).

I set up an other microscope, whichI bought used on eBay for $20. Ship-ping was free. This microscope isheavy, sturdy and the field of view ismuch larger and very pleasing to to theeye. Figure 2 shows this microscope.

Figure 4 shows a picture taken withthis microscope, using the same cameraand the same diatom slide, 10X objec-tive and 10X eyepiece. (I did not workhard on this one. I probably could havedone better)

The difference in image quality isnot even the biggest reason to stay awayfrom this junk. Those cheap, plastictoys are so hard to use that the user willlose interest very soon and thus willmiss out on what could have been a lifelong exciting hobby.

I would venture to say that almostany new microscope with standard size

objectives would be a good gift, espe-cially at the low powers, like 40X and100X. Those are the powers I use 95%of the time.

To contact the author, send an emailto: [email protected]. ■

How well do toy microscopes perform? Wilhelm Resch bought one to give it a test.

Wilhelm Resch

Figure 1: Magnification pow-ers claimed are 75X, 300X,900X.

Quick Guide for buyingMicroscopes

1. Buy microscopes with stan-dardized (ISO) optics.

2. Microscopes should be madeof metal and should be heavy.

3. They should have both acoarse and a fine focus knob.

4. Do not overemphasize magnifi-cation, it is usually the least im-portant aspect. Resolution isimportant. Without it, magnifica-tion is useless.

5. Good microscopes can not bebought in department stores. Talkto a dedicated microscope dealer.

6. Also consider buying stereomicroscopes for young children.They are generally cheaper thancompound microscopes and donot require specimen prepara-tion. (ed.)

REVIEW Toy microscopes


Figure 2: A $20 microscope withstandard ISO objectives. This micro-scope cost as much as the micro-scope shown in figure 1. For thesame price, the image quality is sub-stantially better.

Figures 3 and 4 (bottom): The left im-age was taken with the microscope infigure 1, the right image was takenwith the microscope in figure 2. Thedifference in resolution is significant.

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There are not many specimensthat are suitable for observationusing a 100x oil immersion ob-

jective. Both the depth of field and thefield of view are low. This makes theobservation of moving specimens espe-cially difficult. Unless one wants ofobserve sub-cellular structures (such aschromosomes), the 100x oil immersionobjective, be it bright-fiel or phase con-trast, does not have a particularly widerange of applications. Even the obser-vation of permanent slides is not easilypossible. They become contaminatedwith the immersion oil and must becarefully cleaned after every usage.Otherwise dust may start to collect onthe oily surface. Immersion oil on thepaper label does also not look very nice. The 100x oil immersion objective is,however, very suitable for observingbacteria. Unstained bacteria are bestobserved using phase contrast. Thistechnique will make the transparentbacterial cells appear dark on a brightbackground. Many amateur microsco-

pists and schools use bright-field optics,which are substantially cheaper. It isstill possible to observe live bacteria byclosing the condenser aperture dia-phragm. Diffraction patterns around theindividual cells start to appear, makingthe cells easier to observe. Stained cellscan also be seen with an open dia-phragm. At the same time I also would like tocall to your attention that bacteria areprobably not the most exciting speci-mens to observe. The cells will appearuniform under the microscope and thereare no sub-cellular details visible. Afterall, there are no membrane-bound cellorganelles in bacteria and they com-pletely lack visible cell internal struc-tures.

A warning

Do not spoil food (or other substanc-es) to grow your own bacteria. Do noteven make a hay infusion unless youreally know what you are doing. Enrich-

ing and concentrating unknown bacteriacan be a real health issue. Rather I rec-ommend you to use bacteria found inyogurt for your observations.

A Remark

At this time want to mention that theink that I had available was not verysuccessful in staining the bacteria. Itherefore recommend that you give adifferent substance a try (such as Meth-ylene Blue or Safranin).

Overview of the method

The process of preparing bacteriafor microscopic observation can be di-vided into the following four stages:Making a bacterial suspension: Atthis step the bacteria are brought into asuitable concentration.Heat-fixing the cells: This process im-mobilizes the cells and glues them to theglass slide.

Yogurt bacteria can be observed in a wet mount or after heat-fixing and staining them.

Oliver Kim

Figure 1: Working like a true “ama-teur”. Yoghurt sample (top), suspen-sion of yoghurt bacteria in water (left)and a regular plate for drying thesamples.

HOW TO Safe bacteria to observe


Staining: The chemical stain enter thecells and react with different parts of thecell.Observation: Observation occurs ei-ther at 400x or at 1000x using an oilimmersion and no cover glass.I will address each one of these stagesin the following parts .

Making a bacterial suspension

The bacteria are harvested (take asmall knife tip of yogurt) and are sus-pended in a small amount of water or in0.9% NaCl solution (0.9 g NaCl in100ml of water). Using NaCl solutionprevents the cells from osmoticallybursting, but water will also work in

many cases. If the bacterial suspensionis too concentrated, then many of thecells will overlap on the slide and it willbe difficult to see the individual cells.The suspension should be only slightlyturbid. It is probably best to experimentwith the concentration, as much willdepend on the bacterial concentration ofthe original sample. Ideally the bacterialsuspension should spread over a largersurface on the slide. If you agitate thesuspension to vigorously, then you maybreak apart the chains of Staphylococci,which allow for an easy identification.If you want to make a wet mount, thenit is also possible to directly suspend asmall amount of yogurt on a drop ofwater right on the slide.

Fixing the bacteria

Fixing kills the cells and sticks themto the glass slide so that they can not bewashed off during the staining process.Fixed bacteria are also easier observe,because they do not float in and out ofthe focus. Heat fixing is like frying anegg in a pan without oil. The proteinsstart to denature and the cells stick tothe glass slide much like an egg stickingto the pan. We have already applied a drop ofthe bacterial suspension to the slide.Now we need to allow the slide to air-dry completely without heating theslide. If you heat the slide then thesteam pressure in the cells may causethem to burst. Slight warming is OK,this may indeed speed up the dryingprocess. Be careful because even the

Figure 2: The left and the right sam-ple flow over a larger area. This is thepreferred situation. The slide in thecenter had an oily surface and thedrop may contract during the dryingprocess. This increases the concen-tration of the bacteria and may resultin clumping of the cells. The individu-al cells therefore become more diffi-cult to discern.

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lowest setting of a hot plate is often toohot and the temperature is often difficultto control. Do not put the suspension inthe center of the slide, place the dropfurther towards one end. This way youstill have enough space left to hold theslide without burning yourself whendoing the heat-fixing procedure. The original heat-fixing process re-quires the use of a gas Bunsen burner.Pull the dry slide, specimen on top, twotimes rapidly through the flame of theBunsen burner. The slide should be-come very hot, but you should still beable to just hold it in the open palm ofyour hand without burning yourself. Ifthe temperature is too low (due to ashort heating time), then the bacteriawill not stick to the glass slide and willbe washed off during the staining proce-dure. Under no circumstances shouldthe bacterial sample start to smoke orturn brown. This indicates that there isoxidation taking place and that the spec-imen has been destroyed. As a matter offact, it can be commonly observed thata properly heat fixed bacterial sample isactually slightly brighter, than a speci-men slide which has only been dried inthe air. The place where the bacterialsuspension has been applied becomesslightly more white-ish. While I do notknow the actual causes for this changein color, I do assume that this has to dosomething with protein denaturation,similar to the color change of a fryingegg. I always pull the slide top-downthrough the flame, holding the slide atthe edges with two fingers. I always testthe temperature of the slide by placingit on my open palm (please don't blame

me if you burn yourself). Here it paysoff to have the sample at the far end ofthe slide as you may otherwise also burnthese fingers. Pulling the slide sidewaysthrough the flame may increase the tem-perature too rapidly , possibly crackingthe slide. On the other hand, I guess itshould not really matter much, becausethe temperature should not become toohigh anyway. The total heating durationis about 1-2 seconds. Many microscopy enthusiasts willnot have a Bunsen burner available. In

this case it is possible to briefly placethe slide on a hot plate (I tried this, itworks) or to use a gas-operated lighter(I have never tried this). The small di-ameter of the lighter flame may causean irregular heating of the slide and mayinduce cracking. Be careful. Candlesare probably not as suitable, as they willresult in soot deposits on the slide.

Figures 5 and 6: The images showheat-fixed and stained samples attwo different focus levels. The largeblue cloud-like structures appears tobe stained curdled milk. The roundobjects in short chains are yogurtbacteria.

Figure 7: A long chain of yogurt bac-teria attached to what appears to becurdled milk (bottom). Wet mount,not stained.

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Staining the bacteria

The staining process is relativelysimple, unless one uses differentialstaining (in which more than one stainis used). First, allow the slide to cooldown to room temperature. Severaldrops of the stain are then applied to thefixed bacteria and given time to do itswork. Depending on the affinity of thestain to the cells, this can last severalminutes. It is possible to make a time-series in 5 minute intervals to find theoptimum staining duration. Alternative-

ly it is possible to submerge the wholeslide into a slide-holder filled with thestain, but this may contaminate the stainover time. The stain is carefully removed byrinsing the slide in running water. Holdthe slide at a steep angle and allow athin water stream to run over the slide.The water should not be applied directlyto the fixed bacteria, but above it. Youcan easily see that the stain which didnot come in contact with the bacteria iswashed away quickly, while the bacte-ria themselves start to retain more of the

stain. The washing process can bestopped after there is no more discolor-ation of the washing water. The individ-ual cells should still have retainedenough stain for observation (unlessyou over-wash and completely de-stainthe cells).

The result

To get right to the point: the ink was notable to stain the bacteria well. This maybe due to the small size of the cells,which are not able to retain much ink.The ink did stain the curdled milk,though (figures 5 and 6)! Even underbright field conditions, the Streptococcicould easily be seen as short chains.They had a strong tendency to break thelight and appeared either as dark or verybright spots, depending on the focus.The staining effect of the ink was there-fore not very visible. The curdled milkflakes did not show this tendency andremained uniformly blue. In that sensethe staining process did help to identifythe bacterial cells! The bacteria in wet samples floataround and can be seen as individualcells. During the drying process, manycells may start to aggregate and clumptogether. This effect is particularly pro-nounced when the cell density is toohigh. In this case, it becomes very diffi-cult to see individual cells.There is also the possibility that the longchains of bacteria can be broken apartby the preparation process, makingthem more difficult to identify.

Final thoughts

Heat fixing the bacteria preventsthem from floating around and makes iteasier to find a focus. I found out thatthe ink that I used did not dye the cells.It can also be that the staining was soweak that I was not able to observe it. Inany case, the blue ink seemed to havebeen able to stain the curdled milk,which most certainly also containedbacteria. ■

Figures 8 and 9: Streptococci in a yo-gurt sample. Curdled milk seems tobe the larger flakes.

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Purple-sulfur phototrophic largebacteria stand out clearly to theunaided eyes, when one encoun-

ters their brightly pink-magenta colored'blooms' at the bottom of a stream.These are wonderful stream organismsto enjoy with the 'naked eye' on relaxingstream hikes. Find a stream shore sec-tion with dark black sulfide sediments.You should be able to smell the sulfurwhen you probe into the sediments. Youthen can reasonably expect to find the

bright pink-magenta 'purple-sulfur bac-terial blooms' during some season of thestreams yearly cycle. The location ofmy freshwater inland stream is at anorthern temperate latitude. Each indi-vidual bacterium has a rapid motility.They respond and orient to light levels,sulphide levels, and indirectly to theoxygen levels of the waters they inhabit.These bacteria either rapidly retreat intothe stream floor sediments or form bril-liant sediment surface films. These mi-

crobe-biofilms are dominated by thissingle species! The bacterial bloomsshow brilliant pink-magenta sedimentsurface biofilms.

These are very reactive communi-ties. They possibly respond to diurnalfluctuations and to heavily cloudedskies overhead. These bacteria pose allsorts of questions to a microscopist onan outdoor hike and all sorts of observa-tion possibilities - together with a loyal

Figures 2 and 3: The wa-ter temperature was mea-sured to be 43°F (6°C).Three gauges agree.

A magenta-colored bloom can be an indication for the presence of purplephototrophic bacteria. Aggregates of these bacteria were found ona decaying leaf.

Charles Guevara

Figure 1: Bloom of purple sulfur bac-teria. There are indications of bloomformation on the sediments.

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OBSERVATION Sulfur Bacteria


puppy always “in the thick” of me beingbent scrutinizing the bloom sites. Very recently I encountered a fallentree leaf, soaked and resting in one ofthe few niche-habitats of purple-sulfurphototrophic bacteria. Over the courseof two years these manifested brilliantpink-magenta blooms. This bottom rest-ing tree-leaf offered a hint of this deli-

Figures 5-6: The site of bacterialblooms observed over two years ofthis season.

Figure 7: My puppy at a favorite site!

Figure 4: A repeated 'bloom site'. Thisis a niche-habitat on the stream nearthe bank. This is the location (niche-habitat) for two year’s of purple-sulfurbacterial blooms.

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cate pink-magenta bloom. I transportedit home to my microscopy bench.

I have pleasantly observed, for thefirst time, aggregations of large purple-sulfur bacteria, aggregations of thesewonderfully reactive large bacteria on adecomposing leaf. These I collectedfrom the stream niche-habitat where'blooms' have repeatedly manifested fortwo years.

I still have several simple goals.1) Right at stream site, I want to careful-ly lift a delicate 'bloom sample' to a

wet-mount slide. I want to image thesamples with a field microscope. 2) I want to observe the protists and themeiofauna which occurs with thisunique 'single species dominated bio-film'. 3) I want to observe the morphology ofthe huge bacteria within the biofilm. Iwant to see if patterns of these bacteriaare evident, such as orientations, re-volving cells, morphotype differences,etc.

Delightfully, eukaryotic protists oc-curred within the sample of purple-sul-

fur bacteria. This niche habitat is acomplexity of organisms offering manywonderful microscopy opportunities!

Source materials

"The Phototrophic Bacteria, anerobiclife in the light", John G. Ormerod,1983. ■

Figures 8 and 9: The sample tree leaf at my microscopy bench.

Figure 10: bacteria swarming up into the water colum from the bottomsediment.

Figure 11: : this leaf kept in it's collection jar for days

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Figures 12, 13, 17: Bacteria swarm outof the leaf structure, some encountera diatom.

14: Bacteria gathered in cluster withina decaying leaf

15: DL phase view of swarming pur-ple-sulfur phototrophic bacteria.

16: Dividing bacterium

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Regular fountain-pen will stain epithelial cells. The ink also makes intracellular structures, suchas the cell’s nucleus, visible.

Oliver Kim

Observing and staining epithelialcells from the mouth is one ofthe easiest methods to directly

observe human cells. The cells can bestained with water-based ink and struc-tures inside the cell, such as the nucleus,can be made visible this way. The sim-plicity and quickness of the procedurealso makes it a suitable project for intro-ductory microscopy courses in schools. Epithelial cells from the mouth asuitable for several reasons. First, theycan be obtained easily by scratching theinside surface of the cheek with a cottonswab (or your fingernails). The cellswill come off easily. Second, the cells

are relatively flat and can therefore beobserved without much preparation.

Stains used

For the staining of the epithelialcells, I experimented with two differentkinds of ink: the Royal Blue 4001® inkfrom the company Pelikan and the blackQuink® ink from the company Parker. Isimply happened to have these aroundin my household and decided to givethem a try. These inks are used forfountain pens and are water based. Iconsidered them reasonably safe alsofor classroom usage and therefore gave

them preference over other (more com-monly used) stains like Methylene Blue.The fountain pen inks can also be easilyand cheaply obtained from paper sup-plies companies. A quick research re-vealed that the blue ink belongs to thetriarylmethane dyes.

Collecting and preparing thecells

I used a cotton swab (those that areused for cleaning one's ears) to collectthe epithelial cells. In order to obtain asufficiently large number of cells, it isnecessary to vigorously scratch the in-side of one's cheeks with the cotton

Figure 1 (bottom): A regular wetmount is prepared with only little wa-ter. A small drop of ink is then ap-plied to the edge of the cover glass.The ink is pulled in and stains thecells.

Figure 2 (right): Some of the epitheli-al cells already absorbed much of theink, turning intensely blue. Noticethat the background starts to turnbrighter as more and more cells startto absorb the ink.

HOW TO Safe staining methods


Figures 3 and 4 (top): The nucleus of each cell is visible as a dark blue structure inside each cell.

Figures 5 (top): Large aggregates of epithelial cells start to stain from the outside inwards. Notice that the nuclei start to stainfirst, they seem to have an extremely high affinity for the ink.


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Figure 6 A-H (opposite page): Theseepithelial cells were stained withblack ink. The images show a time-series over several minutes. Theblack ink is evidently composed ofblue and yellow components. Thesecolors seem to separate as they enterthe cell. First the yellow componentof the ink enters the cell, followed byblue. Over time the backgroundstarts to brighten up. This indicatesthat more and more ink is absorbedinto the cells.

Figure 7 (right): The image shows an-other aggregate of cells that werestained with black ink. The bright ar-ea towards the bottom of the image isan air bubble.

swabs. I then streaked the cells on aslide and added a small drop of water tomake a temporary mount. It is also pos-sible to use a small spoon to scratch offsome of the cells (I tried it and it works),but this is less comfortable.

Staining the cells

The slide is placed on the stage andthe cells are then focused. A small (!)drop of ink is then placed at the cornerof the cover glass. Too much ink willstain the background too dark and it willbecome difficult to see the individualcells. The ink is then pulled into thesample, beneath the cover glass, provid-ed that not too much water was used formounting the cells. The ink will gradu-ally spread over the whole specimen. Itried this process with both the blue andthe black ink, and with a surprising re-sult.

The result

The epithelial cells showed a strongaffinity to both the blue and the black

ink. As a matter of fact, the cells en-riched the dye to the extent that theybecame significantly darker than thesurrounding medium. The pictures onthe previous pages and on this pageshow that the background of the cells issignificantly brighter than the cellsthemselves. Also the nuclei are clearlyvisible.

The black ink demonstrated a partic-ularly interesting behavior (figures 6and 7). Black ink seems to be a mixtureof blue and yellow ink. First the yellowcomponent of the ink entered the epi-thelial cells. The blue color was ab-sormed much slower. Figure 6 shows atime series of a collection of cells,which first turn yellow and then blue.

Osmosis test

I could not restrain myself and want-ed to test the response of the cells toosmotic stress. I applied a decent dropof saturated salt water to the corner ofthe cover glass and observed how thesalt water was drawn in. The new fluidquickly washed remaining ink from the

medium away. The cells themselvesretained the color. I hoped to be able toobserve plasmolysis of the cells. I ex-pected the cells to lose much water andconsequently to shrink. I could not ob-serve this, however. Likewise, I applieddistilled water to a different sample ofepithelial cells and hoped to see themswell and burst. This would have madean interesting experiment for schools.Unfortunately I could not observe eitherresponse. I suppose that the epithelialcells are generally more resistant to os-motic extremes. After all, they have towithstand the wide variety of differentfood sources (and liquids) which weconsume on a daily basis.

Conclusion

One of the most surprising resultswas that the cells stained darker than thesurrounding medium. Motivated by theresult I will now attempt to use otherwater-based stains, including food col-oring. Anyone care to try out (and re-port back) on the efficiency of thesestains? ■


What’s this? Answer on page 3.

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