May 2012 - MicrobeHunter Microscopy Magazine

Transcription

Microbe
Hunter
Microscopy Magazine
ISSN 2220-4962 (Print)
ISSN 2220-4970 (Online)
Volume 2, Number 5
May 2012
The Magazine for the
Enthusiast Microscopist
http://www.microbehunter.com
Historical
Stereo Microscopy
Backyard Forensics
Picture Gallery
Making Fluid
Mounts
Desmids:
Micrasterias rotata
Yeast Respiration
History of Stereo
Microscopy
Mystery Object
Fluid Mounting
MicrobeHunter Microscopy Magazine - May 2012 - 1
ABOUT
Microbehunter Microscopy Magazine
The magazine for the enthusiast microscopist
MicrobeHunter Magazine is a non-commercial project.
Volume 2, Number 5, May 2012
ISSN 2220-4962 (Print)
ISSN 2220-4970 (Online)
Download: Microbehunter Microscopy Magazine can be downloaded at: http://www.microbehunter.com
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Publisher and editor:
Oliver Kim, Ziegeleistr. 10-3, A-4490 St.Florian, Austria
Email: editor@microbehunter.com
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Images and Articles by:
Guwak, Mike
Kim, Oliver
Kreindler, R. Jordan
Monzo, Luca
Nassar, R.
Rath, Manfred
Vogel, Johann
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Disclaimer: Articles that are published in Microbehunter Microscopy Magazine and the blog do not necessarily reflect the position or opinion of the publisher. The publication of these articles
does not constitute an endorsement of views they may express.
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Front Cover:
Large image: Oliver Kim (Pollen)
Left image: R. Jordan Kreindler (butterfly)
Middle image: Johann Vogel (Plane tree fiber)
Right image: Oliver Kim
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2 - MicrobeHunter Microscopy Magazine - May 2012
CONTENTS
4
Stereo Microscope: Part 4 - Conclusion
This paper completes a four part series on stereo
microscopes, their history, design and applications.
It presents concluding comments, and offers some
suggestions for potential stereo microscope buyers
4
R. Jordan Kreindler
12
Backyard Forensics: A Case of Mistaken Identity
Asbestos? Fiberglass? Amateur microscopy comes
to the rescue!
Johann Vogel
14
Gallery
Images from Luca Monzo, R. Nassar, Manfred Rath
and Oliver Kim
19
The Difficulty of Making Fluid Permanent Mounts
Here I would like to investigate the making of
permanent slides using liquid mounting media.
23
Micrasterias rotata
Oliver Kim
Mike Guwak
25
Yeast CO2 Production
Carbon dioxide production by yeast can be observed
under the microscope.
Oliver Kim
12
Answers to the crossword puzzle from last
month
Across: 2:mirror 3:apochromatic 4:arm 7:cmount 9:eyepiece 11:focus 13:fine 14:tube
16:condenser 17:base
Down: 1:diopter adjustment 5:diaphragm
6:achromatic 7:cover glass 8:DIN 10:coarse
11:field of view 12:mechanical 15:LEDs
Answer to the puzzle (back cover):
Salt crystals
23
HISTORICAL
MICROSCOPY
Stereo Microscopy
This paper completes a four part series on stereo microscopes, their history, design and
applications. It presents concluding comments, and offers some suggestions for potential
stereo microscope buyers.
R. Jordan Kreindler
Figure 1: Hand dug, small crystals, Mt. Ida, Arkansas USA through stereo Greenough-style trinocular microscope
Stereo Microscopy
HISTORICAL
MICROSCOPY
S
tereo microscope use can be
thought of as belonging to one of
four application areas: (1) biological (including medicine), (2) geological
(including mineralogy and gemology),
(3) industrial, and (4) other specialized
applications (including archaeology,
numismatics, philately, and forensics,
etc.). Although the first is probably the
largest, it's in the last three areas, augmented with some notable additions
from the first, where the stereo microscope finds its greatest applications.
In a compound microscope most
objects to be viewed are first "sliced"
into thin sections, often 1 to 100 micrometers thick. Section thicknesses toward the lower end of this range are
more common. This allows transmitted
light to pass through a specimen. In
these microscopes working distance and
depth of field are shallow, and resolution is relatively high. Most compound
microscopes are built to view objects
under "coverslips". However, stereo microscopes are designed to view specimens directly, without "coverslips".
They can be used to see microscopic
subjects without the need for complex
preparation for viewing.
Stereo microscopes yield quite spectacular views of larger subjects "in context", and with their larger field of view
and greater depth of focus, they can
provide insights that are simply impossible to get with a higher power instrument. Compound microscopes are
usually used at 100x and above, while
stereo microscopes are frequently used
at 40x and below. It's often best for
many microscopic examinations to start
with lower magnifications, the domain
of stereo microscopes.
Stereo microscopes show the true
colors of the objects studied, Figs. 1
through 4, as opposed to the "false"
colors of stained specimens often used
with higher magnification instruments.
Older stereo microscopes can be attractive, and were often very solidly
constructed. These instruments are often
available at relatively low prices. Modern stereo instruments with their computer-designed
lenses,
built-in
high-longevity cool LED illumination,
and strong chemically resistant finishes
are also often available at attractive
Figure 2: Distal region of wasp wing
Figure 3: Black Swallowtail (Papilio polyxenesis) butterfly wings
HISTORICAL
MICROSCOPY
Stereo Microscopy
Figure 4: Microfossil Missouri, USA
prices. These modern stereo microscopes are fully adequate for serious
use.
LEDs generate almost no heat and
last, perhaps, 50,000 to 100,000 hours,
compared to the usually less than 500
hours for traditional tungsten or halogen
bulbs. LEDs also use less power. It may
seem that some of the more compact
models with LED illumination would
be appropriate as field stereo microscopes, as those with batteries can continue to provide functional illumination
through many field trips. Typical modern stereo microscopes with battery
powered LED illumination will last for
over a week of field trips (i.e., over 48
hours of continuous use). Because of
their ability to work with battery power,
microscopes with built-in LEDs need
not be near power outlets to use.
However, some compact and battery
powered LED stereo microscopes have
diminished optical quality compared to
their benchtop cohorts, particularly
those made to sell at low price points.
On some of these compact models,
built-in LED illumination can be insuf-
ficient to provide the full intensity of
light needed for photography. Thus, a
higher quality stereo microscope without built-in lighting, can be a viable, and
a possibly more appropriate option, for
field use than a compact low-cost LED
instrument. If high-longevity LED illumination is desired, it can be provided
by external LED lamps with or without
intensity controls, such as those shown
in Fig. 5. The lamp on the left provides
a gentle diffused light that works well
for visual incident illumination. The
lamp on the right can provide adjustable
intensity spot illumination suitable for
photography though stereo microscopes
using either incident or transmitted illumination.
Although they can function satisfactorily, in general, older stereo models
should be considered primarily as collectibles, in view of the quality of computer-designed lenses, and the relatively
low cost and features of more current
models. However, this is not necessarily true for older compact stereo microscopes without built-in illumination that
may be specially useful in field work,
particularly if work involves screening
of items to bring back for examination
by benchtop instruments.
Many recently discontinued models
by American Optical, Bausch and
Lomb, Haag Streit, Leitz, Olympus
starting with the SZH CMO model, Reichart, and Zeiss are of higher quality
than most of their competition, albeit at
a higher cost. However, as some of
these companies are no longer in the
microscope business, replacement parts
can be harder to obtain. Today Leica,
Nikon, Olympus, and Zeiss make some
exceptional top end stereo microscopes.
Fortunately, with the evolution of
computer-designed lenses, many relatively inexpensive stereo microscopes
sold by AmScope, Barska, Carolina Biological Supply Company (Wolfe),
Swift and other modern stereo microscopes are of good optical quality, and
available at relatively low cost. Although, with inexpensive instruments
care must be taken, as some models
from less well-known vendors, and low
end models from known vendors can be
of diminished quality.
Some factors to consider when selecting a modern stereo microscope, in
addition to image resolution, contrast,
and flatness of field are:
(1) an illumination intensity adjustment rather than just a simple on/off
switch,
(2) the presence of top and bottom
illumination that can be used simultaneously if desired,
(3) "cold light" as provided by fluorescent or LED illuminators (although
this can be added as ring lighting or
external lamps later), it's better if this
lighting is already built-in,
(4) the magnification range (a single fixed magnification is generally not
as desirable),
(5) the presence of zoom capability,
or the number of changeable fixed magnification choices,
(6) the presence of diopter adjustments for the eyepieces, better if you
wear glasses,
Stereo Microscopy
(7) the presence of a column height
adjustment in addition to rack and pinion focusing, this can allow for a greater
vertical range and the examination of
taller objects,
(8) eyepoint, i.e., the distance above
the eyepiece at which you can still see
the entire field of view - high eye relief
eyepieces are particularly important for
eyeglass users,
(9) the field of view (FOV),
(10) height of the focus knob, a
lower knob is usually easier to use.
One of the primary weaknesses of
lower cost instruments is their often
limited field of view (see below), usually measured in millimeters for the diameter of an object seen through the
eyepieces. The field of view for any
combination of optical components in a
stereo microscope can be determined by
placing a millimeter ruler under the microscope and counting the millimeters
across the diameter of the circle seen.
Field Numbers (FNs in mm) are
often placed on eyepiece housings by
stereo microscope makers. Here, the
larger the better. That is, if a larger field
of view (FOV) is desired select eyepieces with a larger field number. To mathematically determine field of view in
millimeters, when a FN is available, just
take:
[Field Number of the eyepiece] /
[(objective magnification) x
(auxiliary lens magnification, if any)],
assuming no other magnification changes.
If your eyepieces have a Field Number of 25, and are used with 5x magnification without auxiliary lenses, the field
of view is 25/5 = 5mm. That is, about
5mm of an object diameter can be seen
without moving the object. (Recall there
are 25.4mm/inch.) This calculation can
be worked in reverse using a millimeter
ruler to determine the FN if not available for any eyepieces (see below). If an
eyepiece FN is available, the magnification of the eyepiece is not needed to
determine the FOV in the formula
above. So, a 12.5X/23 and a 10X/23
would have the same field of view.
Today, common wide field stereo
eyepiece field numbers are plus or minus:
26 for 5x eyepieces,
23 for 10x eyepieces,
20 for 12.5,
15 for 15x, and
7 for 30x.
The field number is always slightly
less than the diameter of the eyepiece
tube. Some stereo binocular heads may
prevent the full realization of an eye-
HISTORICAL
MICROSCOPY
Figure 5: LED lamps. Left: Diffuse
lighting with 12 LEDs, on/off only.
Right: Single bulb, spot with on/off
and intensity control
piece's field number. This is not the case
for stereo microscopes and their companion eyepieces made by the major
manufacturers.
One way to conceal poor optics is to
reduce the eyepiece field of view, so
FOV should always be considered before a purchase. Fortunately, most lowcost stereo microscopes are fully satisfactory, except for more demanding research applications. Even inexpensive
Chinese microscopes can have reasonable FOVs. I recently measured the
FOV for an inexpensive Chinese trinocular with WF10X eyepieces using an
objective magnification of 1. It yielded
an object diameter of 20mm. So, it had
a FN of 20 (20/1 = 20mm).
Before the 1970s about 3/4ths of
stereomicroscope applications were in
the life sciences. The 1970s saw the
rapid growth of the semiconductor industry and the use of zoom stereo microscopes for the examination of thin
sheets of semiconductor material,
called wafers, and integrated circuits,
Fig. 6, and circuit boards. The most
HISTORICAL
MICROSCOPY
Stereo Microscopy
Figure 6: Portion of a damaged integrated circuit (IC) as seen through a
Greenough-style stereo microscope
Stereo Microscopy
common zoom microscopes used by the
semiconductor industry were of the
Greenough type. Bausch and Lomb StereoZooms, in particular, became popular with the growing technology
companies in Silicon Valley, and were
sold in significant numbers. They are
still widely available, e.g., almost any
week on eBay, although their production stopped at the beginning of this
century.
Many, if not most, of the stereo
zoom microscopes were industrial purchases. They were often used extensively, and tend to be well worn both
visually and mechanically, and often
have optical problems
(Kreindler,
2012). Potential buyers should carefully
examine/consider any possible acquisition of a stereo zoom microscope, particularly if it's to be purchased for use
rather than display. If an instrument's
stage shows extensive wear, this is almost always an immediate indication of
heavy prior use, and these instruments
should usually be avoided. Many later
model stereo instruments were build as
pods, i.e., the microscope itself without
a stand or other components, so they
could be used with a variety of stands
and other components. Figs. 7, 8 show
examples of pods from American Optical and Bausch and Lomb.
Today, one often sees stereo zoom
instruments sold as "pods" only, which
can mask heavy use, as it's not possible
to determine stage or frame wear.
It's probably best to avoid the purchase of used stereo pods that have been
removed from their stands, particularly
if they show signs of wear and rough
handling.
Some of the used stereo pods I've
recently handled have had minor or
major optical problems (including image alignment issues, or a failure to
focus both eyes simultaneously, or produced blurred images).
If you are considering the purchase
of a stereo pod, if available for sale
online, ask the seller if images through
the instrument come into sharp simultaneous focus with both eyes, and about
their return policy.
Avoid purchasing any "As Is" stereo
pods, unless you're comfortable clean
HISTORICAL
MICROSCOPY
Figure 7: AO StereoStar Zoom pod
ing and aligning optical elements,
should this be a problem. Remember,
the cost to fix some problems can easily
exceed the current value of the pod.
Be particularly cautious of stereo
microscopes sold at government or university surplus sales. These microscopes were often roughly handled, as
they were not the personal property of
the user(s). School and University instruments often have station or acquisi-
tion numbers stenciled or engraved on
the stands or the microscopes. The presence of a station or acquisition number
is usually a "red flag". Also, many government microscopes were roughly
handled, particularly after they were
declared surplus, often simply placed or
just tossed into a storage carton before
sale. These should be carefully examined before possible purchase. Think
HISTORICAL
MICROSCOPY
very carefully before bidding for a stereo instrument on the basis of pictures
alone.
New stereo microscopes range in
cost from relatively low prices, less than
$50 USD, to quite expensive instruments, above $6,000. More expensive
models often come with higher quality
objectives, high build quality, multiple
pairs of objectives on a rotating turret or
large zoom ratios, trinocular arrangements, longer working distances, and
built-in and adjustable high-intensity
illumination for photography with
transmitted or reflected light.
Stereo microscopes make great gifts
for beginners, as objects can be placed
under the microscope, without prepara-
Stereo Microscopy
tion, and the viewer is usually amazed
with the magnified 3D view they find.
The relatively long working distance
and large distances possible between the
stage and the objectives allow for larger
objects to be examined, if desired. Stereo microscopes are also excellent tools
for some household activities such as
the repair of small objects, examining
plumbing contamination, checking
knife edges, or looking more closely at
coins, stamps, or jewelry, etc.
With the wide availability of modern and relatively inexpensive stereo
microscopes, there is now a stereo microscope to fit almost any budget.
Figure 8: Bausch and Lomb StereoZoom stand and pod with coaxial
lighting option
©2011, 2012 Text and photographs by
the author.
The author welcomes suggestions for
corrections or improvement. He can be
reached at:
R. Jordan Kreindler:
leona111@bellsouth.net
■
Stereo Microscopy
HISTORICAL
MICROSCOPY
Combined References and End Notes
Allen, R. M., (1940), The Microscope. Boston:
D. Van Nostrand Company, Inc., p87.
Bryant, Dr. Mark L., (2012) Thanks to Dr. Bryant and his staff for permission to photograph their Topcon slit lamp.
Bausch & Lomb Optical Co, (1929) Microscopes
& Accessories: Photomicrographic and Micro- Projection Apparatus Microtomes . Colorimeters Optical Measuring Instruments
and Refractometers. Bausch & Lomb" New
York, p 81.
Carpenter, William (with revisions by Rev. W.
H. Dallinger) , (1901), The Microscope and
Its Revelations. Eighth Edition. Philadelphia: P. Blakiston's Son & Company, p 96.
Carl Zeiss, Jena (1937) Microscope catalog.
Cherubin, d'Orleans. Père, (1677), La Dioptrique
Oculaire ou La vision parfait ou le concours
des deux axes de la vision en un seul point
de l'objet , Paris: S. Mabre-Cramoisy
Davis, George E., F.R.M. S. (1882) Practical Microscopy. David Bogue, London
Encyclopaedia Britannica, (1910), - A Dictionary
of Arts, Sciences, Literature and General
Information, 11th Edition, Volume 3, Binocular Instrument. New York, p 950.
Ferraglio, Paul L., (2008), The Riddell-Stephenson Binocular Microscope. The Journal of
the Microscope Historical Society. Volume
16. - The author thanks Dr. Ferraglio, a
leading authority on Prof. Riddell's microscope and its successors, for providing reprints of his papers, and his helpful
comments on the draft section for the Riddell microscope. However, the content of
this section is the sole responsibility of the
author.
Ford, Brian, (1973), The Optical Microscope
Manual. Past and Present Uses and Techniques. New York: Crane, Russet & Company, Inc.
Goren, Yuval, The author's thanks to Dr. Goren
for his continued emphasis on the importance of setting microscopes in their historical context, rather than just discussing their
physical characteristics, and for the many
exchanges we've had on historical microscopes.
Gubas, Lawrence J., (2008) A Survey of Zeiss
Microscopes 1846-1945. Las Vegas: Graphics 2000, Color photographs of Model XV
and its storage case can be found on page
253 of this book. This book can be recommended for its detailed discussions, illustrations, and photographs of Zeiss microscopes.
Hagan, Kevin (private correspondence, 2011)
Thanks to Mr. Hagan of ALA industries
Limited, Valparaiso, Indiana for providing
a Contamikit brochure and PDF of the "Instruction Manual".
Journal of the Society of Arts, Vol XXXIV,
(Nov 1886). London: George Bell and Sons,
for the Society of Arts, Fig. 16, p 1014.
Kreindler, R.J. and Yuval Goren, (March 2011),
Comparison of the Swift FM-31 Portable
Field Microscope and an FM-31 Clone, Micscape, Figs. 11, 12, and 13.
Kreindler, R.J. and Yuval Goren, (May 2011),
Baker's Traveller's Microscope, Micscape
Kreindler, R.J. and Yuval Goren, (November
2011), The TWX-1 Folded-Optics Microscope, Micscape
Kreindler, R. J. (2012) The author worked in Silicon Valley for a number of years and often
saw the extensive use, and occasional abuse,
stereo microscopes in high-tech companies
were often subjected to.
Maertin, Rainer , www.photosrsenal.com for permission to use the photo of the Brewster
type stereo viewer.
Moe, Harald, (2004), The Story of the Microscope. Denmark: Rhodes International Science and Art Publishers with the
Collaboration of The Royal Microscopical
Society, p. 176.
Nikon Microscopy U (undated) "Introduction to
Stereomicroscopy" states, "The first modern
stereomicroscope was introduced in the
United States by the American Optical Company in 1957. Named the Cycloptic®, this
breakthrough design...". This was a landmark in American stereomicroscopes. However, the common objective concept was
first used by Riddell in 1850s, while the
common large objective was later implemented by Zeiss in their 20th century Citoplast design, considerably before the
Cycloptic® introduced.
Orlowski, Kristen and Dr. Michael Zölffel (private correspondence, 2012) - The author's
thanks to both Kristen Orlowski, Product
Marketing Manager, Light Microscopes,
Carl Zeiss Microscopy, LLC and Dr. Michael Zölffel, Carl Zeiss MicroImaging
GmbH, Jena, Germany for information and
materials they provided regarding Zeiss history.
Phillips, Jay. (private correspondence, (2011,
2012) Provided a copy of Zeiss' catalog
"Mikroskope für Wissenschaft und Technologie" (Prob. 1951).
Purtle, Helen R. (Second Edition), (1987 reprint). The Billings Microscope Collection.
Second Edition. Washington, D.C.: Armed
Forces Institute of Pathology, p 228, Figure
458 (Catalog number: M- 030.00541, AFIP
accession number: 518,969, MIS photograph: 73-3899)
Riemer, Marvin F., (1962) Microscope and the
World of Science. New York: SCOPE Instrument Corp.
Sander, Klaus. (1994) An American in Paris and
the origins of the stereomicroscope. Institut
für Biologie I (Zoologie). Freiburg, Germany: Springer-Verlag,
Schulze, Fritz , (2011, 2012), The author would
like to thank Mr. Schulze, former head of
the Historical Microscopical Society of
Canada, for our extended exchanges on stereo microscopes.
Schwidefsky, Kurt,( 1950) Grundriss der Photogrammetrie, Verlag für Wissenschaft und
Fachbuch: 1950 (Reference from Fritz
Schulze).
Wade Nicolas , (1998) A Natural History of Vision. Cambridge, Mass: MIT press,p 301.
Waldsmith, John (1991) Stereo Views: An Illustrated History and Price Guide. WallaceHomestead Book Company: Radnor, Pennsylvania.
Walker, David (undated) . This is a short no frills
introduction to stereo microscopes.
http://www.microscopyuk.org.uk/dww/novice/choice3.htm
Wheatstone, Charles. (1838) Contributions to the
Physiology of Vision.—Part the First. On
some remarkable, and hitherto unobserved,
Phenomena of Binocular Vision, June 21,
1838
Wise, F. C., Francis Edmund Jury Ockenden, P.
K.Sartory, (1950) The binocular microscope:
its development, illumination and manipulation. (Quekett Microscopical Club Monograph) London: Williams & Norgate.
Zeiss, (Microscopy, LLC, MicroImaging GmbH,
Jena)
- Carl Zeiss catalog (1937)
- Zeiss catalog (1951) "Mikroskope für Wissenschaft und Technologie"
- Zeiss Citoplast brochure (undated, East
Germany)
- Zeiss Opton catalog (undated, West Germany)
- Zeiss Stemi DR, Stemi DV4, Stemi Stereomicroscopes brochure (undated)
Gubas, Lawrence J., (2012) Personal communication exchanges. The author thanks Mr.
Gubas for both his pictures and information on Zeiss instruments.
OBSERVATIONS
Plant Fibers
Asbestos? Fiberglass? Amateur microscopy comes to the rescue!
Johann Vogel
T
his past winter has been one of
the mildest I remember. Those
who keep tabs on the weather say we've had the fourth warmest winter
and the warmest March on record.
It was then little surprise that home
improvement jobs never quite stopped
for the season, as they usually would.
With them came noise, dust, and some
strange fibers. I was often able to enjoy
the unseasonable warmth out on the
deck, happy that there were no mosquitos to chase me back indoors. To my
great dismay, though, these pesky little
fibers appeared everywhere: short, slim,
brownish, they were like hundreds of
tiny little needles.
Some time ago, a major international airport's use of airspace has been
redesigned, with takeoffs fanned out
over residential areas, so now low-flying jumbos were clouding my evening
sky. However, I highly doubted these
fibers would be from the planes. For
one thing, they were abundant even during the morning hours; for another, I
haven't noticed these fibers during the
previous fall and summer.
Under the scope, they seemed almost too symmetrical to be natural. I
supposed that such symmetry was a
telltale sign of them being man-made,
something that machines put out with
programmed precision. That they were
so easily wind-borne was evident from
their apparently hollow structure: the
tubes seemed to "fill up" in a wet mount.
Next I turned to the web, certain that
this would be an easy job for the search
engines. Asbestos needles were fast
ruled out, as where various more mundane fibers like those from carpets, rugs
and alike. For some time there, I was
almost hoping I was on the path to discovering something new. Caroline of
Warwick, NY, came to mind, who at
age 14 is the second youngest person to
discover a supernova (SN 2008ha).
Then I remembered about the ongoing home remodeling job a few houses
over on the other side of the street. A
team of fast-moving, handy workers
had been busy skinning the walls and
tearing off the roof, and dressing them
up in new insulation and fresh siding
and shingles. That was it, the smoking
gun! I was now sure the little fibers
would turn out to be the familiar fiberglass. Back to the search engines and...
coming up empty again! I then thought
this might just be some new type of
insulation, but all searches were just not
turning up anything quite like my bristles.
I took a piece of black felt and laid
it out on the deck railing again, waiting
for more and better samples. The fibers
collected fast and they were very similar
to each other, with slight variations in
length.
Unable to identify them yet wary
they might be somewhat dangerous, and
unwilling to inhale them or have them
stuck to my hair and clothing, I decided
to limit my exposure pending more investigations. At this point I realized that
in the back of my mind I was perhaps
starting to hope for some spectacular
find, on par with the small fortune invested in the scientific gear.
The warm and dry weather kept on
tempting me outdoors and soon enough
I decided to take short walks in a nearby
wooded park surrounding a stocked
pond. Always interested in collecting
specimens for observation, I picked up
1
Plant Fibers
OBSERVATIONS
2
a torn ball of London Plane. Once back
home, I quickly separated one of the the
tufted achenes and laid it on a slide,
paying special attention to the bristles.
Surprise! These were the very fibers I
have not until then been able to identify...
So then, what a relief! No asbestos.
No fiberglass! No colonizers from outer
space, but the homely London Plane or
perhaps American Sycamore. Content
with the outcome, I resumed my occasional recesses on my backyard deck. I
continue to maintain an array of clean
slides and investigate whatever tiny particles they collect, which are mostly
pollen this time of year.
Mixed in with the tiny grains, I keep
noticing irregularly shaped dark little
blobs of... I don't yet know what. Could
it be soot from the airplanes? Or
clumped-up dust and dirt? What else?
I'll research this next and find out for
sure.
3
Text and images are copyrighted by the
author. The author welcomes your comments.
■
Figure 1 (dry mount) and Figure 2 (wet mount) of the yet unidentified fibers.
Figure 3: Tufted achenes on a Plane tree ball (globose head).
GALLERY
Flatworms
Gyratrix sp. from my acquarium.
Equipment: Canon EOS 350D at
200x
Gyratrix belongs to the flatworms
(Platyhelminthes).
By Luca Monzo
14 - MicrobeHunter Microscopy Magazine - May
March
2012
2012
- Send images to editor@microbehunter.com
and
GALLERY
Top: Haemotococcus sp. from a
bird's bath. This is a focus stack
(18 images at 1 µm increments,
CombineZP), DIC, 100x oil-immersion objective, 1.25x intermediate
magnification, 2.5x projection
lens; Olympus E-P1 camera, exposure 1/8 s at ISO 200.
Bottom: From a drop of muddy
water. This is a stack of 20 images (taken at 2 µm increments with
a 20x objective, 1.25x intermediate
magnification, 2.5x projection
lens) formed using CombineZP;
exposure was 1/80 s at ISO 200. I
think this is Arcella, but correction
of the identification will be received with many thanks.
By R. Nassar
Send images to editor@microbehunter.comMicrobeHunter
- MicrobeHunter
Microscopy
Microscopy
Magazine
Magazine
- March
- May 2012 - 15
GALLERY
Leaf Veins
Veins of a maple leaf. A maple
leaf was boiled for several
hours to soften the tissue.
The leaf was placed on a flat
surface and the soft tissue
was carefully removed with a
soft brush to expose the leafveins. This procedure does
not work with all types of
leaves and experimenting is
necessary. Remaining
chlorophyll was removed
from the veins with alcohol
and the leaf was re-hydrated
with water to compensate the
shrinking. The veins were
then scanned with a flatbed
scanner at high resolution.
By Oliver Kim
March
2012
2012
Wasp and Hornet
GALLERY
Top: image of a wasp.
Bottom: Portrait of a
hornet. Stack of 20
images made with
Combine ZP ("all
methods"). Equipment:
Stereo microscope
(Lomo MBS10).
By Manfred Rath
I would like to thank all
contributors for giving
their permission to
republish their images.
The copyright of the
images remains with the
photographer (ed.)
Send images to editor@microbehunter.comMicrobeHunter
- MicrobeHunter
Microscopy
Microscopy
Magazine
Magazine
- March
- May 2012 - 17
GALLERY
Citric Acid
A small amount of citric acid was
placed between cover glass and
slide and then carefully heated.
Re-crystallization took place over
several days. The crystals were
observed in polarized light.
By Oliver Kim
March
2012
2012
Slide Mounting
LABWORK
The vast majority of permanently mounted slides are made with mounting media which solidify.
Here I would like to investigate the making of permanent slides using liquid mounting media.
Oliver Kim
P
ermanent mounts made with liquid mounting media (which remain liquid) are a rather
uncommon way of making a permanent
slide. The slide is prepared much like a
regular wet mount: The specimen is
placed in liquid mounting medium of
one’s choice on the slide. A cover glass
is also applied, which is then held in
place with a sealing medium. The sealing medium both prevents dehydration
and also physically stabilizes the cover
glass.
In his book „The Microtomist's Formulary and Guide“, Peter Gray (1954)
stated that liquid mounting media
should not be used when solidifying
alternatives are available. I assume that
the disadvantages of making and storing
these mounts outweigh the advantages
in most cases. Making such slides is
time consuming and requires more patience. Lack of physical stability is also
an issue. The liquid medium will not
support the cover glass if pressure is
applied. It is also necessary to store the
slides horizontally, in order to prevent
the specimens from sinking to the side
of the cover glass.
There are occasions, however, when
there are no other suitable alternatives
to using liquid mounting media. The
three media have been used extensively
in the past are glycerol, bromonaphthalene, and liquid petrolatum (petroleum
jelly). Gray stated that certain specimens, such as nematode worms, shrink
when mounted in solidifying mounting
media and can result in artifacts that can
complicate the identification of the
worm. Liquid glycerol is therefore the
mounting media of choice for these
specimens. In the case of diatoms, the
high refractive index of Bromonaphthalene is the main advantage. Bromonaphthalene allows for the resolving of very
fine details: “Нe one who has ever ex
amined diatoms mounted in bromonaphthalene will ever wish to use
any other medium and, though the process is tedious, the end result justifies
the trouble taken.” (Gray, 1954, p. 37).
For sealing the cover glass Gray
suggests three possibilities: Dichromate
Gelatin (in combination with an additional external varnish), molten resinous medium and petrolatum. I had none
of these sealing media at my disposal
and therefore decided to experiment
with clear nail polish. Nail polish is
sometimes used to seal some solidifying
mounting media (such as glycerine gelatin), and I wondered if it is also useful
for the sealing of liquids. Nail polish
dries quickly and I considered this a
significant practical aspect.
As mentioned, fluid permanent
mounts are a bit difficult to make. The
specimen is mounted using glycerol (or
other fluids) much like a regular wet
mount. There is an important difference, however. The specimen with the
glycerol drop must be sufficiently small
so that it is easily possible to seal the
cover slip with clear nail polish. Liquid
mounting medium which emerges beneath the cover slip is a problem. It will
contaminate the glass so that the sealing
medium may not adhere to the glass
anymore. For this reason the maximum
size of the specimen is limited, or one
Figure 1: Flower pollen mounted in
pure glycerine.
LABWORK
Slide Mounting
Figures 2-4: Various attempts of creating a space between the slide and
the cover glass.
Figure 2: I first glued several cover
glasses to a slide with nail polish.
The small square in the center is the
place for the mounting medium. The
drying process resulted in some
shrinking of the nail polish and the
formation of air spaces beneath the
cover glass. I discarded this approach as too time consuming and
tedious.
Figure 3: I applied nail polish to the
edges of a cover glass. The result
was rather uneven and irregular. This
resulted in an uneven flow during the
sealing process.
Figure 4: Here I attempted to stamp a
round spacer on the slide. This too
resulted in an irregular pattern.
has to use an extra-large cover glass.
Specimens which are thick will require
much glycerol and also much sealing
fluid. This may result in stability problems and easier breaking of the cover
glass when pressure is applied.
The Method
I started to experiment the making
of permanent wet mounts without much
previous knowledge or research. In the
following section, I simply want to
present my findings and some of the
difficulties that I encountered. Instead
of mounting real specimens, I occasionally used a drop of diluted ink instead of
glycerine. The color of the ink allowed
me to distinguish it more clearly from
the nail polish that I used for sealing the
cover glass.
Trial 1: On the first trial I directly
attempted to mount some house dust in
liquid glycerol. I placed a small drop of
glycerol on a slide, added some dust and
then placed a cover glass on top. The
drop was compressed beneath the cover
slip and, due to capillary action, the
glycerol quickly spread beneath the
whole cover slip. I tried to hold the
Slide Mounting
5
clouding
6
Water
(with ink)
bubbles
Nail Polish
Figures 5 and 6: The sealing medium (clear nail polish) started to react with the
water. This resulted in the clouding of the medium and the formation of many
small bubbles . A similar reaction could be observed when using glycerol as a
mounting medium instead of water.
LABWORK
cover glass as I applied the nail polish
(this prevents the brush of the nail polish to from moving the cover glass. The
application of pressure on the cover
glass resulted in some of the glycerol to
emerge and made it impossible for me
to apply the nail polish without contacting the glycerol. Evidently I used too
much glycerol. It came as no surprise
that the nail polish did not stick to the
glass at those areas which were contaminated with glycerol.
Trial 2: I now significantly decreased the size of the drop of mounting
medium. The glycerol now formed a
thin film of liquid beneath the cover
glass. I could now apply the nail polish
even without holding the cover glass.
The result was quite satisfactory and
appeared to be physically stable (fig. 8)
The disadvantage is that only very thin
specimens can be mounted like this.
Trial 3: I still further reduced the
drop of glycerol. There was now much
air surrounding the mounting medium
and the weight of the cover glass could
not spread the medium over a large area.
I then applied clear nail polish. The nail
polish was quickly drawn in and spread
beneath the cover glass to fill the air
spaces. I also added more nail polish to
the edges of the cover glass. In my view
this should be enough to hold the cover
glass nicely in place. A quick microscopic check revealed, however, that
the glycerol and the nail polish started
to react with each other. There were
many small bubbles forming. It appeared as if there is some kind of emulsification process going on. If some of
the nail polish solvent can enter the
glycerol, then this may also affect the
specimen in an unknown way. I therefore considered it necessary to apply the
nail polish in such a way that there is a
ring of protective air separating the
glycerol from the sealant. I repeated the
experiment, but the capillary forces
were too strong and always pulled the
nail polish to the center of the cover
glass. Maybe the nail polish was too
liquid and should be thickened by allowing some of the solvent to evaporate.
Trial 4: I then contemplated on
making a spacer. This should prevent
the cover glass from squashing the
specimen and should form a small cavi-
Slide Mounting
LABWORK
Figure 7: Here the drop of glycerine
moved from center of the slide to the
side. The specimen (pollen) stayed in
the center.
7
Nail Polish
Specimen
Glycerine
Air
8
Figure 8: So far the best solution. A
very small amount of glycerine was
used. Naturally this works only for
very small or thin specimens.
ring around the cover glass prevented
the nail polish from completely flowing
beneath the cover glass. The drop of
glycerine with the specimen was nicely
separated from the sealing medium by
several mm of air. After a day or two,
however, I could see that the glycerine
moved from the center to the side, leaving much of the flower pollen behind
(fig. 7).
Trial 6: Finally I tried to “stamp” a
spacer onto the slide. I covered the edge
of a plastic cap with sealing medium
and then transferred the medium to the
slide by pressing the cap against the
slide. This too was not satisfactory and
the result was too irregular.
Lessons Learned
Glycerine
Nail Polish
ty for the liquid mounting medium. The
edges of the cover glass were first
ringed with nail polish and allowed to
dry (fig 3). Instead of using glycerol, I
decided to use diluted ink instead as my
mounting medium. Any gaps or breaks
in the sealing would allow water to
evaporate and this would serve as a
clear warning sign that something was
not tight. The ink also allowed me to
visually keep the two liquids apart. This
trial turned out to be even more diffi-
cult. The spacer was uneven and this
resulted in the nail polish to unevenly
flow beneath the cover glass. There was
also some unacceptable mixing of the
nail polish and the ink, which resulted
in significant clouding (figs. 5, 6).
Trial 5: I used a prepared cover
glass from trial 4 and mounted some
flower pollen in liquid glycerine. I used
a very small drop of glycerine to prevent mixing of the liquids. Initially everything seemed to work out fine! The
I have to admit that making fluid
permanent mounts turned out to be
more difficult than anticipated. The best
result was obtained in trial 2 (fig. 8),
where a thin film of glycerol was used
between the cover glass and the slide.
What should one do if a thicker specimen requires the use of more mounting
medium? This is still something that I
have to figure out. A possible chemical
reaction between the mounting medium
and the sealing fluid is another issue
that must be addressed. I can imagine
that the use of slides with concave depressions may be a possible solution
here.
References
Gray, Peter (1954). The microtomist's
formulary and guide. Blakiston, New
York.
http://archive.org/details/
microtomistsform00gray
■
Reference Plate
DESMIDS
Micrasterias rotata
Mike Guwak
1
Name: Micrasterias rotata (GREV.) RALFS ex RALFS (var. rotata)
Synonyms: Cosmarium striolatum var. nordstedtii and Pleurotaeniopsis tessellata var. nordstedtii
Literature: Ralfs, J. (1848).
Origin of name: From Greek mikros, "small" and aster, "star"
Occurrence: This is a fresh water species and occurs in weakly acidic waters.
DESMIDS
Reference Plate
3
4
Size: 200-300 by 190-270 μm. 45-70μm thick
Shape: The cells are large and slightly oval to round
(fig. 1). The center of the cell is strongly constricted and
the cell walls are covered with numerous short spines.
The external lobes towards the sides are larger and divided. The lobes terminate with 2-3 teeth (fig 2). The cell
wall contains numerous small densely packed pores.
References
Lenzenweger, Rupert (1996). Desmidiaceenflora von Österreich: Teil 1. Berlin, Stuttgart: J. Cramer
Image Credit
Image copyright Mike Guwak (2012).
http://www.mikroskopie-main-taunus.de
mike.guwak@mikroskopie-main-taunus.de
Lab ideas for Schools
LABWORK
1-5
Carbon dioxide production by yeast can be observed
microscopically - an easy experiment for the classroom.
Oliver Kim
B
aker’s yeast (Saccharomyces
cerevisiae) has been widely
used in biotechnology, both for
baking bread and for brewing. The yeast
takes up sugar and converts it, in a
process called cell respiration, to carbon
dioxide gas (CO2) and water. The formation of CO2 can be easily observed
(and even quantified) with the help of a
microscope. The procedure is simple
enough to be conducted in a school
laboratory.
Take some fresh yeast and suspend
the cells in warm (but not hot) water.
You need a dense yeast suspension.
Add a small amount of table sugar and
mix well. Take a drop of the mixture
and make a wet mount to observe the
formation and growth of CO2 bubbles.
The speed of the bubbles forming
and growing depends on several factors.
Temperature is critical. Too low a temperature and the metabolism of the yeast
will be too slow. Bubbles take much
longer to form. A high temperature can
result in the denaturation of enzymes
and also a lower respiration rate. If the
sugar concentration either too high or
too low, then the formation of bubbles
also takes much longer. High concentrations of sucrose lowers the respiration
rate possibly due to osmotic imbalance.
The speed of CO2 production can be
determined by taking photographs of a
bubble every few minutes. The area of
the bubble corresponds to the amount of
CO2 produced (the bubble is not a
sphere, but compressed between cover
glass and slide).
There are several possibilities of
modifying the experiment. CO2 production can be measured depending on
yeast concentration, sugar concentration or temperature. Provided that one
designs an appropriate set-up, it may
even be possible to compare CO2 production under aerobic and anaerobic
conditions.
It takes a few minutes for the yeast
to start producing CO2. Once production has started, the growth of the bubbles can be quite rapid. A growing CO2
bubble pushes the yeast cells ahead as it
grows. Figures 1-5 show this accumulation of cells around the bubble. The gas
production can be so high that bubbles
start to overtake the whole field of view
(fig. 6).
■
6
Figures 1-6: Growing CO2 bubbles
formed by respiring yeast.
What’s this? Answer on page 3.

May 2012 - MicrobeHunter Microscopy Magazine

Transcription

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