January 2014 - MicrobeHunter Microscopy Magazine - 1

Microbe
Hunter
Microbe
Hunter
ISSN 2220-4962 (Print)
ISSN 2220-4970 (Online)
Volume 4, Number 1
January 2014
The Magazine for the
Enthusiast Microscopist
http://www.microbehunter.com Microscopy Magazine
DIY Polarization Observing Animal
Droppings
Osmosis of
Red Onion Cells
2 - MicrobeHunter Microscopy Magazine - January 2014
Microbehunter Microscopy Magazine
The magazine for the enthusiast microscopist
The Magazine is a non-commercial project.
Volume 4, Number 1, January 2014
ISSN 2220-4962 (Print)
ISSN 2220-4970 (Online)
Download: Microbehunter Microscopy Maga-
zine can be downloaded from:
http://www.microbehunter.com
Print version: The printed version can be or-
dered at: http://microbehunter.magcloud.com
Publisher and editor: Oliver Kim, Ziegeleistr.
10-3, A-4490 St.Florian, Austria
Email: editor@microbehunter.com
Web: http://www.microbehunter.com
Tel.: +43 680 2115051
Images and Articles by: Oliver Kim, Neill Tuck-
er, Mazen Fakih, MD
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the authors have confirmed that they are the full
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Front Cover: Red onion cells (Oliver Kim)
Left image: Mazen Fakih, MD
Middle image: Neill Tucker
Right image: Oliver Kim
3 This Month on the Web: Webpages to check out!
Whats going on on the web? Here is a summary of
microscopy related publications.
Oliver Kim
4 A Microscopic Look at Animal Droppings
Animal droppings can reveal quite much about the diet
of the animals. Plant material, insect parts and an
occasional parasite make these specimens interesting
to look at.
Neill Tucker
12 A DIY Approach to Polarized Light Microscopy
A cheap alternative to professional linear polarizing
filters and their limitations.
Mazen Fakih, MD
14 Plasmolysis of Red Onion Cells
By adding concentrated salt water to the cells of red
onions, it is possible to make the cells contents shrink.
Oliver Kim
CONTENTS
B a c k c o v e r : S a l t c r y s t a l
( d i s s o l v e d i n w a t e r a n d
e v a p o r a t i o n o n t h e s l i d e )
Gold Chloride
(Page 12)
Animal Droppings
(Page 4)
Plasmodesmata of
red onions (Page 14)
January 2014 - MicrobeHunter Microscopy Magazine - 3
MICROSCOPY
NEWS Microscopy on the Web
Entanglement Enhanced
Microscope
Takafumi Ono and colleagues at
Hokkaido University in Japan devel-
oped the first entanglement enhanced
microscope. These are variants of the
differential interference contrast micro-
scopes and use entangled photons in-
stead of regular light. This way they
were able to increase the resolution of
the microscope by 1.35 times over the
ordinary system. This microscope is
able to detect extremely small differ-
ences of sample height by measuring
the interference pattern of the light that
exits the specimen. In the setup that
they used, the letter Q was etched into a
sample. The letter was only 17nm high-
er than the surrounding. Using normal
light microscopic techniques, this small
difference in height is almost not detect-
able. Entangles photons carry more in-
formation and therefore could resolve
the specimen without problems.
http://www.technologyreview.com/view/
524521/worlds-first-entanglement-enh-
ancedmicroscope/
Super Resolution Microscopy
The Journal of Optics has selected
super resolution microscopy as the
2013 Highlight. The resolution of
conventional microscopes is deter-
mined by the so-called diffraction limit.
Super resolution microscopy over-
comes this limit by using fluorescent
labeling and computer modeling. The
cells are first labeled with a fluorescent
dye, which lights up in UV light. The
bright spot of the dye is quite large
(adhering to the diffraction limit). Com-
puters are now used to calculate the
center of each spot to obtain the high
resolution. In order to prevent the prob-
lem of overlapping spots, the fluores-
cent dyes can be switched on and off so
that only one spot is visible at a time.
http://www.npl.co.uk/news/super-resolut-
ionresearch-highlight
3D Visualization of Spermatozoa
Scientists trapped living sperm cells
between two lasers and were able to
determine their three dimensional posi-
tion in space, based on the interference
pattern that they made. The researchers
were also able to record their 3D move-
ment on video, which provided more
information on the effect of sperm ab-
normalities. So far, only the two dimen-
sional movement of the sperm could be
observed. The new technique gives fer-
tility scientist new insight on the effect
of sperm defects on the movement.
http://www.sci-tech
today.com/story.xhtml
?story_id=030001P2KCT6
New Surgical Microscope
Sometimes tumor removal opera-
tions have to be repeated, because the
edges of the tumor (which contain can-
cerous cells) was not removed and con-
tinues to grow. A new generation
surgical microscope now addresses this
problem. It allows for real-time obser-
vation of cells during a surgery. The
microscope several tiny microscopes
(each one about 1 mm) arranged next to
each other. This way it is possible to
obtain both high magnification of about
1000x together with a large field of
view. One main difficulty in the devel-
opment of this microscope was the min-
iaturization of the focusing lenses.
http://www.scienceworldreport.com/
articles/12903/20140214/next-gen-
surgical-microscopes-see-tumor-cells
in-real-time.htm
In-vivo Microscopy
Eye problems in horses can now be
detected more easily with a new in vivo
corneal confocal microscope. This mi-
croscope has a focal distance of 1.5 mm
and is therefore able to completely cov-
er the depth of a horses cornea of 1
mm. The cornea, the outermost part of
the eye, might be harmed by fungal
infections, microscopic injuries or other
problems. These damages can now be
diagnosed much faster by taking a di-
rect picture of the eye.
http://horsetalk.co.nz/2014/02/18/new-
method-detecting-eye-problems
horses/#.UwPbooWZhGA
SEM images of Arthropods
I found a new FaceBook page with
scanning electron micrographs of Ar-
thropods and other Organisms. It is
worth a visit, the images are indeed im-
pressive.
https://www.facebook.com/
shfkhsdfjhdsnf

Whats going on on the web?
Here is a summary of microscopy
related publications of the last
few weeks.
By Oliver Kim
4 - MicrobeHunter Microscopy Magazine - January 2014
O
ne of the more common practi-
cal uses of the microscope is to
monitor parasitic infestations
of larger animals. Some parasites such
as mites and lice are external, while
others such as bots and worms are inter-
nal; eggs of the parasites appearing in
the dung or droppings signal some level
of infestation. There are already numer-
ous articles and forums on the web de-
scribing the techniques for diagnosing
and treating different types of parasitic
problems, so I will not reiterate them
here. My interest was more general in
nature, a case of, I wonder what things
look like after they have passed through
an animal and are there any interesting
associated observations that can be
made. For the higher magnification pic-
tures (600x), I have used a blue filter to
improve the contrast of the images.
Donkey Dung
Since we have donkeys (Figure 1),
my first port of call was a nice fresh
lump of dung. The main volume of the
dung is pretty coarse and hardly needed
a microscope to identify its constituents,
chewed up hay and grass mainly. How-
ever, I was still keen to see what there
was at a microscopic level, so I mixed a
small quantity of dung with water to
make a sort of soup. I let it stand for a
few minutes to let the larger pieces set-
tle, I then put a couple of drops of the
finer suspension on a slide and viewed
it at 40x magnification (Figure 2). I
suppose the thing that struck me the
most was the sheer amount of plant
material that was still recognisable,
even at a microscopic level. It makes
you realise what a tough time ruminants
must have in extracting anything nutri-
tious from their very high fibre diet.
Closer inspection at 100x revealed
finer plant matter and a Strongyle para-
site egg (Figure 3, centre), which we
knew would be present in small num-
bers, despite a regular worming regime.
At 600x there was a uniform scattering
of very fine plant material (Figure 4),
but not much evidence of bacteria,
which I found surprising since at least
Investigating Animal Droppings
Figure 1: Our Donkeys
Figure 2: Donkey Dung 40x
Animal droppings can reveal
quite a lot about the diet of the
animals. Plant material, insect
parts and an occasional parasite
make these specimens interest-
ing to look at.
By Neill Tucker
1
2
OBSERVATIONS
January 2014 - MicrobeHunter Microscopy Magazine - 5
OBSERVATIONS Investigating Animal Droppings
some types should be visible at this
magnification. In relation to the parasite
problem, my intention is to acquire a
McMasters slide (slide divided into
sections of known volume) and learn
how to do a proper faecal egg count.
Worming products are expensive and I
dont think they are overly good for the
natural environment. I noticed that dung
beetles and larvae of other winged in-
sects rapidly break down dung from
untreated animals. By contrast, dung
from recently wormed animals tends to
hang around for much longer and I fre-
quently see dead insects around the
dung. It is also documented that overuse
of worming products can result in resis-
tance in the parasites and make the
treatment less effective; so a little lab
work on my part may prove to be good
for my pocket and the environment.
Bird Droppings
On the way back from the donkey
field I passed some of the bird feeders
we have around (Figure 5), a rich hunt-
ing ground for some fresh guano. The
first thing I noticed about the bird drop-
pings was how different the consistency
was compared to the donkey dung.
When I added some water, it was dis-
tinctly reluctant to dissolve into a soup
and took some considerable agitation
before it did so. This insoluble nature
may explain its ability to stick and re-
main stuck to car paintwork, despite any
rain.
Viewed at 40x magnification (Fig-
ure 6), the remnants of a white grain
interior (endosperm) is clearly visible
together with fine hairs from wild seeds
and some other plant material. At 100x
there was finer plant material as in the
donkey dung. However, viewing at
600x revealed a fine suspension of
droplets (Figure 7), somewhat like the
fat droplets you see in homogenised
milk (Figure 8)
Thinking about it, this is probably
not so surprising. Small birds such as
the ones that come to our feeders have
huge energy requirements for their
flight muscles and also to keep warm in
the winter months. As a result, they will
search out the highest energy seeds and
grains, generally those with the highest
fat content e.g. peanuts and sunflower
seeds. Not only that, they will take the
time to remove outer husks or skin so as
not to fill their stomachs with low ener-
gy fibre. The result is that they have a
very fatty diet, some of which ends up
undigested and in the droppings. This
fat will not mix with water but will form
an emulsion if suitably agitated, like the
milk.
Bat Droppings
My next port of call was going to be
mouse droppings, and I found what I
thought would be some good candidates
at the base of the south-side wall of our
Figure 3: Donkey Dung 100x (with
strongyle egg)
Figure 4: Donkey Dung 600x
3
4
6 - MicrobeHunter Microscopy Magazine - January 2014
Figure 5: Gold finches on our feeder
Figure 6: Bird droppings at 40x
(above and sub-stage lighting)
OBSERVATIONS Animal Droppings
5
6
January 2014 - MicrobeHunter Microscopy Magazine - 7
farmhouse. I collected a few, added
some water as before and got a nice
suspension of fine particles to put on a
slide. I have to say I was somewhere
between confused, surprised and excit-
ed at what I saw (Figure 9). The drop-
pings looked like an encyclopaedia of
insect body parts; I thought mice ate
predominantly seeds and young plants,
clearly further investigation was re-
quired. Keeping a look out over the next
few days revealed the answer, a small
bat (Figure 10), a Pipistrelle I think, had
taken up residence in the wall.
Although normally nocturnal, this
little chap does appear to be partial to a
bit of afternoon sun and isnt exactly
camera shy. Not being a qualified ento-
mologist I couldnt say exactly what my
little friend had been eating but there
seemed to be parts of legs, thoraxes,
compound eyes and moth wing-scales.
I felt like I should have be doing some-
thing a little more scientific with my
observations but just looking around the
slide was fantastic, a bit like walking
round a car-boot sale, you never know
what youre going to see next. Viewing
at 600x is really too high a magnifica-
tion for the majority of the features,
except perhaps to see the detail on the
moth wing-scales. A good example of
where higher magnification is not al-
ways a good thing. If you know of any
bat roosts and want to collect droppings
yourself, take care not to disturb them.
Bats are protected in many countries
and it might be better to contact your
local conservation group for help or
advice. Bizarrely, bats are called
chauve-souris here in France, which
translates to shaved mice, so I had
found mouse droppings of a sort!
In order to find some actual mouse
droppings I ventured into the basement,
which was once used as the cattle shed.
In amongst some loose stones I soon
found what I was looking for, some nice
fresh droppings. Examination under the
microscope at 100x (Figure 11) showed
pretty much what I was expecting,
namely small pieces of plant material.
The piece in the centre could be a fairly
well digested young leaf; some of the
brick like cell structure is still visible.
The material on the right looks like wild
grass, with some of the stoma and silica
edge-teeth still in evidence. Im not sure
what the small starfish centre right is,
Ive seen them infrequently in the other
animal droppings as well. It might be a
very small seed and since Ive never
seen one move, I dont think its a para-
site? Examination of the sample at 600x
(Figure 12) proved very interesting, re-
vealing very large numbers of bacteria.
Whilst I had noticed small clumps of
bacteria in the other samples, I had not
seen them in anything like the same
numbers as in the mouse droppings. A
good reason to make sure they stay
outside and not let them get in to where
food is stored or prepared.
Fungus, Fireworks and Cow
Dung
During my hunt for the mouse drop-
pings I did get somewhat distracted by
what appeared to be some sort of fungus
Figure 7: Bird dropping, 600x
Figure 8: Homogonised milk, 600x
OBSERVATIONS Animal Droppings
7 8
8 - MicrobeHunter Microscopy Magazine - January 2014
growing on the stone walls of the base-
ment (Figure 13). The fungus was pre-
dominantly on a wet/dry transition
region of the wall. Old stone walls do
not have waterproof damp courses in
them but rely on the wall to dry natural-
ly by evaporation. The lower section
stays damp (the dark stones in the pho-
to) but as you move up the wall it be-
comes increasingly drier (light coloured
stones).
In winter the proximity of natural
ground water prevents freezing while
evaporation cools the room in summer.
The result is a surprisingly stable tem-
perature of around 8C, all year round.
This is probably why the peasant farm-
ers used to reserve part of the old cattle
sheds for maturing cheeses, charcuterie
and wine. Anyway, I digress, back to
the possible fungus problem and a clos-
er look (Figure 14). With the naked eye
it had a slightly woolly appearance and
was easily dislodged with a small brush.
I took a small sample up to the micro-
scope for examination; at 40x in dark-
field illumination (Figure 15) it was
clear that my fungus was more crystal-
line than organic. Viewed at the same
magnification using polarised light
(Figure 16), the crystals looked to be
birefringent (their refractive index var-
ies with the direction and polarisation of
the incident light), producing the char-
acteristic rainbow of colours. What was
interesting was that only some of the
sample was actually in single crystal
form, the thin rods, other parts of the
sample appear to have melted and re-
solidified. The melted parts looked a bit
like snow that had sun on it during the
day and had then re-frozen at night.
I remember reading somewhere that
saltpeter (Potassium Nitrate KNO3) was
often found on the walls of caves where
animals had dwelled in the past. The
Figure 9: Bat dropping, 100x
Figure 10: Our little bat
Investigating Animal Droppings
9
10
OBSERVATIONS
January 2014 - MicrobeHunter Microscopy Magazine - 9
nitrate in the dung and urine gets
washed into the soil and then wicks up
any porous stone in the walls. The con-
tinual evaporation increases the concen-
tration of the dissolved salts until they
begin to crystallise out. In my search for
various different animal droppings I
may have stumbled across the chemical
relics of some really old cow dung. The
apparent melting of the crystals was
probably due to changes in local humid-
ity levels causing some of the crystals to
go back into solution.
I didnt have the equipment for full
blown chemical analysis but there were
a few simple tests that I could do to try
Figure 11 Mouse Dropping 100x
Figure 12 Mouse Dropping 600x
Investigating Animal Droppings
11
12
OBSERVATIONS
10 - MicrobeHunter Microscopy Magazine - January 2014
and identify my white crystals. The first
was a flame test, which involved dis-
solving some of the crystals in distilled
water and then dipping a small wire
loop into the solution. The loop was
then introduced into a gas flame; as the
loop heated up the gas flame burnt lilac
in colour (Figure 17), which is charac-
teristic of the element Potassium, so far
so good.
The next test was to see if the nitrate
part was present. Most group-1 nitrates
(except Lithium) will decompose when
heated, into a nitrite (KNO2 in this
case) and Oxygen (O2), the oxygen will
then react readily with any combustible
material in close proximity. The bal-
anced chemical equation is 2KNO3 =>
2KNO2 + O2. My crude test involved
grinding up some charcoal, which is
essentially just Carbon (C), into a fine
powder and mixing it with an equal
volume of the crystals. I then applied a
glowing splint, which gave spectacular
results (Figure 18) and show that ex-
treme care and small quantities should
be used if doing this yourself. The result
was certainly indicative of a violent
oxidation reaction (C+O2 => CO2 ) and
therefore the probable presence of a
nitrate compound. A safer experiment
would be to put the crystals in a test
tube, heat them over a gas flame and
then put the glowing splint in the test
tube. If the splint started to glow bright-
ly, it would indicate the presence of
Oxygen given off by the decomposition
of the Nitrate. Perhaps a little more
scientific but it wouldnt have made
such a nice photo.
The main thing I have taken away
from my little investigation of animal
droppings, is that if you have just the
germ of an idea to investigate some-
thing, have a go, you never know what
you might find out along the way.

Figure 13: Basement wall
Figure 14: Basement wall, close-up
Investigating Animal Droppings
13
14
OBSERVATIONS
January 2014 - MicrobeHunter Microscopy Magazine - 11
Figure 15: Crystals from wall, dark-field 40x
Figure 16: Crystal from wall, polarised light 40x
Figure 17: Flame test
Figure 18: Oxidation test
Investigating Animal Droppings
15 16
17
18
OBSERVATIONS
12 - MicrobeHunter Microscopy Magazine - January 2014
A DIY Approach to
Polarized Light Microscopy
W
hile high quality linear po-
larizers can be expensive,
the widespread of 3D the-
aters worldwide has allowed the ama-
teur microscope user to acquire such
filters for a much reduced cost (Glasses
can sell for as low as one US Dollar).
The 3D glasses that are given at the
movies can be easily adapted for use
with a microscope. All what needs to be
done is to break the plastic frames while
making sure not to damage the filters.
Before using the pair, try aligning them
(the right eye filter with the left eye
filter) perpendicular to each other until
the light is blocked. This will give you
an idea about the proper alignment to
use. If all you see is a shift from yellow
to blue when turning the filters, then try
flipping one of the filters. If you are
using a daylight filter on your micro-
scope, it can scatter any polarized light,
so make sure that the daylight filter is
between the light source and the polar-
izer. The second filter is placed right
above the slide you are viewing.
One of the advantages of using this
DIY option, apart from the low cost, is
the curvature of the filters. If you have
specimens on slides without any cover
glasses, the filters will only touch the
extreme sides of the slide.
A cheap alternative to profession-
al linear polarizing filters and
their limitations.
By Mazen Fakih, MD
1
DIY Polarized Light Microscopy
January 2014 - MicrobeHunter Microscopy Magazine - 13
Figure 1: Gold Chloride
crystals 100x magnification
Figure 2: Zenkers Fixative
400x
Figure 3: Sugar crystal 100x
magnification
2
3
Keep in mind that the filters are
made from thin sheets of plastic. Expo-
sure to the heat from the light may in-
crease their curvature. Try to place them
a centimeter or two above the light
source if possible.
Author contact: mff06@aub.edu.lb

DIY Polarized Light Microscopy
14 - MicrobeHunter Microscopy Magazine - January 2014
P
lasmolysis of red onions belong
to one of the easiest introductory
microscopy experiments. By
adding salt water to the onion cells, the
cells contents starts to shrink to the
point that the cell membrane starts to
separate from the surrounding cell wall.
Plasmolysis is due to the diffusion of
water out of the cell, by a process called
osmosis. The salt ions (Na+ and Cl-)
bind the water molecules and immobi-
lize them. For this reason the probabili-
ty of a water molecules diffusing into
the cells is smaller than the probability
of one diffusing out, which results in a
net movement of water out of the cell.
Obtaining onion skin
To make the process visible, it is
necessary to use red onions. The natural
pigmentation of the cells makes the pro-
cess visible the best.
Cut out a square piece of a red onion
and tear off (do not cut) the strongly
pigmented epidermis. Tearing will re-
sult in a single layer of cells. I found it
easiest to use a dull knife to first cut into
the meat of the onion and then tear off
the red epidermis. This process will
break many of the cells open and these
cells will lose their cell contents (and
their red color). In Figure 1 you can see
the torn epidermis on the bottom. Some
parts of the epidermis are clear, these
are the parts that are not useful for ob-
servation. The thick parts of the onion
were cut away and the remaining onion
epidermis was then placed on a micro-
scope slide with water and cover glass
and observed normally at low to medi-
um power.
By adding concentrated salt wa-
ter to the cells of red onions, it is
possible to make the cells con-
tents shrink. Plasmodesmata
may become visible as well.
By Oliver Kim
Figure 1: The skin of the red onion
peeled off by tearing.
Figure 2: High magnification image of
plasmolyzed red onion cells. You can
see the strands of cytoplasm extend-
ing from the dark red region towards
the cell wall. These plasma strands
are referred to as plasmodesmata
and are responsible for cell-to-cell
communication. During plamolysis,
the shape of the cell wall is not af-
fected.
Observing Osmosis OBSERVATIONS
January 2014 - MicrobeHunter Microscopy Magazine - 15
Figure 3: Original onion cells before
addition of salt water. Some cells
were pigmented red, others were not.
This was because the tearing pro-
cess destroyed some of the cells and
caused the red pigment to spill out.
On other parts of the onion epider-
mis, all cells were pigmented.
Figure 4: The plasmolyzed cells. The
cell membrane (surrounding the pig-
mented contents) remained attached
to the cell wall at certain places, the
plasmodesmata.
Plasmolyzing the cells
I first made a saturated salt water
solution. I did not weigh the salt, but
simply added excess salt to some water.
It is then necessary to allow the solution
to settle in order to minimize the dis-
turbing effect of small salt crystals on
the image.
Excess water is then removed from
the wet mounted slide with some tissue
paper. Several small drops of salt water
are then added to the edge of the cover
glass. The salt water is pulled beneath
the cover glass by capillary action and
comes into contact with the onion cells.
Observing plasmodesmata
The red cell content started to shrink
quickly. The color of the cell content
became more intense, an indicator that
the cell membrane did not allow the
pigment to pass (Figures 3 and 4). The
cell membrane started to separate from
the cell wall, but continued to be at-
tached on some placed to the cell wall.
Thin strands extended from the cyto-
plasm of the cell towards the wall. This
phenomenon can be seen well in Figure
2. These plasma strands are important
for cell to cell communication and con-
nect at the cell wall at places called
plasmodesmata.
After some time the process came to
a halt, because the salt solution became
more and more dilute. In this case it was
necessary to add more salt water to the
cells.

Observing Osmosis OBSERVATIONS
16 - MicrobeHunter Microscopy Magazine - January 2014
Whats this? Answer on page 2.