Hi smitty3360,
I?m still not completely sure what you are asking, but I?m going to
tackle what I think you mean. Please ask for an Answer Clarification,
before rating, if my answer is not what you were hoping for.
?The typical compound light microscope is capable of increasing our
ability to see detail by 1000 times so that objects as small as 0.1
micrometer (um) or 100 nanometers (nm) can be seen.?
http://abacus.bates.edu/~ganderso/biology/resources/microscopy.html
With a compound microscope, using an oil immersion lens, you would be
able to see things as small as .1 um (micrometer),and discern things
that are 0.1 um apart.
With a 10x compound light microscope, such as used in clinical
laboratories (Usually Zeiss or Nikon), you would typically use a:
10x lens for scanning a slide for the presence of bacteria, tissue,
Fern tests, a quick WBC estimate before doing a manual differential,
etc.
Using the 40x lens (high dry) you can enumerate cells, scan for
abnormal WBC and RBC, RBC morphology, perform a manual platelet count,
manual WBC count, sperm counts, etc., do a manual diff (if you are
experienced), do a urine microscopic, etc.
With the 100x oil immersion lens you perform platelet estimates,
morphology (WBC) and determine bacterial species (Gram stain), Tzanck
prep, etc. 100X oil immersion if for fine detail. You can see granules
in stained eosinophils and basophils. You can see some WBC cell
division (mitosis) in immature WBC, see iron deposits in siderocytes,
see malarial parasites in an RBC, and even an Auer rod in a
myeloblast, indicating myelocytic leukemia. Because good microscope
lenses are parfocal, you can quickly switch from 10x to 40x to 100x
without losing your field of view.
Auer Rod
http://udel.edu/~rmaser/hematology/wbc/AUERROD.JPG
Here is a picture of several WBCs, using 100x oil
http://www-medlib.med.utah.edu/WebPath/HEMEHTML/HEME100.html
?Determining Field-of-View Diameter
You may wish to estimate the size of the specimens (e.g., cells) you
will see in lab. The best way to do this is with an ocular micrometer,
a precision ocular lens insert that has a ruler etched into glass. The
monocular scopes we use in the introductory courses are not so
equipped, so we will use an alternative method based upon knowing the
field-of-view diameter for your particular microscope. To do this, you
must determine:
·the approximate diameter of your low magnification field-of-view for
your particular microscope.
·the total magnification for each of your other objective lenses.
Knowing this for each objective lens, you can compare the size of the
specimen against the known field diameter and make a reasonable
esimate of size. This technique works for any microscope.
1. Obtain a slide scale and position it on your scope. A transparent
metric ruler will work as well.
2. Bring it into focus using the 10x objective (100x total). The scale
bars are increments of 1mm as shown in the figure below. Thus, a black
bar = 0.5mm as does a space.
3. Move the slide such that the edge of an outside black bar is just
tangent to the lighted field (see point "A" above).
4. Starting at that edge, estimate how many bars and spaces it takes
to cross the field-of-view. You will probably have to estimate the
last fraction of a space or bar. In the figure above, it is
approximately 1.8mm wide.
5. Record your scope's ID number and field diameter at 100x in your
lab notebook for future reference.
6. Next, calculate the field width at 430x total magnification using
the following formula (we refer to the 100x mag as "low power" and
430x as "high power"):
(low power mag/ high power mag) x low power field diameter (in mm)
For the example above,
(100 / 430) x 1.8 mm = 0.418 mm = 418 um (micrometers)
Note that the field diameter at high power is proportional to the
ratio of the low to high power objectives. That is, as you increase
magnification, the actual field of view becomes proportionally
smaller.?
http://abacus.bates.edu/~ganderso/biology/resources/microscopy.html
Bacteria are about 2 micrometers in size. A healthy RBC (Red blood
cell) is about 7 micrometers across. Platelets are 1-4 micrometers in
size. (It is unknown how big knifelets and forklets are - sorry, I
couldn?t resist some clinical humor) Using a compound light
microscope, you would be able to determine if bacteria had a
capsule(faintly), if it were gram negative or gram positive, some
flagella, and the size of bacteria, but you would not be able to see
ribosomes, DNA, or much separation of cell wall and cytoplasm. For
that you'd need an electron microscope.
Bacteria
http://www.disknet.com/indiana_biolab/b003.htm
RBC
http://www.wadsworth.org/chemheme/heme/microscope/rbc.htm
Platelets
http://www.wadsworth.org/chemheme/heme/microscope/platelets.htm
http://www.dmacc.cc.ia.us/instructors/scottie.htm
See all the parts of a compound microscope here:
http://www.cas.muohio.edu/~mbi-ws/microscopes/microscopeparts.html
This site gives you an idea of the size of microscopic things
http://www.cellsalive.com/howbig.htm
More about compound microscopes
http://www.mansfield.ohio-state.edu/~sabedon/biol4030.htm
As you can see, of course you can discern bacteria that are 3 micrometers apart.
This page has a rather blurry picture of bacteria in a high dry(400x)
vs. oil immersion fields (100x).
http://biology.clc.uc.edu/fankhauser/Labs/Microscope/Oil_Immersion/07_compare_400x_1000x.jpg
?Use an oil immersion lens when you have a fixed (dead - not moving)
specimen that is no thicker than a few micrometers. Even then, use it
only when the structures you wish to view are quite small - one or two
micrometers in dimension. Oil immersion is essential for viewing
individual bacteria or details of the striations of skeletal muscle.
It is nearly impossible to view living, motile protists at a
magnification of 1000x, except for the very smallest and slowest.?
?Note that you can see some detail at 400x, but the shapes and colors
of the bacteria are somewhat distorted.
Move the 400x lens out of the way, place a drop of immersion oil
directly on the smear where the objective was, and swing the oil
immersion lens in place. Move the fine focus up and down slightly to
ensure that the lens is in contact with the oil. Now watch the end of
the objective and bring it as close to the slide surface as you can
without touching it. Note in which direction you must focus to move
the objective away from the slide, look in the eyepieces, and slowly
rotate the fine focus control until the image is focused.
Most bacterial species are rod-shaped or round (cocci), although some
are curved, spiral-shaped, or irregularly shaped. The gram stain
leaves some cell types pink (Gram negative) and others dark blue (Gram
positive) depending on cell wall characteristics. Gram stain results
are a major criterion for identification of species. Note how much
more clear the image is at 1000x with oil than it was at 400x without
oil.?
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/oilimm.html
Here you can see the difference in magnification between electron and
light microscopy.
e.coli as seen in an electron microscope
http://www.microbelibrary.org/images/mketterer/Images/mketng.jpg
e.coli using a compound light microscope, 100x oil immersion
http://www.courses.ahc.umn.edu/medical-school/IDis/Images/E.coli.gif
This page contrasts electron and light microscopy
http://www.bact.wisc.edu/Bact330/lecturenf
Principals of Light Microscopy
http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html
Using a microscope is one of the most enjoyable aspects of laboratory
science. I found it exhilarating to be the one to find the leukemic
cell, the malarial parasite, or the two-headed sperm. (When you get to
the point where you can identify trichomonas in urine or a wet prep,
try this trick: add a drop of KOH under the cover glass, and watch
through the oculars as the KOH floods the field. Within a few seconds,
the trichomonads will swell and burst! (Sort of like watching an old
Space Invaders game!)
Hope this helps you increase your knowledge of microscopy!
Regards
crabcakes
Oh, don't forget to wipe off the oil immediately after using an oil
immersion lens!
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