2.1 Ocular
Micrometer
INTRODUCTION
Ocular Microscope
Microscopes are often
used to measure small objects. For instance, forensic scientists use microscopy
to measure the distance between microscopic tool marks or the diameter of
fibers. Compound microscopes have ocular micrometers, or rulers that can help
you measure items under the microscope. However, the scale on the ocular
micrometer changes with total magnification, and thus has no absolute value. Therefore,
the ocular micrometer does not have units and it needs to be calibrated prior
to use.
We use a stage micrometer
to calibrate the ocular micrometer. A stage micrometer is essentially a ruler
that is mounted on a microscope slide that does have units (millimeters (mm) or
micrometers (mm)). When calibrating, we will line up the
stage micrometer with the ocular micrometer and count the number of divisions
on the ocular micrometer per millimeter or micrometer on the staged micrometer.
The number of divisions will change as the magnification changes.
OBJECTIVE
To measure and count cells using a microscope
Results
l.Lactobacillus sp.
400x magnification
1000x magnification
2. Yeast
400x magnification
1000x magnification
calibration:
400x:
5 x 0.01 = 0.05
0.05/ 20 = 0.0025 mm
1000x:
5 x 0.01 = 0.05mm
0.05/ 47 = 0.0011 mm
·
Lactobacillus sp.
400x magnification :
2
division x 0.0025 mm = 0.005 mm
1000x magnification :
5 division x 0.0011 mm = 0.0055 mm
·
Yeast
400x magnfication :
5 division x 0.0025 mm = 0.0125 mm
1000x magnification :
7 division x 0.0011 mm = 0.0077 mm
DISCUSSION
An ocular micrometer is a
small glass disk with thin lines and numbering etched in the glass.
An ocular micrometer was
placed into one ocular on your microscope so that the lines superimposed on the
image will allow to measure the specimen.
For each magnification, must
compare the lines on the ocular micrometer to the lines on a stage micrometer.
The stage micrometer is a
glass slide with precisely space lines etched at known intervals.
The vertical distance of an
object that is in focus. When
magnification is increased, less of the object is in focus (depth of field
decreases) – but greater detail of the area in focus can be seen.
We can adjust the focus of our
eyepiece in order to make the scale as sharp as possible. If we do that, also
adjust the other eyepiece to match the focus. Any ocular scale must be
calibrated, using a device called a stage micrometer
The ocular lenses usually
magnify 10X. Thus the total
magnification observed is the multiplication of the power of magnification of
the ocular times the objective. For
example an object magnified by the ocular and the 40X high-dry objective is
viewed at 4002 times its real size. Most
ocular lenses can be moved back and forth to adjust to the interpupillary
distance of the student. When
first using the microscope, adjust the ocular lenses back and forth until a
circular field is viewed with both eyes open. Additionally, many microscopes allow
the ocular lenses to be adjusted up and down (mechanical tube length
adjustment) and there is a scale alongside the tube. After adjusting the interpupillary
distance, read the distance off the scale and adjust the tube length of the
ocular lens to the same value. Now
the ocular lenses are adjusted to our eyes.
Conclusion
Ocular micrometer a glass disk that fits in a microscope eyepiece and
that has a ruled scale when calibrated with a slide micrometer, direct
measurements size of a microscopic object can be made.
Reference
2.2 Neubauer Chamber
INTRODUCTION
Neubauer Chamber
Neubauer chamber or hemocytometer are
more convenient for counting microbes. It is a heavy glass slide with two
counting areas separated by a H-shaped through figure. To prepare the counting
chamber the mirror-like polished surface is carefully cleaned with lens paper.
The coverslip is also cleaned. Coverslips for counting chambers are specially
made and are thicker than those for conventional microscopy, since they must be
heavy enough to overcome the surface tension of a drop of liquid. The coverslip
is placed over the counting surface prior to putting on the cell suspension.
The suspension is introduced into one of the V-shaped wells with a pasteur or
other type of pipet. The area under the coverslip fills by capillary action.
Enough liquid should be introduced so that the mirrored surface is just
covered. The charged counting chamber is then placed on the microscope stage
and the counting grid is brought into focus at low power.
It is essential to be extremely careful with higher power objectives, since
the counting chamber is much thicker than a conventional slide. The chamber or
an objective lens may be damaged if the user is not not careful. One entire
grid on standard hemacytometers with Neubauer rulings can be seen at 40x (4x
objective). The main divisions separate the grid into 9 large squares (like a
tic-tac-toe grid). Each square has a surface area of one square mm, and the
depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a
volume of 0.9 mm-cubed.
Objective
1.
To count cells using microscope.
RESULT
sum of the cell 10 box = 642
average = 642/10 = 64.2
volume box (16 box) = 0.2 mm x 0.2 mmx 0.1 mm
= 4 x 10^-3 mm
= 4 x 10^-6 cm^3
64.2 cell in 4 x 10 ^-6 ml
1 ml = 642/ 4 x 10 ^-6 = 1.61 x 10^8 cell/ml
DISCUSSION
To prepare the
counting chamber the mirror-like polished surface is carefully cleaned with
lens paper. The coverslip is also cleaned.
Coverslips for
counting chambers are specially made and are thicker than those for
conventional microscopy, since they must be heavy enough to overcome the
surface tension of a drop of liquid.
The coverslip
is placed over the counting surface prior to putting on the cell suspension.
The suspension is introduced into one of the H-shaped wells with a pasteur or
other type of pipet.
The area under
the coverslip fills by capillary action. Enough liquid should be introduced so
that the mirrored surface is just covered.
The charged
counting chamber is then placed on the microscope stage and the counting grid
is brought into focus at low power.
Conclusion
Neubauer chamber is a device
used for determining the number of cells per unit volume of a suspension. Hemocytometer
was widely used since it was originally designed for performing blood cell
counts.
Reference
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