2.1
OCULAR MICROMETER
INTRODUCTION
Your microscope may be equipped with a
scale (called a reticule) that is built into one eyepiece. The reticule can be
used to measure any planar dimension in a microscope field since the ocular can
be turned in any direction and the object of interest can be repositioned with
the stage manipulators. To measure the length of an object note the number of
ocular divisions spanned by the object. Then multiply by the conversion factor
for the magnification used. The conversion factor is different at each magnification.
Therefore, when using a reticule for the first time, it is necessary to
calibrate the scale by focusing on a second micrometer scale (a stage
micrometer) placed directly on the stage.
Instructions
Measure the actual size of the
letter on the microscope slide using the millimeter ruler. This measurement
will help you calibrate the ocular micrometer to determine if it is giving you
accurate measurements.
Attach the ocular micrometer to
the microscope eyepiece by unscrewing the eyepiece cap, placing the ocular
micrometer over the lens and screwing the eyepiece cap back into place. Some
microscopes may have an ocular micrometer pre-installed, allowing you to skip
this step.
Slide the stage micrometer onto
the microscope slide stage. Adjust the microscope to the lowest possible
magnification, which should bring the grid on the stage micrometer into focus.
Move the stage micrometer until the
measurement marks on the ocular micrometer align with the measurement marks on
the stage micrometer. The measurement "0" on the ocular micrometer
should line up with the measurement "0.0" on the stage micrometer.
Count the number of measurement marks
until the measurements of both the micrometers line up again. At 4x
magnification (the lowest setting on most microscopes), the two micrometers
will line up again at "3" on the ocular micrometer and
"0.3" on the stage micrometer.
Write down the number of measurement
marks between the aligning measurements for the two micrometers. The distance
between measurement marks is 0.01 mm, so you can now determine the distance
between coinciding measurement marks. Repeat the exercise at higher
magnifications (10x, 40x and 100x), and record these values as well.
Use the calibrated ocular micrometer to
measure the dimensions of the letter printed on your slide. Compare the
dimensions to the dimensions you measured with the millimeter ruler to ensure
that the ocular micrometer is functioning properly.
OBJECTIVE
To
measure and count cells using a microscope
RESULTS
1A)
lactobacillus
2B)
yeast
400x magnification
|
1000x magnification
|
|||
calibrate
|
Stage
micrometer
|
Ocular
division
|
Stage
micrometer
|
Ocular
divison
|
0.03
mm
|
39
division
|
0.09
mm
|
96
division
|
|
7.69
x10⁻⁴ mm
|
1
division
|
9.38x10⁻⁴ mm
|
1
division
|
|
0.769
µm
|
1
division
|
0.94
µm
|
1
division
|
|
YEAST :
400x magnification
4 division x 0.769 µm
=3.076 µm
1000x magnification
7 division x 0.94 µm
= 6.58 µm
LACTOBACILLUS
:
400x magnification
2 division x 0.769 µm
=1.538 µm
1000x magnification
5
division x 0.94 µm
=4.7 µm
DISCUSSION
Suppose
that a stage micrometer scale has divisions that are equal to 0.1 mm, which is
100 micrometers (µm). Suppose that the scale is lined up with the ocular scale,
and at 100x it is observed that each micrometer division covers the same
distance as 10 ocular divisions. Then one ocular division (smallest increment
on the scale) = 10 µm at 100 power. The conversion to other magnifications is
accomplished by factoring in the difference in magnification. In the example,
the calibration would be 25 µm at 40x, 2.5 µm at 400x, and 1 µm at 1000x.
Some
stage micrometers are finely divided only at one end. These are particularly
useful for determining the diameter of a microscope field. One of the larger
divisions is positioned at one edge of the field of view, so that the fine part
of the scale ovelaps the opposite side. The field diameter can then be
determined to the maximum available precision.
CONCLUSION
A
specimen such as Chaos (Pelomyxa) carolinensis represents a real challenge.
Ameoboid organisms are irregularly shaped most of the time. Is it flat on the
slide, or does it extend up toward the coverslip? Perhaps it is attached to
both. What model do you use as a basis for volume estimation? Is it best to
assume a particular shape and take measurements at different times? Is it best
to estimate a maximum and minimum for each possible dimension and obtain a
range of possible volumes? Remember, you are only asked to estimate. Sometimes
the best estimates have a potential error of more than an order of magnitude.
When
you change magnifications, it appears as though the size of the stage
micrometer is changing while the ocular micrometer remains fixed. Both
micrometers actually stay fixed, but the view of the stage micrometer becomes
distorted as the magnification changes.
REFERENCE
2.2 NEUBAUER
CHAMBER
INTRODUCTION
For microbiology, cell culture, and many
applications that require use of suspensions of cells it is necessary to
determine cell concentration. One can often determine cell density of a
suspension spectrophotometrically, however that form of determination does not
allow an assessment of cell viability, nor can one distinguish cell types.
A device used for determining the number
of cells per unit volume of a suspension is called a counting chamber. The most
widely used type of chamber is called a hemocytometer, since it was originally
designed for performing blood cell counts.
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.
OBJECTIVE
To
measure and count cells using a microscope
RESULTS
experiment is as shown below :
390 ÷ 10 = 39
Volume
= 0.02 x 0.02 x 0.01 mm
= 4 x 10⁻3 mm
= 4 x 10⁻6 cm
= 4 x 10-6 ml
1ml
= 39 ÷ (4 x 10-6 )
= 9.75 x 10-5 cells/ml
3
x 0.01 = 0.03 nm
39
divisions = 0.03 ÷ 39
= 7.69 x 10-4 mm
per division
= 0.769 µm per division
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.
Suspensions should be dilute enough so
that the cells or other particles do not overlap each other on the grid, and
should be uniformly distributed. To perform the count, determine the
magnification needed to recognize the desired cell type. Now systematically
count the cells in selected squares so that the total count is 100 cells or so
(number of cells needed for a statistically significant count). For large cells
this may mean counting the four large corner squares and the middle one. For a
dense suspension of small cells you may wish to count the cells in the four
1/25 sq. mm corners plus the middle square in the central square. Always decide
on a specific counting patter to avoid bias. For cells that overlap a ruling,
count a cell as "in" if it overlaps the top or right ruling, and
"out" if it overlaps the bottom or left ruling.
CONCLUSION
The original suspension must be mixed
thoroughly before taking a sample. This ensures the sample is representative,
and not just an artifact of the particular region of the original mixture it
was drawn from.
An
appropriate dilution of the mixture with regard to the number of cells to be
counted should be used. If the sample is not diluted enough, the cells will be
too crowded and difficult to count. If it is too dilute, the sample size will
not be enough to make strong inferences about the concentration in the original
mixture.
By
performing a redundant test on a second chamber, the results can be compared.
If they differ greatly, the method of taking the sample may be unreliable
(e.g., the original mixture is not mixed thoroughly).
REFERENCE
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