OCULAR MICROMETER
Introduction
An
ocular micrometer is a glass disk that attaches to a microscope's eyepiece. An
ocular micrometer has a ruler that allows the user to measure the size of
magnified objects. The distance between the marks on the ruler depends upon the
degree of magnification. The ruler on a typical ocular micrometer has between
50 to 100 individual marks, is 2 mm long and has a distance of 0.01 mm between
marks.
The
main purpose of ocular micrometer is to measuer the size of microorganism. The
ocular micrometer consists of 2 main scales that are stage scale and ocular
scales.
To
use this micrometer, we must loacte the ocular scale at the out microscope
eyepiece to allow for measurements of objects being viewed. The other scale
called stage scales locate at the special slide that contains scales.
Objective
To measure and count cells using a microscope
Result
1) lactobacillus
2) 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
|
|
LACTOBACILLUS :
400x magnification
2 division x 0.769 µm
=1.538 µm
1000x magnification
5 division x 0.94 µm
=4.7 µm
YEAST :
400x magnification
4
division x 0.769 µm
=3.076 µm
1000x magnification
7
division x 0.94 µm
=
6.58 µm
Discussion
An ocular micrometer is a glass disk that attaches to a microscope's eyepiece. An ocular micrometer has a ruler that allows the user to measure the size of magnified objects. The distance between the marks on the ruler depends upon the degree of magnification. The ruler on a typical ocular micrometer has between 50 to 100 individual marks, is 2 mm long and has a distance of 0.01 mm between marks.
How to use a ocular micrometer
1. 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.
2. 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
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
Before using an ocular micrometer, we must calibrated it first. A typical scale consists of 50 - 100 divisions. You may have to adjust the focus of your eyepiece in order to make the scale as sharp as possible. If you do that, also adjust the other eyepiece to match the focus. Any ocular scale must be calibrated, using a device called a stage micrometer.A stage micrometer is simply a microscope slide with a scale etched on the surface. A typical micrometer scale is 2 mm long and at least part of it should be etched with divisions of 0.01 mm (10 µm).
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
1. This report has identified the correct way to calibrate ocular micrometer. Ocular micrometer has a ruler that allows the user to measure the size of magnified objects. A special slides which contains scales also used to place the objects being observed. Besides, this report also show how to calculate the scale using stage scale and ocular eyepiece. By learning these, small particles such as microorganisms or cell can be measure and the size can be compared.
NEUBAUER CHAMBER
Introduction
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.
It is essential to be
extremely careful with higher power objectives, since the counting chamber is
much thicker than a conventional slide. 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.
Result
 |
Figure 1 : 400x magnification
|
Result from the 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
Discussion
Using a Counting Chamber
1. To
prepare the counting chamber the mirror-like polished surface is carefully
cleaned with lens paper. The coverslip is also cleaned.
2. 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.
3. 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.
4. The
area under the coverslip fills by capillary action. Enough liquid should be
introduced so that the mirrored surface is just covered.
5. The
charged counting chamber is then placed on the microscope stage and the
counting grid is brought into focus at low power.
6. Here
is a way to determine a particle count using a Neubauer hemocytometer. Suppose
that you conduct a count as described above, and count 187 particles in the
five small squares described.
Conclusion
This report has
identified how to use the Neubauer Chamber. This special chamber is a heavy
glass slide with two counting areas separated by a H-shaped trough. A special
coverslip is placed over the counting areas. When the slide observed via
microscope, the sample was viewed on the many grids. These grids helps to
count the cells under the microscope.
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