ocular micrometer
introduction
An ocular micrometer is a small glass disc on which uniformly spaced lines of
undesignated distance are etched. The ocular micrometer, located inside one of the
ocular lenses of the microscope is calibrated against a stage micrometer slide that has
uniformly spaced lines of known distances. Stage micrometers vary but most contain a
line 2mm long that is subdivided into 0.01mm (10um) lengths. After calibration with the
stage micrometer, the ocular micrometer can be used as a ruler to measure the actual size
of the objects in the microscope's field of view.
A Stage Micrometer is simply a microscope slide with a finely divided
scale marked on the surface. The scale is of a known true length and
is used for calibration of optical systems with eyepiece graticule
patterns. This is particularly important when alternating between
objectives on one microscope or when using the same graticule in
different microscopes.
Result
40 magnification
Observation | Length,mm | Width, mm |
1 | 3.21 | 1.75 |
2 | 3.22 | 1.50 |
3 | 3.45 | 1.25 |
4 | 3.65 | 1.75 |
5 | 3.15 | 1.50 |
6 | 3.46 | 1.25 |
7 | 3.47 | 1.50 |
8 | 3.44 | 1.75 |
9 | 3.70 | 1.50 |
10 | 3.30 | 1.25 |
Total | 34.05 | 15.00 |
100 magnification
Observation | Length,mm | Width,mm |
1 | 3.35 | 1.0 |
2 | 3.32 | 1.1 |
3 | 3.31 | 1.2 |
4 | 3.46 | 1.1 |
5 | 3.28 | 1.1 |
6 | 3.37 | 1.1 |
7 | 3.35 | 1.0 |
8 | 3.33 | 1.2 |
9 | 3.34 | 1.1 |
10 | 3.46 | 1.0 |
Total | 33.57 | 10.9 |
400 magnification
Observation | Length,mm | Width,mm |
1 | 2.870 | 0.950 |
2 | 2.918 | 0.925 |
3 | 2.918 | 0.900 |
4 | 2.923 | 0.925 |
5 | 2.919 | 0.950 |
6 | 2.867 | 0.900 |
7 | 2.905 | 0.925 |
8 | 2.903 | 0.900 |
9 | 2.918 | 0.925 |
10 | 2.908 | 0.925 |
Total | 29.049 | 9.225 |
Calculation
Average length | = Total of exact distance Number of observation |
= 34.05 10 | |
= 3.405 |
Average width | = Total of exact distance Number of observation |
= 15.00 10 | |
= 1.50 |
For 100 power of magnification,
Average length | = Total of exact distance Number of observation |
= 33.57 10 | |
= 3.357 |
Average width | = Total of exact distance Number of observation |
= 10.9 10 | |
= 1.09 |
For 400 power of magnification,
Average length | = Total of exact distance Number of observation |
= 29.049 10 | |
= 2.9049 |
Average width | = Total of exact distance Number of observation |
= 9.225 10 | |
= 0.9225 |
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.
In this experiment, we take some time to move the stage until we are able to superimpose the lines of the ocular micrometer upon those of the stage micrometer. With the lines of the two micrometers coinciding at one end of the field, we can count the spaces of each micrometer to a point at which the lines of the micrometers coincide again.
To make observations on living specimens we need to work very quickly, or you have to prepare sealed wet mounts so that the specimens do not dry up in the heat of the microscope lamp. We did it several times because sometimes the coverslip on the slip contains bubbles.We also spend a lot of time to observe the sample of yeast cells because we failed to do a good wet amount and inexperienced in using the micrometer.
Instructions on how to use ocular micrometer:
1Measure 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.
2Attach 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.
3Slide 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.
4Move 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.
5Count 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.
6Write 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.
7Use 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.
Conclusion:
The size of the microorganism can be measured by using ocular micrometer and stage micrometer.
References:
neubauer chamber
introduction
The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm). Additionally, a grid is etched into the glass of the hemocytometer. This grid, an arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to determine the number of cells in a specified volume.
Result
11 | 12 | ||
7 | |||
0 | 9 |
1 small box =1mm/4
=0.25mm
Average of cell no. =(11+12+0+9+7)/5
=8 cells
Volume of 1 small box =area x depth
=1.25mm x 0.25mm x 0.1mm
=6.25 x 10-6 mL
Concentrations of cells =average of cells/volume
=8 cells / 6.25 x 10-6 mL
=1.28 x 106 cells / mL
Discussions
Be careful not to scratch the surface of the cover slip, and a "light touch" so not to break it into pieces. Place the cover slip over the counting platform, pressing on the elevated ridges of the hematocytometer, not the center.
To get the best result, the mirror-like polished surfaced must be cleaned with lens paper. The cover slip is also cleaned to prevent unwanted particles that may affect the counting result. Also, enough liquid should be introduced so that the mirrored surface is just covered. Finally, when placing the cover slip we should make sure no air bubble is trapped inside.
There is no need to count the whole chamber. If there are lot of cells, perform a count in a section of the chamber and use the grid to determine what proportion of the chamber that is. Then estimate how many cells are in the chamber to continue with the calculation.
Conclusion:
By using neubauer chamber, we can estimate and hence calculate the number of microorganisms.
Reference
- http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html
- http://medicalshopping.akasdoctor.com/detail.cfm?ID=MEDICARECNR&storeid=1
- http://www.tpub.com/corpsman/232.htm
- http://www.uni-greifswald.de/~immuteach/methods/counting_chamber/counting_chamber.html
- http://www.mohfw.nic.in/listfac/Microbiology%20R/IMPROVED%20NEUBAUER%20COUNTING%20CHAMBER.htm
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