2.1: Ocular
Micrometer
1.Introduction
An ocular micrometer makes it very simple to measure
the size of micro-organisms that are mounted on a slide. An ocular micrometer
consists of a circular disk of glass which has graduations engraved on one
surface. In some microscopes, the ocular has to be disassembled so that the
disk can be placed on a shelf in the ocular tube between the two lenses;
however, in most microscopes the ocular micrometer is simply inserted into the
bottom of the ocular. Before an ocular
micrometer can be used, it is necessary to calibrate it for each of the
objectives by using a stage micrometer. The principle purpose of the exercise
is to show you how to calibrate an ocular micrometer for the various objectives
on a microscope. The distance between the lines of an ocular micrometer is an
arbitrary measurement that only has meaning if the ocular micrometer is
calibrated for the objective being used. A stage micrometer, also known as an
objective micrometer, has scribed lines on it that are exactly 0.01mm (10
micrometers) apart. Illustration C reveals the appearance of these graduations.
2. Materials:
Microscope fitted with an ocular micrometer
Slide micrometer
Stained preparation of yeast and bacteria
3. Procedure:
1. The stage micrometer is
placed on the stage
2. The microscope is focused
using the lowest power objective until the image on the stage micrometer is
observed superimposed on the eyepiece scale.
3. The amount of the
divisions of the eyepiece scale corresponding top a definite number of
divisions on the stage scale is determined.
4. The measurement of an
eyepiece division in micrometer is calculated.
5. The process is repeated by
using the high-power and oil immersion objective.
6. An example is shown as
below:
Each division of the stage micrometer = 10 µm.
If 100 eyepiece divisions = 11 stage division = 110
µm then:
1 eyepiece division = 110/100 = 1.1 µm
7. The diameter of the field for each objective is
calculated and recorded for further reference.
8. The average dimensions (in µm) of a sample of yeast
cells is determined and the process is repeated using a sample of bacterial
cells.
RESULT
Total Magnification = objective lens power x eyepiece
lens power (10x)
The ratio magnification was calculated by:
Number divisions on stage micrometer/number of
divisions on ocular micrometer
Hence, the observed diameter of yeast =0.9mm ocular
By using 10x magnification, 1mm on the stage
micrometer represent 9.5mm on the eyepiece
|
Magnification
|
Ocular(mm)
|
Stage(mm)
|
|
4x
|
3.7
|
1
|
|
10x
|
9.5
|
1
|
Discussion
1.
An ocular micrometer is a glass disc which is inside the
ocular lens. However, to view the specimen, the distance between the etched
lines is dependent on the objective lens.
2.
To calibrate the micrometer, the ocular and stage are
superimposed.
3.
The stage micrometer is act as a fixed ruler that fixed by
the certain distance. It is used to be tell the distance that far apart marks on
ocular micrometer.
Conclusion
By using both of the ocular micrometer and stage
micrometer, the actual size of the microorganisms can be measured accurately.
Reference
2.2 Neubauer Chamber
Introduction
In this experiment, we are going to use the
hemocytometer or known as counting chamber. It is a device used in manual blood
cell counts consisting of a counting chamber of uniform that is covered by a
ruler cover glass so that region under ruled square contains a known volume of
the diluted blood specimen. The cover glass which is placed on the sample
containing grid with an arrangement of squares of different sizes that make the
counting of cells easier. It makes the counting number of cells in specified
volume become possible.
Materials and
reagents:
Serial dilutions of bacteria cultures
Neubauer and coverslip
70% ethanol
Sterile Pasteur pipettes
Procedure:
1.
By using the
sterile dropper, the yeast culture (use 10-3 or 10-4) dilution is added to
space between the coverslip and the counting chamber.
2. The neubauer with the yeast culture is observed
3. Under the microscope again with the same
magnification.
4. The number of yeast cells in the 16 randomly chosen
squares are recorded.
Counting:
1. The large middle square of the neubauer
hemocytometer is chosen.
2. The 16 smaller squares are randomly chosen from the
large square.
3. The number of yeast cells is counted from the 16
small squares.
4. The average number of yeast cells per small squares
is calculated (Only the cells inside a square and the cells that touch the
upper and left grids are counted. For example, there are 7 yeast cells
counted in a small square with red grids on the top and left in the diagram
below.)
Result
Calculations
Volume of a big box =1mm X 1mm X 0.1mm
=0.1mm3
Hence, 0.1mm3 = 0.0001cm3=0.0001ml
One big box =256 small boxes
Volume of one small box = 0.0001 / 256 =3.90625 x 10-7
Average of cells in 10 small boxes
=(25+27+17+15+23+20+19+24+26+17)/10
=213 / 10
=21.3cells
Concentration =21.3 /(3.90625 x 10-7)
=54528000cell/ml
Discussion
1.
The Neubauer chamber is a device to determine the number of
particles per volume unit of a liquid which is made of special optical glass.
2.
The central square millimeter is
ruled into 25 groups of 16 small squares. However, triple lines seperates each
groups and the middle one is boundary.
3.
Furthermore, the suspension used
should be diluted to prevent those cells overlapping with each other. Hence, it
easier to be counted.
.
Conclusion
By using the Neubauer chamber, we can estimate the number
of the microorganisms and calculated the concentration of the cells.
Reference
http://www.hawksley.co.uk/cell-count_glassware/05a_cell-counting/
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